323,659 results on '"PHOSPHORYLATION"'
Search Results
2. Fast and deep phosphoproteome analysis with the Orbitrap Astral mass spectrometer.
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Lancaster, Noah, Sinitcyn, Pavel, Forny, Patrick, Peters-Clarke, Trenton, Fecher, Caroline, Smith, Andrew, Shishkova, Evgenia, Arrey, Tabiwang, Pashkova, Anna, Robinson, Margaret, Arp, Nicholas, Fan, Jing, Hansen, Juli, Galmozzi, Andrea, Serrano, Lia, Rojas, Julie, Gasch, Audrey, Westphall, Michael, Stewart, Hamish, Hock, Christian, Damoc, Eugen, Pagliarini, David, Zabrouskov, Vlad, and Coon, Joshua
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Humans ,Phosphoproteins ,Animals ,HeLa Cells ,Phosphorylation ,Mice ,Proteome ,Mass Spectrometry ,Proteomics - Abstract
Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method, we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology is benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We apply this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detect 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of phosphorylation events relevant to mitochondrial and brain biology.
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- 2024
3. GRK2 kinases in the primary cilium initiate SMOOTHENED-PKA signaling in the Hedgehog cascade.
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Walker, Madison, Zhang, Jingyi, Steiner, William, Ku, Pei-I, Zhu, Ju-Fen, Michaelson, Zachary, Yen, Yu-Chen, Lee, Annabel, Long, Alyssa, Casey, Mattie, Poddar, Abhishek, Nelson, Isaac, Arveseth, Corvin, Nagel, Falko, Clough, Ryan, LaPotin, Sarah, Kwan, Kristen, Schulz, Stefan, Stewart, Rodney, Tesmer, John, Caspary, Tamara, Subramanian, Radhika, Ge, Xuecai, and Myers, Benjamin
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Animals ,Cilia ,Smoothened Receptor ,Hedgehog Proteins ,G-Protein-Coupled Receptor Kinase 2 ,Signal Transduction ,Mice ,Cyclic AMP-Dependent Protein Kinases ,Zebrafish ,Phosphorylation ,Zebrafish Proteins ,NIH 3T3 Cells - Abstract
During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.
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- 2024
4. TMEM16F exacerbates tau pathology and mediates phosphatidylserine exposure in phospho-tau-burdened neurons.
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Zubia, Mario, Yong, Adeline, Holtz, Kristen, Huang, Eric, Jan, Yuh, and Jan, Lily
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P301S (PS19) ,TMEM16F ,lipid scrambling ,phosphatidylserine exposure ,tauopathy ,Animals ,Anoctamins ,Phosphatidylserines ,Neurons ,tau Proteins ,Mice ,Tauopathies ,Humans ,Microglia ,Phosphorylation ,Mice ,Transgenic ,Disease Models ,Animal ,Phospholipid Transfer Proteins ,Brain ,Mice ,Knockout - Abstract
TMEM16F is a calcium-activated phospholipid scramblase and nonselective ion channel, which allows the movement of lipids bidirectionally across the plasma membrane. While the functions of TMEM16F have been extensively characterized in multiple cell types, the role of TMEM16F in the central nervous system remains largely unknown. Here, we sought to study how TMEM16F in the brain may be involved in neurodegeneration. Using a mouse model that expresses the pathological P301S human tau (PS19 mouse), we found reduced tauopathy and microgliosis in 6- to 7-mo-old PS19 mice lacking TMEM16F. Furthermore, this reduction of pathology can be recapitulated in the PS19 mice with TMEM16F removed from neurons, while removal of TMEM16F from microglia of PS19 mice did not significantly impact tauopathy at this time point. Moreover, TMEM16F mediated aberrant phosphatidylserine exposure in neurons with phospho-tau burden. These studies raise the prospect of targeting TMEM16F in neurons as a potential treatment of neurodegeneration.
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- 2024
5. A designed ankyrin-repeat protein that targets Parkinsons disease-associated LRRK2.
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Dederer, Verena, Sanz Murillo, Marta, Karasmanis, Eva, Hatch, Kathryn, Chatterjee, Deep, Preuss, Franziska, Abdul Azeez, Kamal, Nguyen, Landon, Galicia, Christian, Dreier, Birgit, Plückthun, Andreas, Versees, Wim, Mathea, Sebastian, Leschziner, Andres, Reck-Peterson, Samara, and Knapp, Stefan
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DARPin ,LRRK2 ,Parkinson’s disease ,Rab8a ,WD40 ,cryo-electron microscopy ,kinase ,kinase inhibitor ,microtubule ,Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ,Humans ,Ankyrin Repeat ,Parkinson Disease ,HEK293 Cells ,rab GTP-Binding Proteins ,Phosphorylation ,Cryoelectron Microscopy ,Protein Binding - Abstract
Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinsons disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.
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- 2024
6. Multi-step control of homologous recombination via Mec1/ATR suppresses chromosomal rearrangements
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Xie, Bokun, Sanford, Ethan James, Hung, Shih-Hsun, Wagner, Mateusz, Heyer, Wolf-Dietrich, and Smolka, Marcus B
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Biochemistry and Cell Biology ,Biological Sciences ,Genetics ,Prevention ,Human Genome ,Saccharomyces cerevisiae Proteins ,Saccharomyces cerevisiae ,Protein Serine-Threonine Kinases ,Homologous Recombination ,Intracellular Signaling Peptides and Proteins ,Cell Cycle Proteins ,Checkpoint Kinase 2 ,RecQ Helicases ,Signal Transduction ,Phosphorylation ,Chromosome Aberrations ,Gene Rearrangement ,Mec1 ,Sgs1 ,Resection ,Chromosomal Rearrangement ,Information and Computing Sciences ,Medical and Health Sciences ,Developmental Biology ,Biological sciences ,Biomedical and clinical sciences - Abstract
The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.
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- 2024
7. A RAB7A phosphoswitch coordinates Rubicon Homology protein regulation of Parkin-dependent mitophagy.
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Tudorica, Dan, Basak, Bishal, Puerta Cordova, Alexia, Khuu, Grace, Rose, Kevin, Lazarou, Michael, Holzbaur, Erika, and Hurley, James
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Mitophagy ,Humans ,rab7 GTP-Binding Proteins ,Phosphorylation ,Ubiquitin-Protein Ligases ,Protein Serine-Threonine Kinases ,rab GTP-Binding Proteins ,HeLa Cells ,Protein Binding ,Intracellular Signaling Peptides and Proteins ,Autophagy-Related Proteins ,Mitochondria ,HEK293 Cells - Abstract
Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.
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- 2024
8. Growth factor–dependent phosphorylation of Gαi shapes canonical signaling by G protein–coupled receptors
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Roy, Suchismita, Sinha, Saptarshi, Silas, Ananta James, Ghassemian, Majid, Kufareva, Irina, and Ghosh, Pradipta
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Biochemistry and Cell Biology ,Biological Sciences ,1.1 Normal biological development and functioning ,Generic health relevance ,Phosphorylation ,Signal Transduction ,Humans ,HEK293 Cells ,GTP-Binding Protein alpha Subunits ,Gi-Go ,Receptors ,CXCR4 ,Epidermal Growth Factor ,Receptors ,G-Protein-Coupled ,Animals ,Biochemistry and cell biology - Abstract
A long-standing question in the field of signal transduction is how distinct signaling pathways interact with each other to control cell behavior. Growth factor receptors and G protein-coupled receptors (GPCRs) are the two major signaling hubs in eukaryotes. Given that the mechanisms by which they signal independently have been extensively characterized, we investigated how they may cross-talk with each other. Using linear ion trap mass spectrometry and cell-based biophysical, biochemical, and phenotypic assays, we found at least three distinct ways in which epidermal growth factor affected canonical G protein signaling by the Gi-coupled GPCR CXCR4 through the phosphorylation of Gαi. Phosphomimicking mutations in two residues in the αE helix of Gαi (tyrosine-154/tyrosine-155) suppressed agonist-induced Gαi activation while promoting constitutive Gβγ signaling. Phosphomimicking mutations in the P loop (serine-44, serine-47, and threonine-48) suppressed Gi activation entirely, thus completely segregating growth factor and GPCR pathways. As expected, most of the phosphorylation events appeared to affect intrinsic properties of Gαi proteins, including conformational stability, nucleotide binding, and the ability to associate with and to release Gβγ. However, one phosphomimicking mutation, targeting the carboxyl-terminal residue tyrosine-320, promoted mislocalization of Gαi from the plasma membrane, a previously uncharacterized mechanism of suppressing GPCR signaling through G protein subcellular compartmentalization. Together, these findings elucidate not only how growth factor and chemokine signals cross-talk through the phosphorylation-dependent modulation of Gαi but also how such cross-talk may generate signal diversity.
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- 2024
9. Baseline unfolded protein response signaling adjusts the timing of the mammalian cell cycle.
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Chowdhury, Soham, Solley, Sabrina, Polishchuk, Elena, Bacal, Julien, Conrad, Julia, Gardner, Brooke, Acosta-Alvear, Diego, and Zappa, Francesca
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Unfolded Protein Response ,eIF-2 Kinase ,Signal Transduction ,Humans ,Cell Cycle ,Endoplasmic Reticulum ,Phosphorylation ,Eukaryotic Initiation Factor-2 ,Activating Transcription Factor 6 ,Protein Serine-Threonine Kinases ,Endoribonucleases ,Animals ,HeLa Cells ,Endoplasmic Reticulum Stress - Abstract
The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, suggesting coordination between the mechanisms overseeing ER integrity and those controlling the cell cycle to maintain organelle inheritance. The Unfolded Protein Response (UPR) is a conserved signaling network that regulates ER homeostasis. Here, we show that pharmacological and genetic inhibition of the UPR sensors IRE1, ATF6, and PERK in unstressed cells delays the cell cycle, with PERK inhibition showing the most penetrant effect, which was associated with a slowdown of the G1-to-S/G2 transition. Treatment with the small molecule ISRIB to bypass the effects of PERK-dependent phosphorylation of the translation initiation factor eIF2α had no such effect, suggesting that cell cycle timing depends on PERKs kinase activity but is independent of eIF2α phosphorylation. Using complementary light and electron microscopy and flow cytometry-based analyses, we also demonstrate that the ER enlarges before mitosis. Together, our results suggest coordination between UPR signaling and the cell cycle to maintain ER physiology during cell division.
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- 2024
10. Arabidopsis α-Aurora kinase plays a role in cytokinesis through regulating MAP65-3 association with microtubules at phragmoplast midzone.
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Deng, Xingguang, Xiao, Yu, Tang, Xiaoya, Liu, Bo, and Lin, Honghui
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Arabidopsis ,Cytokinesis ,Microtubules ,Arabidopsis Proteins ,Microtubule-Associated Proteins ,Phosphorylation ,Mutation ,Spindle Apparatus ,Protein Serine-Threonine Kinases ,Plants ,Genetically Modified ,Mitosis - Abstract
The α-Aurora kinase is a crucial regulator of spindle microtubule organization during mitosis in plants. Here, we report a post-mitotic role for α-Aurora in reorganizing the phragmoplast microtubule array. In Arabidopsis thaliana, α-Aurora relocated from spindle poles to the phragmoplast midzone, where it interacted with the microtubule cross-linker MAP65-3. In a hypomorphic α-Aurora mutant, MAP65-3 was detected on spindle microtubules, followed by a diffuse association pattern across the phragmoplast midzone. Simultaneously, phragmoplast microtubules remained belatedly in a solid disk array before transitioning to a ring shape. Microtubules at the leading edge of the matured phragmoplast were often disengaged, accompanied by conspicuous retentions of MAP65-3 at the phragmoplast interior edge. Specifically, α-Aurora phosphorylated two residues towards the C-terminus of MAP65-3. Mutation of these residues to alanines resulted in an increased association of MAP65-3 with microtubules within the phragmoplast. Consequently, the expansion of the phragmoplast was notably slower compared to wild-type cells or cells expressing a phospho-mimetic variant of MAP65-3. Moreover, mimicking phosphorylation reinstated disrupted MAP65-3 behaviors in plants with compromised α-Aurora function. Overall, our findings reveal a mechanism in which α-Aurora facilitates cytokinesis progression through phosphorylation-dependent restriction of MAP65-3 associating with microtubules at the phragmoplast midzone.
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- 2024
11. Temporal dynamics of the multi-omic response to endurance exercise training
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Bae, Dam, Dasari, Surendra, Dennis, Courtney, Evans, Charles R, Gaul, David A, Ilkayeva, Olga, Ivanova, Anna A, Kachman, Maureen T, Keshishian, Hasmik, Lanza, Ian R, Lira, Ana C, Muehlbauer, Michael J, Nair, Venugopalan D, Piehowski, Paul D, Rooney, Jessica L, Smith, Kevin S, Stowe, Cynthia L, Zhao, Bingqing, Clark, Natalie M, Jimenez-Morales, David, Lindholm, Malene E, Many, Gina M, Sanford, James A, Smith, Gregory R, Vetr, Nikolai G, Zhang, Tiantian, Almagro Armenteros, Jose J, Avila-Pacheco, Julian, Bararpour, Nasim, Ge, Yongchao, Hou, Zhenxin, Marwaha, Shruti, Presby, David M, Natarajan Raja, Archana, Savage, Evan M, Steep, Alec, Sun, Yifei, Wu, Si, Zhen, Jimmy, Bodine, Sue C, Esser, Karyn A, Goodyear, Laurie J, Schenk, Simon, Montgomery, Stephen B, Fernández, Facundo M, Sealfon, Stuart C, Snyder, Michael P, Adkins, Joshua N, Ashley, Euan, Burant, Charles F, Carr, Steven A, Clish, Clary B, Cutter, Gary, Gerszten, Robert E, Kraus, William E, Li, Jun Z, Miller, Michael E, Nair, K Sreekumaran, Newgard, Christopher, Ortlund, Eric A, Qian, Wei-Jun, Tracy, Russell, Walsh, Martin J, Wheeler, Matthew T, Dalton, Karen P, Hastie, Trevor, Hershman, Steven G, Samdarshi, Mihir, Teng, Christopher, Tibshirani, Rob, Cornell, Elaine, Gagne, Nicole, May, Sandy, Bouverat, Brian, Leeuwenburgh, Christiaan, Lu, Ching-ju, Pahor, Marco, Hsu, Fang-Chi, Rushing, Scott, Walkup, Michael P, Nicklas, Barbara, Rejeski, W Jack, Williams, John P, Xia, Ashley, Albertson, Brent G, Barton, Elisabeth R, Booth, Frank W, Caputo, Tiziana, Cicha, Michael, De Sousa, Luis Gustavo Oliveira, Farrar, Roger, Hevener, Andrea L, Hirshman, Michael F, Jackson, Bailey E, Ke, Benjamin G, Kramer, Kyle S, Lessard, Sarah J, Makarewicz, Nathan S, Marshall, Andrea G, and Nigro, Pasquale
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Health Sciences ,Sports Science and Exercise ,Prevention ,Human Genome ,Cardiovascular ,Behavioral and Social Science ,Genetics ,Physical Activity ,2.1 Biological and endogenous factors ,1.1 Normal biological development and functioning ,Generic health relevance ,Inflammatory and immune system ,Good Health and Well Being ,Animals ,Female ,Humans ,Male ,Rats ,Acetylation ,Blood ,Cardiovascular Diseases ,Databases ,Factual ,Endurance Training ,Epigenome ,Inflammatory Bowel Diseases ,Internet ,Lipidomics ,Metabolome ,Mitochondria ,Multiomics ,Non-alcoholic Fatty Liver Disease ,Organ Specificity ,Phosphorylation ,Physical Conditioning ,Animal ,Physical Endurance ,Proteome ,Proteomics ,Time Factors ,Transcriptome ,Ubiquitination ,Wounds and Injuries ,MoTrPAC Study Group ,Lead Analysts ,MoTrPAC Study Group ,General Science & Technology - Abstract
Regular exercise promotes whole-body health and prevents disease, but the underlying molecular mechanisms are incompletely understood1-3. Here, the Molecular Transducers of Physical Activity Consortium4 profiled the temporal transcriptome, proteome, metabolome, lipidome, phosphoproteome, acetylproteome, ubiquitylproteome, epigenome and immunome in whole blood, plasma and 18 solid tissues in male and female Rattus norvegicus over eight weeks of endurance exercise training. The resulting data compendium encompasses 9,466 assays across 19 tissues, 25 molecular platforms and 4 training time points. Thousands of shared and tissue-specific molecular alterations were identified, with sex differences found in multiple tissues. Temporal multi-omic and multi-tissue analyses revealed expansive biological insights into the adaptive responses to endurance training, including widespread regulation of immune, metabolic, stress response and mitochondrial pathways. Many changes were relevant to human health, including non-alcoholic fatty liver disease, inflammatory bowel disease, cardiovascular health and tissue injury and recovery. The data and analyses presented in this study will serve as valuable resources for understanding and exploring the multi-tissue molecular effects of endurance training and are provided in a public repository ( https://motrpac-data.org/ ).
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- 2024
12. APOE4/4 is linked to damaging lipid droplets in Alzheimers disease microglia.
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Haney, Michael, Pálovics, Róbert, Munson, Christy, Long, Chris, Johansson, Patrik, Yip, Oscar, Dong, Wentao, Rawat, Eshaan, Tsai, Andy, Guldner, Ian, Lamichhane, Bhawika, Smith, Amanda, Schaum, Nicholas, Calcuttawala, Kruti, Shin, Andrew, Wang, Yung-Hua, Wang, Chengzhong, Koutsodendris, Nicole, Serrano, Geidy, Beach, Thomas, Reiman, Eric, Glass, Christopher, Abu-Remaileh, Monther, Enejder, Annika, Huang, Yadong, Wyss-Coray, Tony, Schlachetzki, Johannes, and West, Elizabeth
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Animals ,Female ,Humans ,Male ,Mice ,Alzheimer Disease ,Amyloid beta-Peptides ,Apolipoprotein E4 ,Induced Pluripotent Stem Cells ,Lipid Droplets ,Microglia ,Triglycerides ,tau Proteins ,Culture Media ,Conditioned ,Phosphorylation ,Genetic Predisposition to Disease - Abstract
Several genetic risk factors for Alzheimers disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimers disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimers disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimers disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aβ induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimers disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimers disease.
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- 2024
13. A helical fulcrum in eIF2B coordinates allosteric regulation of stress signaling.
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Lawrence, Rosalie, Shoemaker, Sophie, Deal, Aniliese, Sangwan, Smriti, Anand, Aditya, Wang, Lan, Walter, Peter, and Marqusee, Susan
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Eukaryotic Initiation Factor-2B ,Allosteric Regulation ,Cryoelectron Microscopy ,Guanine Nucleotide Exchange Factors ,Signal Transduction ,Phosphorylation - Abstract
The integrated stress response (ISR) enables cells to survive a variety of acute stresses, but chronic activation of the ISR underlies age-related diseases. ISR signaling downregulates translation and activates expression of stress-responsive factors that promote return to homeostasis and is initiated by inhibition of the decameric guanine nucleotide exchange factor eIF2B. Conformational and assembly transitions regulate eIF2B activity, but the allosteric mechanisms controlling these dynamic transitions and mediating the therapeutic effects of the small-molecule ISR inhibitor ISRIB are unknown. Using hydrogen-deuterium exchange-mass spectrometry and cryo-electron microscopy, we identified a central α-helix whose orientation allosterically coordinates eIF2B conformation and assembly. Biochemical and cellular signaling assays show that this switch-helix controls eIF2B activity and signaling. In sum, the switch-helix acts as a fulcrum of eIF2B conformational regulation and is a highly conserved actuator of ISR signal transduction. This work uncovers a conserved allosteric mechanism and unlocks new therapeutic possibilities for ISR-linked diseases.
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- 2024
14. Phosphorylation regulates taus phase separation behavior and interactions with chromatin.
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Abasi, Lannah, Elathram, Nesreen, Movva, Manasi, Deep, Amar, Corbett, Kevin, and Debelouchina, Galia
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Humans ,Phosphorylation ,tau Proteins ,Chromatin ,Nucleosomes ,Heterochromatin ,Phase Separation ,DNA - Abstract
Tau is a microtubule-associated protein often found in neurofibrillary tangles (NFTs) in the brains of patients with Alzheimers disease. Beyond this context, mounting evidence suggests that tau localizes into the nucleus, where it may play a role in DNA protection and heterochromatin regulation. The molecular mechanisms behind these observations are currently unclear. Using in vitro biophysical experiments, here we demonstrate that tau can undergo liquid-liquid phase separation (LLPS) with DNA, mononucleosomes, and reconstituted nucleosome arrays under low salt conditions. Low concentrations of tau promote chromatin compaction and protect DNA from digestion. While the material state of samples at physiological salt is dominated by chromatin oligomerization, tau can still associate strongly and reversibly with nucleosome arrays. These properties are driven by taus strong interactions with linker and nucleosomal DNA. In addition, tau co-localizes into droplets formed by nucleosome arrays and phosphorylated HP1α, a key heterochromatin constituent thought to function through an LLPS mechanism. Importantly, LLPS and chromatin interactions are disrupted by aberrant tau hyperphosphorylation. These biophysical properties suggest that tau may directly impact DNA and chromatin accessibility and that loss of these interactions could contribute to the aberrant nuclear effects seen in tau pathology.
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- 2024
15. Role of the leucine-rich repeat protein kinase 2 C-terminal tail in domain cross-talk
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Sharma, Pallavi Kaila, Weng, Jui-Hung, Manschwetus, Jascha T, Wu, Jian, Ma, Wen, Herberg, Friedrich W, and Taylor, Susan S
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Biochemistry and Cell Biology ,Biological Sciences ,Parkinson's Disease ,Neurodegenerative ,Neurosciences ,Brain Disorders ,2.1 Biological and endogenous factors ,Aetiology ,Neurological ,Protein Serine-Threonine Kinases ,Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ,Leucine-Rich Repeat Proteins ,Protein Domains ,Mutation ,Peptides ,Phosphorylation ,C-terminal helix ,GaMD simulation ,LRRK2 ,Parkinson's disease ,peptide array ,Chemical Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biochemistry and cell biology - Abstract
Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.
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- 2024
16. Manuka Honey Inhibits Human Breast Cancer Progression in Preclinical Models
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Márquez-Garbán, Diana C, Yanes, Cristian D, Llarena, Gabriela, Elashoff, David, Hamilton, Nalo, Hardy, Mary, Wadehra, Madhuri, McCloskey, Susan A, and Pietras, Richard J
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Biomedical and Clinical Sciences ,Oncology and Carcinogenesis ,Women's Health ,Breast Cancer ,Cancer ,5.1 Pharmaceuticals ,Humans ,Honey ,Breast Neoplasms ,Female ,Animals ,Apoptosis ,MCF-7 Cells ,Cell Proliferation ,Signal Transduction ,Mice ,Xenograft Model Antitumor Assays ,Mice ,Nude ,Leptospermum ,TOR Serine-Threonine Kinases ,Proto-Oncogene Proteins c-akt ,Antineoplastic Agents ,STAT3 Transcription Factor ,Disease Progression ,AMP-Activated Protein Kinases ,Cell Line ,Tumor ,Phosphorylation ,Manuka honey ,breast cancer ,estrogen receptor-positive breast cancer ,triple-negative breast cancer ,in vivo xenografts ,AMP kinase signaling ,mTOR ,STAT3 ,Food Sciences ,Nutrition and Dietetics ,Clinical sciences ,Nutrition and dietetics ,Public health - Abstract
Manuka honey (MH) exhibits potential antitumor activity in preclinical models of a number of human cancers. Treatment in vitro with MH at concentrations ranging from 0.3 to 5.0% (w/v) led to significant dose-dependent inhibition of proliferation of human breast cancer MCF-7 cells, but anti-proliferative effects of MH were less pronounced in MDA-MB-231 breast cancer cells. Effects of MH were also tested on non-malignant human mammary epithelial cells (HMECs) at 2.5% w/v, and it was found that MH reduced the proliferation of MCF-7 cells but not that of HMECs. Notably, the antitumor activity of MH was in the range of that exerted by treatment of MCF-7 cells with the antiestrogen tamoxifen. Further, MH treatment stimulated apoptosis of MCF-7 cells in vitro, with most cells exhibiting acute and significant levels of apoptosis that correlated with PARP activation. Additionally, the effects of MH induced the activation of AMPK and inhibition of AKT/mTOR downstream signaling. Treatment of MCF7 cells with increased concentrations of MH induced AMPK phosphorylation in a dose-dependent manner that was accompanied by inhibition of phosphorylation of AKT and mTOR downstream effector protein S6. In addition, MH reduced phosphorylated STAT3 levels in vitro, which may correlate with MH and AMPK-mediated anti-inflammatory properties. Further, in vivo, MH administered alone significantly inhibited the growth of established MCF-7 tumors in nude mice by 84%, resulting in an observable reduction in tumor volume. Our findings highlight the need for further research into the use of natural compounds, such as MH, for antitumor efficacy and potential chemoprevention and investigation of molecular pathways underlying these actions.
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- 2024
17. Evolved histone tail regulates 53BP1 recruitment at damaged chromatin
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Kelliher, Jessica L, Folkerts, Melissa L, Shen, Kaiyuan V, Song, Wan, Tengler, Kyle, Stiefel, Clara M, Lee, Seong-Ok, Dray, Eloise, Zhao, Weixing, Koss, Brian, Pannunzio, Nicholas R, and Leung, Justin W
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Biochemistry and Cell Biology ,Biological Sciences ,Genetics ,1.1 Normal biological development and functioning ,Generic health relevance ,Humans ,Adaptor Proteins ,Signal Transducing ,BRCA1 Protein ,Camptothecin ,Cell Cycle Proteins ,Chromatin ,DNA Damage ,DNA Repair ,HEK293 Cells ,Histones ,Phosphorylation ,Tumor Suppressor p53-Binding Protein 1 ,Ubiquitin-Protein Ligases ,Ubiquitination - Abstract
The master DNA damage repair histone protein, H2AX, is essential for orchestrating the recruitment of downstream mediator and effector proteins at damaged chromatin. The phosphorylation of H2AX at S139, γH2AX, is well-studied for its DNA repair function. However, the extended C-terminal tail is not characterized. Here, we define the minimal motif on H2AX for the canonical function in activating the MDC1-RNF8-RNF168 phosphorylation-ubiquitination pathway that is important for recruiting repair proteins, such as 53BP1 and BRCA1. Interestingly, H2AX recruits 53BP1 independently from the MDC1-RNF8-RNF168 pathway through its evolved C-terminal linker region with S139 phosphorylation. Mechanistically, 53BP1 recruitment to damaged chromatin is mediated by the interaction between the H2AX C-terminal tail and the 53BP1 Oligomerization-Tudor domains. Moreover, γH2AX-linker mediated 53BP1 recruitment leads to camptothecin resistance in H2AX knockout cells. Overall, our study uncovers an evolved mechanism within the H2AX C-terminal tail for regulating DNA repair proteins at damaged chromatin.
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- 2024
18. Comprehensive Characterization of fucAO Operon Activation in Escherichia coli
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Zhang, Zhongge, Huo, Jialu, Velo, Juan, Zhou, Harry, Flaherty, Alex, and Saier, Milton H
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Biochemistry and Cell Biology ,Biological Sciences ,Genetics ,fucAO operon ,operon promoter ,Crp ,FucR ,SrsR ,transcriptional activation ,transcriptionally silent region ,protein-protein interactions ,Escherichia coli ,Fucose ,Binding Sites ,Phosphorylation ,Operon ,fucAO operon ,Other Chemical Sciences ,Other Biological Sciences ,Chemical Physics ,Biochemistry and cell biology ,Microbiology ,Medicinal and biomolecular chemistry - Abstract
Wildtype Escherichia coli cells cannot grow on L-1,2-propanediol, as the fucAO operon within the fucose (fuc) regulon is thought to be silent in the absence of L-fucose. Little information is available concerning the transcriptional regulation of this operon. Here, we first confirm that fucAO operon expression is highly inducible by fucose and is primarily attributable to the upstream operon promoter, while the fucO promoter within the 3'-end of fucA is weak and uninducible. Using 5'RACE, we identify the actual transcriptional start site (TSS) of the main fucAO operon promoter, refuting the originally proposed TSS. Several lines of evidence are provided showing that the fucAO locus is within a transcriptionally repressed region on the chromosome. Operon activation is dependent on FucR and Crp but not SrsR. Two Crp-cAMP binding sites previously found in the regulatory region are validated, where the upstream site plays a more critical role than the downstream site in operon activation. Furthermore, two FucR binding sites are identified, where the downstream site near the first Crp site is more important than the upstream site. Operon transcription relies on Crp-cAMP to a greater degree than on FucR. Our data strongly suggest that FucR mainly functions to facilitate the binding of Crp to its upstream site, which in turn activates the fucAO promoter by efficiently recruiting RNA polymerase.
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- 2024
19. Screening of herbal extracts binding with vascular endothelial growth factor by applying HerboChip platform.
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Liu, Yang, Liang, Jia-Ming, Guo, Guo-Xia, Qiu, Yu-Huan, Yu, Le-Le, Tsim, Karl Wah-Keung, Qin, Qi-Wei, Chan, Gallant Kar-Lun, and Hu, Wei-Hui
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CHINESE medicine , *VASCULAR endothelial growth factors , *PHARMACEUTICAL technology , *PHOSPHORYLATION , *DATA analysis , *HERBAL medicine , *CELL proliferation , *IMMUNODIAGNOSIS , *DESCRIPTIVE statistics , *ENDOTHELIAL cells , *WESTERN immunoblotting , *ANALYSIS of variance , *STATISTICS , *CELL survival , *DATA analysis software , *NEOVASCULARIZATION , *CELL surface antigens - Abstract
Background: Traditional Chinese medicine (TCM) has been hailed as a rich source of medicine, but many types of herbs and their functions still need to be rapidly discovered and elucidated. HerboChip, a target-based drug screening platform, is an array of different fractions deriving from herbal extracts. This study was designed to identify effective components from TCM that interact with vascular endothelial growth factor (VEGF) as a target using HerboChip. Methods: Selected TCMs that are traditionally used as remedies for cancer prevention and wound healing were determined and extracted with 50% ethanol. Biotinylated-VEGF was hybridized with over 500 chips coated with different HPLC-separated fractions from TCM extracts and straptavidin-Cy5 was applied to identify plant extracts containing VEGF-binding fractions. Cytotoxicity of selected herbal extracts and their activities on VEGF-mediated angiogenic functions were evaluated. Results: Over 500 chips were screened within a week, and ten positive hits were identified. The interaction of the identified herbal extracts with VEGF was confirmed in cultured endothelial cells. The identified herbs promoted or inhibited VEGF-mediated cell proliferation, migration and tube formation. Results from western blotting analysis demonstrated the identified herbal extracts significantly affected VEGF-triggered phosphorylations of eNOS, Akt and Erk. Five TCMs demonstrated potentiating activities on the VEGF response and five TCMs revealed suppressive activities. Conclusions: The current results demonstrated the applicability of the HerboChip platform and systematically elucidated the activity of selected TCMs on angiogenesis and its related signal transduction mechanisms. [ABSTRACT FROM AUTHOR]
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- 2024
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20. Tyrosine phosphorylation and palmitoylation of TRPV2 ion channel tune microglial beta-amyloid peptide phagocytosis.
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Yang, Shaobin, Du, Yaqin, Li, Yanhong, Tang, Qi, Zhang, Yimeng, and Zhao, Xiaoqian
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TRPV cation channels , *PROTEIN-tyrosine kinases , *ION channels , *PEPTIDES , *ALZHEIMER'S disease - Abstract
Alzheimer's disease (AD) is the leading form of dementia, characterized by the accumulation and aggregation of amyloid in brain. Transient receptor potential vanilloid 2 (TRPV2) is an ion channel involved in diverse physiopathological processes, including microglial phagocytosis. Previous studies suggested that cannabidiol (CBD), an activator of TRPV2, improves microglial amyloid-β (Aβ) phagocytosis by TRPV2 modulation. However, the molecular mechanism of TRPV2 in microglial Aβ phagocytosis remains unknown. In this study, we aimed to investigate the involvement of TRPV2 channel in microglial Aβ phagocytosis and the underlying mechanisms. Utilizing human datasets, mouse primary neuron and microglia cultures, and AD model mice, to evaluate TRPV2 expression and microglial Aβ phagocytosis in both in vivo and in vitro. TRPV2 was expressed in cortex, hippocampus, and microglia.Cannabidiol (CBD) could activate and sensitize TRPV2 channel. Short-term CBD (1 week) injection intraperitoneally (i.p.) reduced the expression of neuroinflammation and microglial phagocytic receptors, but long-term CBD (3 week) administration (i.p.) induced neuroinflammation and suppressed the expression of microglial phagocytic receptors in APP/PS1 mice. Furthermore, the hyper-sensitivity of TRPV2 channel was mediated by tyrosine phosphorylation at the molecular sites Tyr(338), Tyr(466), and Tyr(520) by protein tyrosine kinase JAK1, and these sites mutation reduced the microglial Aβ phagocytosis partially dependence on its localization. While TRPV2 was palmitoylated at Cys 277 site and blocking TRPV2 palmitoylation improved microglial Aβ phagocytosis. Moreover, it was demonstrated that TRPV2 palmitoylation was dynamically regulated by ZDHHC21. Overall, our findings elucidated the intricate interplay between TRPV2 channel regulated by tyrosine phosphorylation/dephosphorylation and cysteine palmitoylation/depalmitoylation, which had divergent effects on microglial Aβ phagocytosis. These findings provide valuable insights into the underlying mechanisms linking microglial phagocytosis and TRPV2 sensitivity, and offer potential therapeutic strategies for managing AD. [ABSTRACT FROM AUTHOR]
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- 2024
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21. Phosphorylated Tau at T181 accumulates in the serum of hibernating Syrian hamsters and rapidly disappears after arousal.
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León-Espinosa, G., Murillo, A. M. M., Turegano-Lopez, M., DeFelipe, J., and Holgado, M.
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NEUROFIBRILLARY tangles , *GOLDEN hamster , *TAU proteins , *ALZHEIMER'S disease , *BLOOD proteins , *NEURODEGENERATION - Abstract
The search for biomarkers for the early diagnosis of neurodegenerative diseases is a growing area. Numerous investigations are exploring minimally invasive and cost-effective biomarkers, with the detection of phosphorylated Tau (pTau) protein emerging as one of the most promising fields. pTau is the main component of the paired helical filaments found in the brains of Alzheimer's disease cases and serves as a precursor in the formation of neurofibrillary tangles (NFTs). Recent research has revealed that analysis of p-Tau181, p-Tau217 and p-Tau231 in blood may be an option for detecting the preclinical stage of Alzheimer's disease. In this study, we have analyzed the values of pTau 181 in the serum of Syrian hamsters during hibernation. Naturally, over the course of hibernation, these animals exhibit a reversible accumulation of pTau in the brain tissue, which rapidly disappears upon awakening. A biosensing system based on the interferometric optical detection method was used to measure the concentration of pTau181 protein in serum samples from Syrian hamsters. This method eliminates the matrix effect and amplifies the signal obtained by using silicon dioxide nanoparticles (SiO2 NPs) biofunctionalized with the αpTau181 antibody. Our results indicate a substantial increase in the serum concentration of pTau in threonine-181 during hibernation, which disappears completely 2–3 h after awakening. Investigating the mechanism by which pTau protein appears in the blood non-pathologically may enhance current diagnostic techniques. Furthermore, since this process is reversible, and no tangles are detected in the brains of hibernating hamsters, additional analysis may contribute to the discovery of improved biomarkers. Additionally, exploring drugs targeting pTau to prevent the formation of tangles or studying the outcomes of any pTau-targeted treatment could be valuable. [ABSTRACT FROM AUTHOR]
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- 2024
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22. Phosphorylation activates master growth regulator DELLA by promoting histone H2A binding at chromatin in Arabidopsis.
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Huang, Xu, Zentella, Rodolfo, Park, Jeongmoo, Reser, Larry, Bai, Dina L., Ross, Mark M., Shabanowitz, Jeffrey, Hunt, Donald F., and Sun, Tai-ping
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GROWTH regulators ,GENETIC transcription ,TRANSCRIPTION factors ,PLANT development ,PHOSPHORYLATION - Abstract
DELLA proteins are conserved master growth regulators that play a central role in controlling plant development in response to internal and environmental cues. DELLAs function as transcription regulators, which are recruited to target promoters by binding to transcription factors (TFs) and histone H2A via their GRAS domain. Recent studies showed that DELLA stability is regulated post-translationally via two mechanisms, phytohormone gibberellin-induced polyubiquitination for its rapid degradation, and Small Ubiquitin-like Modifier (SUMO)-conjugation to increase its accumulation. Moreover, DELLA activity is dynamically modulated by two distinct glycosylations: DELLA-TF interactions are enhanced by O-fucosylation, but inhibited by O-linked N-acetylglucosamine (O-GlcNAc) modification. However, the role of DELLA phosphorylation remains unclear as previous studies showing conflicting results ranging from findings that suggest phosphorylation promotes or reduces DELLA degradation to others indicating it has no effect on its stability. Here, we identify phosphorylation sites in REPRESSOR OF ga1-3 (RGA, an AtDELLA) purified from Arabidopsis by mass spectrometry analysis, and show that phosphorylation of two RGA peptides in the PolyS and PolyS/T regions enhances RGA activity by promoting H2A binding and RGA association with target promoters. Notably, phosphorylation does not affect RGA-TF interactions or RGA stability. Our study has uncovered a molecular mechanism of phosphorylation-induced DELLA activity. Huang and colleagues uncover a key role of phosphorylation in enhancing the activity of Arabidopsis master growth regulator DELLA by promoting its interaction with histone H2A at target chromatin. [ABSTRACT FROM AUTHOR]
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- 2024
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23. Two KaiABC systems control circadian oscillations in one cyanobacterium.
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Köbler, Christin, Schmelling, Nicolas M., Wiegard, Anika, Pawlowski, Alice, Pattanayak, Gopal K., Spät, Philipp, Scheurer, Nina M., Sebastian, Kim N., Stirba, Florian P., Berwanger, Lutz C., Kolkhof, Petra, Maček, Boris, Rust, Michael J., Axmann, Ilka M., and Wilde, Annegret
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GENE clusters ,CIRCADIAN rhythms ,BACKSCATTERING ,SYNECHOCYSTIS ,PHOSPHORYLATION - Abstract
The circadian clock of cyanobacteria, which predicts daily environmental changes, typically includes a standard oscillator consisting of proteins KaiA, KaiB, and KaiC. However, several cyanobacteria have diverse Kai protein homologs of unclear function. In particular, Synechocystis sp. PCC 6803 harbours, in addition to a canonical kaiABC gene cluster (named kaiAB1C1), two further kaiB and kaiC homologs (kaiB2, kaiB3, kaiC2, kaiC3). Here, we identify a chimeric KaiA homolog, named KaiA3, encoded by a gene located upstream of kaiB3. At the N-terminus, KaiA3 is similar to response-regulator receiver domains, whereas its C-terminal domain resembles that of KaiA. Homology analysis shows that a KaiA3-KaiB3-KaiC3 system exists in several cyanobacteria and other bacteria. Using the Synechocystis sp. PCC 6803 homologs, we observe circadian oscillations in KaiC3 phosphorylation in vitro in the presence of KaiA3 and KaiB3. Mutations of kaiA3 affect KaiC3 phosphorylation, leading to growth defects under both mixotrophic and chemoheterotrophic conditions. KaiC1 and KaiC3 exhibit phase-locked free-running phosphorylation rhythms. Deletion of either system (∆kaiAB1C1 or ∆kaiA3B3C3) alters the period of the cellular backscattering rhythm. Furthermore, both oscillators are required to maintain high-amplitude, self-sustained backscatter oscillations with a period of approximately 24 h, indicating their interconnected nature. The cyanobacterial circadian clock typically includes a standard oscillator consisting of proteins KaiA, KaiB and KaiC, but some cyanobacteria have additional homologous proteins of unclear function. Here, the authors show that a KaiABC homolog system contributes, together with the canonical oscillator, to the control of circadian rhythms in the model cyanobacterium Synechocystis sp. PCC 6803. [ABSTRACT FROM AUTHOR]
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- 2024
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24. Chk2 homolog Mek1 limits exonuclease 1–dependent DNA end resection during meiotic recombination in Saccharomyces cerevisiae.
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Krystosek, Jennifer T and Bishop, Douglas K
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DNA metabolism , *PHOSPHORYLATION , *CELL cycle proteins , *RESEARCH funding , *CELL physiology , *DNA , *ESTERASES , *PROTEIN-tyrosine kinases , *MASS spectrometry , *YEAST - Abstract
The conserved Rad2/XPG family 5′–3′ exonuclease, exonuclease 1 (Exo1), plays many roles in DNA metabolism including during resolution of DNA double-strand breaks via homologous recombination. Prior studies provided evidence that the end resection activity of Exo1 is downregulated in yeast and mammals by Cdk1/2 family cyclin-dependent and checkpoint kinases, including budding yeast kinase Rad53 which functions in mitotic cells. Here, we provide evidence that the master meiotic kinase Mek1 , a paralog of Rad53 , limits 5′–3′ single-strand resection at the sites of programmed meiotic DNA breaks. Mutational analysis suggests that the mechanism of Exo1 suppression by Mek1 differs from that of Rad53. [ABSTRACT FROM AUTHOR]
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- 2024
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25. Mck1-mediated proteolysis of CENP-A prevents mislocalization of CENP-A for chromosomal stability in Saccharomyces cerevisiae.
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Zhang, Tianyi, Au, Wei-Chun, Ohkuni, Kentaro, Shrestha, Roshan L, Kaiser, Peter, and Basrai, Munira A
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ENZYME metabolism , *PROTEIN metabolism , *RESEARCH funding , *PHOSPHORYLATION , *CELL cycle proteins , *CELL physiology , *CHROMOSOME abnormalities , *CELLULAR signal transduction , *PLANT extracts , *GENE expression , *IMMUNOHISTOCHEMISTRY , *PROTEOLYTIC enzymes , *HISTOLOGICAL techniques , *SACCHAROMYCES , *YEAST , *DNA-binding proteins , *PHENOTYPES - Abstract
Centromeric localization of evolutionarily conserved CENP-A (Cse4 in Saccharomyces cerevisiae) is essential for chromosomal stability. Mislocalization of overexpressed CENP-A to noncentromeric regions contributes to chromosomal instability in yeasts, flies, and humans. Overexpression and mislocalization of CENP-A observed in many cancers are associated with poor prognosis. Previous studies have shown that F-box proteins, Cdc4 and Met30 of the Skp, Cullin, F-box ubiquitin ligase cooperatively regulate proteolysis of Cse4 to prevent Cse4 mislocalization and chromosomal instability under normal physiological conditions. Mck1 -mediated phosphorylation of Skp, Cullin, F-box- Cdc4 substrates such as Cdc6 and Rcn1 enhances the interaction of the substrates with Cdc4. Here, we report that Mck1 interacts with Cse4 , and Mck1 -mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability. Our results showed that mck1 Δ strain overexpressing CSE4 (GAL- CSE4) exhibits lethality, defects in ubiquitin-mediated proteolysis of Cse4 , mislocalization of Cse4 , and reduced Cse4 – Cdc4 interaction. Strain expressing GAL- cse4 -3A with mutations in three potential Mck1 phosphorylation consensus sites (S10, S16, and T166) also exhibits growth defects, increased stability with mislocalization of Cse4 -3A, chromosomal instability, and reduced interaction with Cdc4. Constitutive expression of histone H3 (Δ16H3) suppresses the chromosomal instability phenotype of GAL- cse4 -3A strain, suggesting that the chromosomal instability phenotype is linked to Cse4 -3A mislocalization. We conclude that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4 – Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and chromosomal instability. These studies advance our understanding of pathways that regulate cellular levels of CENP-A to prevent mislocalization of CENP-A in human cancers. [ABSTRACT FROM AUTHOR]
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- 2024
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26. An acidic loop in the forkhead-associated domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.
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Weng, Qixuan, Wan, Lihong, Straker, Geburah C, Deegan, Tom D, Duncker, Bernard P, Neiman, Aaron M, Luk, Ed, and Hollingsworth, Nancy M
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PROTEIN metabolism , *CYTOGENETICS , *IN vitro studies , *RESEARCH funding , *PHOSPHORYLATION , *CELL physiology , *TRANSCRIPTION factors , *CELLULAR signal transduction , *RECOMBINANT proteins , *TRANSFERASES , *YEAST , *MICROBIAL genetics , *DNA-binding proteins - Abstract
The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at double-strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover-specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80 , regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a 5-amino acid sequence, RPSKR, located between the DNA-binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full-length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a noncanonical interaction with this motif. A second protein, the 5′-3′ helicase Rrm3 , similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt 2-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint and, in certain circumstances, exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif. [ABSTRACT FROM AUTHOR]
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- 2024
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27. GSK3s promote the phyB‐ELF3‐HMR complex formation to regulate plant thermomorphogenesis.
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Yang, Ruizhen, Dong, Huixue, Xie, Xianzhi, Zhang, Yunwei, and Sun, Jiaqiang
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GLYCOGEN synthase kinase , *PLANT growth , *PLANT development , *PHYTOCHROMES , *PHOSPHORYLATION - Abstract
Summary Although elevated ambient temperature causes many effects on plant growth and development, the mechanisms of plant high‐ambient temperature sensing remain unknown. In this study, we show that GLYCOGEN SYNTHASE KINASE 3s (GSK3s) negatively regulate high‐ambient temperature response and oligomerize upon high‐temperature treatment. We demonstrate that GSK3 kinase BIN2 specifically interacts with the high‐temperature sensor phytochrome B (phyB) but not the high‐temperature sensor EARLY FLOWER 3 (ELF3) to phosphorylate and promote phyB photobody formation. Furthermore, we show that phosphorylation of phyB by GSK3s promotes its interaction with ELF3. Subsequently, we find that ELF3 recruits the phyB photobody facilitator HEMERA (HMR) to promote its association with phyB. Taken together, our data reveal a mechanism that GSK3s promote the phyB‐ELF3‐HMR complex formation in regulating plant thermomorphogenesis. [ABSTRACT FROM AUTHOR]
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- 2024
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28. A sophisticated mechanism governs Pol ζ activity in response to replication stress.
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Li, Chun, Fan, Shuchen, Li, Pan, Bai, Yuzhen, Wang, Ye, Cui, Yueyun, Li, Mengdi, Wang, Ruru, Shao, Yuan, Wang, Yingying, Zheng, Shuo, Wang, Rong, Gao, Lijun, Li, Miaomiao, Zheng, Yuanyuan, Wang, Fengting, Gao, Sihang, Feng, Shiguo, Wang, Jianing, and Qu, Xinqi
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MUTAGENESIS ,MUTAGENS ,DNA polymerases ,PHOSPHORYLATION ,DNA ,HUMAN beings - Abstract
DNA polymerase ζ (Pol ζ) plays an essential role in replicating damaged DNA templates but contributes to mutagenesis due to its low fidelity. Therefore, ensuring tight control of Pol ζ's activity is critical for continuous and accurate DNA replication, yet the specific mechanisms remain unclear. This study reveals a regulation mechanism of Pol ζ activity in human cells. Under normal conditions, an autoinhibition mechanism keeps the catalytic subunit, REV3L, inactive. Upon encountering replication stress, however, ATR-mediated phosphorylation of REV3L's S279 cluster activates REV3L and triggers its degradation via a caspase-mediated pathway. This regulation confines the activity of Pol ζ, balancing its essential role against its mutations causing potential during replication stress. Overall, our findings elucidate a control scheme that fine tunes the low-fidelity polymerase activity of Pol ζ under challenging replication scenarios. DNA polymerase ζ (Pol ζ) maintains continuity but impairs accuracy during DNA replication. Here, the authors report a mechanism by which human cells precisely control the essential, yet mutagenic, activity of Pol ζ to ensure both continuation and fidelity during DNA replication challenges. [ABSTRACT FROM AUTHOR]
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- 2024
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29. TaNAC1 boosts powdery mildew resistance by phosphorylation‐dependent regulation of TaSec1a and TaCAMTA4 via PP2Ac/CDPK20.
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Liu, Yuanming, You, Hongguang, Li, Hanping, Zhang, Chujun, Guo, Huan, Huang, Xueling, Zhang, Qiong, Zhang, Xiangyu, Ma, Chuang, Wang, Yajuan, Li, Tingdong, Ji, Wanquan, Kang, Zhensheng, and Zhang, Hong
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TRANSCRIPTION factors , *NUCLEAR proteins , *PHOSPHOPROTEIN phosphatases , *POWDERY mildew diseases , *NUCLEIC acids - Abstract
Summary The integrity of wheat (Triticum aestivum) production is increasingly jeopardized by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), particularly amid the vicissitudes of climate change. Here, we delineated the role of a wheat transcription factor, TaNAC1, which precipitates cellular apoptosis and fortifies resistance against Bgt. Utilizing BiFC, co‐immunoprecipitation, protein quantification, luciferase report assays, we determined that cytoplasmic TaNAC1‐7A undergoes phosphorylation at the S184/S258 sites by TaCDPK20, facilitating its nuclear translocation. This migration appears to prime further phosphorylation by TaMPK1, thereby enhancing transcriptional regulatory activity. Notably, the apoptotic activity of phosphorylated TaNAC1‐7A is negatively modulated by the nuclear protein phosphatase PP2Ac. Furthermore, activation of TaNAC1 phosphorylation initiates transcription of downstream genes TaSec1a and TaCAMTA4, through binding to the C[T/G]T[N7]A[A/C]G nucleic acid motif. Suppression of TaNAC1, TaCDPK20, and TaMPK1 in wheat compromises its resistance to Bgt strain E09, whereas overexpression of TaNAC1 and silencing of PP2Ac markedly elevate resistance levels. Our results reveal the pivotal role of TaNAC1 in basal resistance which is mediated by its effects on homotypic fusion, vacuolar protein sorting, and the expression of defense‐related genes. The findings highlight the potential through targeting TaNAC1 and its regulators as a strategy for improving wheat's resistance to fungal pathogens. [ABSTRACT FROM AUTHOR]
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- 2024
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30. Differential contribution of Arabidopsis chitin receptor complex components to defense signaling and ubiquitination‐dependent endocytotic removal from the plasma membrane.
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Mittendorf, Josephine, Niebisch, Jule Meret, Pierdzig, Leon, Sun, Siqi, Petutschnig, Elena Kristin, and Lipka, Volker
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PROTEIN kinases , *CELL membranes , *UBIQUITINATION , *AUTOPHOSPHORYLATION , *CHITIN , *ENDOCYTOSIS - Abstract
Summary In Arabidopsis, the enzymatically active lysin motif‐containing receptor‐like kinase (LysM‐RLK) CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) and the pseudokinases LYSIN MOTIF‐CONTAINING RECEPTOR‐LIKE KINASE 5 (LYK5) and LYK4 are the core components of the canonical chitin receptor complex. CERK1 dimerizes and autophosphorylates upon chitin binding, resulting in activation of chitin signaling. In this study, we clarified and further elucidated the individual contributions of LYK4 and LYK5 to chitin‐dependent signaling using mutant (combination)s and stably transformed Arabidopsis plants expressing fluorescence‐tagged LYK5 and LYK4 variants from their endogenous promoters. Our analyses revealed that LYK5 interacts with CERK1 upon chitin treatment, independently of LYK4 and vice versa. We show that chitin‐induced autophosphorylation of CERK1 is predominantly dependent on LYK5, whereas chitin‐triggered ROS generation is almost exclusively mediated by LYK4. This suggests specific signaling functions of these two co‐receptor proteins apart from their redundant function in mitogen‐activated protein kinase (MAPK) signaling and transcriptional reprogramming. Moreover, we demonstrate that LYK5 is subject to chitin‐induced and CERK1‐dependent ubiquitination, which serves as a signal for chitin‐induced internalization of LYK5. Our experiments provide evidence that a combination of phosphorylation and ubiquitination events controls LYK5 removal from the plasma membrane via endocytosis, which likely contributes to receptor complex desensitization. [ABSTRACT FROM AUTHOR]
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- 2024
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31. Phospho-code of a conserved transcriptional factor underpins fungal virulence.
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Yang, Jiyun, Li, Bing, Pan, Yu-Ting, Wang, Ping, Sun, Mei-Ling, Kim, Ki-Tae, Sun, Hui, Ye, Jian-Ren, Jiao, Zhen, Lee, Yong-Hwan, and Huang, Lin
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TRANSCRIPTION factors , *MITOGEN-activated protein kinases , *CHITIN synthase , *PHYTOPATHOGENIC fungi , *FUNGAL virulence - Abstract
Background: Cell wall integrity (CWI) is crucial for fungal growth, pathogenesis, and adaptation to extracellular environments. Calcofluor white (CFW) is a cell wall perturbant that inhibits fungal growth, yet little is known about how phytopathogenic fungi respond to the CFW-induced stress. Results: In this study, we unveiled a significant discovery that CFW triggered the translocation of the transcription factor CgCrzA from the cytoplasm to the nucleus in Colletotrichum gloeosporioides. This translocation was regulated by an interacting protein, CgMkk1, a mitogen-activated protein kinase involved in the CWI pathway. Further analysis revealed that CgMkk1 facilitated nuclear translocation by phosphorylating CgCrzA at the Ser280 residue. Using chromatin immunoprecipitation sequencing, we identified two downstream targets of CgCrzA, namely CgCHS5 and CgCHS6, which are critical for growth, cell wall integrity, and pathogenicity as chitin synthase genes. Conclusions: These findings provide a novel insight into the regulatory mechanism of CgMkk1-CgCrzA-CgChs5/6, which enables response of the cell wall inhibitor CFW and facilitates infectious growth for C. gloeosporioides. [ABSTRACT FROM AUTHOR]
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- 2024
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32. A Novel tsRNA, m7G‐3′ tiRNA LysTTT, Promotes Bladder Cancer Malignancy Via Regulating ANXA2 Phosphorylation.
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Ying, Xiaoling, Hu, Wenyu, Huang, Yapeng, Lv, Yifan, Ji, Ding, Chen, Cong, Yang, Baotong, Zhang, Chengcheng, Liang, Yaomin, Zhang, Haiqing, Liu, Mingrui, Yuan, Gang, Wu, Wenqi, and Ji, Weidong
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TRANSFER RNA , *BLADDER cancer , *RNA modification & restriction , *CHEMICAL models , *PHOSPHORYLATION , *NON-coding RNA - Abstract
Emerging evidence indicates that transfer RNA (tRNA)‐derived small RNAs (tsRNAs), originated from tRNA with high abundance RNA modifications, play an important role in many complex physiological and pathological processes. However, the biological functions and regulatory mechanisms of modified tsRNAs in cancer remain poorly understood. Here, it is screened for and confirmed the presence of a novel m7G‐modified tsRNA, m7G‐3′‐tiRNA LysTTT (mtiRL), in a variety of chemical carcinogenesis models by combining small RNA sequencing with an m7G small RNA‐modified chip. Moreover, it is found that mtiRL, catalyzed by the tRNA m7G‐modifying enzyme mettl1, promotes bladder cancer (BC) malignancy in vitro and in vivo. Mechanistically, mtiRL is found to specifically bind the oncoprotein Annexin A2 (ANXA2) to promote its Tyr24 phosphorylation by enhancing the interactions between ANXA2 and Yes proto‐oncogene 1 (Yes1), leading to ANXA2 activation and increased p‐ANXA2‐Y24 nuclear localization in BC cells. Together, these findings define a critical role for mtiRL and suggest that targeting this novel m7G‐modified tsRNA can be an efficient way for to treat BC. [ABSTRACT FROM AUTHOR]
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- 2024
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33. Alzheimer's disease: a review on the current trends of the effective diagnosis and therapeutics.
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Abdul Manap, Aimi Syamima, Almadodi, Reema, Sultana, Shirin, Sebastian, Maheishinii Grace, Kavani, Kenil Sureshbhai, Lyenouq, Vanessa Elle, and Shankar, Aravind
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ALZHEIMER'S disease treatment ,BRAIN physiology ,ALZHEIMER'S disease diagnosis ,SALIVA analysis ,GASTROINTESTINAL system physiology ,TAU proteins ,HETEROCYCLIC compounds ,ANTI-inflammatory agents ,ALZHEIMER'S disease ,MILD cognitive impairment ,BEHAVIOR modification ,PHOSPHORYLATION ,SENSORY stimulation ,CLINICAL trials ,MICRORNA ,ENZYME inhibitors ,IMMUNOTHERAPY ,EICOSAPENTAENOIC acid ,EXERCISE therapy ,GOAL (Psychology) ,PHYTOCHEMICALS ,POSITRON emission tomography ,MAGNETIC resonance imaging ,NEURODEGENERATION ,DRUG approval ,EARLY diagnosis ,DRUG development ,MACROLIDE antibiotics ,STEM cells ,COGNITIVE therapy ,BIOMARKERS ,AMYLOID beta-protein precursor ,TEARS (Body fluid) ,SYMPTOMS - Abstract
The most prevalent cause of dementia is Alzheimer's disease. Cognitive decline and accelerating memory loss characterize it. Alzheimer's disease advances sequentially, starting with preclinical stages, followed by mild cognitive and/or behavioral impairment, and ultimately leading to Alzheimer's disease dementia. In recent years, healthcare providers have been advised to make an earlier diagnosis of Alzheimer's, prior to individuals developing Alzheimer's disease dementia. Regrettably, the identification of early-stage Alzheimer's disease in clinical settings can be arduous due to the tendency of patients and healthcare providers to disregard symptoms as typical signs of aging. Therefore, accurate and prompt diagnosis of Alzheimer's disease is essential in order to facilitate the development of disease-modifying and secondary preventive therapies prior to the onset of symptoms. There has been a notable shift in the goal of the diagnosis process, transitioning from merely confirming the presence of symptomatic AD to recognizing the illness in its early, asymptomatic phases. Understanding the evolution of disease-modifying therapies and putting effective diagnostic and therapeutic management into practice requires an understanding of this concept. The outcomes of this study will enhance indepth knowledge of the current status of Alzheimer's disease's diagnosis and treatment, justifying the necessity for the quest for potential novel biomarkers that can contribute to determining the stage of the disease, particularly in its earliest stages. Interestingly, latest clinical trial status on pharmacological agents, the nonpharmacological treatments such as behavior modification, exercise, and cognitive training as well as alternative approach on phytochemicals as neuroprotective agents have been covered in detailed. [ABSTRACT FROM AUTHOR]
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- 2024
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34. Mechanisms for controlling Dorsal nuclear levels.
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McGehee, James and Stathopoulos, Angelike
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POST-translational modification ,DROSOPHILA melanogaster ,GENE expression ,ECTODERM ,TRANSCRIPTION factors - Abstract
Formation of the Dorsal nuclear-cytoplasmic gradient is important for the proper establishment of gene expression patterns along the dorsal-ventral (DV) axis during embryogenesis in Drosophila melanogaster. Correct patterning of the DV axis leads to formation of the presumptive mesoderm, neurogenic ectoderm, dorsal ectoderm, and amnioserosa, which are tissues necessary for embryo viability. While Toll signaling is necessary for Dorsal gradient formation, a gradient still forms in the absence of Toll, suggesting there are additional mechanisms required to achieve correct nuclear Dorsal levels. Potential mechanisms include post-translational modification, shuttling, and nuclear spacing. Post-translational modification could affect import and export rates either directly through modification of a nuclear localization sequence or nuclear export sequence, or indirectly by affecting interactions with binding partners that alter import and export rates. Shuttling, which refers to the facilitated diffusion of Dorsal through its interaction with its cytoplasmic inhibitor Cactus, could regulate nuclear levels by delivering more Dorsal ventrally. Finally, nuclear spacing could result in higher nuclear levels by leaving fewer nuclei in the ventral domain to uptake Dorsal. This review details how each of these mechanisms may help establish Dorsal nuclear levels in the early fly embryo, which serves as a paradigm for understanding how the dynamics of graded inputs can influence patterning and target gene expression. Furthermore, careful analysis of nuclear Dorsal levels is likely to provide general insights as recent studies have suggested that the regulation of nuclear import affects the timing of gene expression at the maternal-to-zygotic transition. [ABSTRACT FROM AUTHOR]
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- 2024
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35. Electroacupuncture negatively regulates the Nesfatin-1/ERK/CREB pathway to alleviate HPA axis hyperactivity and anxiety-like behaviors caused by surgical trauma.
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Zheng, Jiayuan, Wang, Yu, Zhang, Chi, Zhang, Anjing, Zhou, Yuxiang, Xu, Yunhua, Yu, Jin, and Tian, Zhanzhuang
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TRAUMA surgery , *IN vitro studies , *PHOSPHORYLATION , *MENTAL illness , *CELLULAR signal transduction , *ANXIETY , *IN vivo studies , *ELECTROACUPUNCTURE , *MICE , *SURGICAL complications , *CORTICOTROPIN releasing hormone , *NEUROPEPTIDES , *HYPOTHALAMIC-pituitary-adrenal axis , *ANIMAL behavior , *ANIMAL experimentation , *ADRENOCORTICOTROPIC hormone , *GENE expression profiling , *ELECTRIC stimulation , *HEPATECTOMY - Abstract
Background: Hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis constitutes a pivotal response by surgical trauma, manifesting as a critical aspect of the acute stress reaction. This hyperactivity resulted in adverse surgical outcomes and is often associated with increased postoperative anxiety. Increased evidence suggests that Nesfatin-1 plays a crucial role in stress responses and stress-related psychiatric disorders. Electroacupuncture (EA) is widely used to alleviate stress responses and anxiety, although its mechanism of action remains unclear. This study aimed to assess the mechanisms by which hypothalamic Nesfatin-1 contribute to the alleviation of HPA axis hyperactivity and anxiety by EA. Methods: Partial hepatectomy (HT) was performed to simulate surgical trauma, and EA was applied at Zusanli (ST36) and Sanyinjiao (SP6). The levels of hypothalamic Nesfatin-1, c-Fos, and corticotropin-releasing hormone (CRH) were detected, and serum adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were regarded as indicators of HPA axis activity. Anxiety levels were assessed through open field tests (OFT), elevated plus maze (EPM), and light–dark box tests (LDBT). To investigate the role of Nesfatin-1, its expression was modulated using stereotactic viral injections or plasmid transfections. Transcriptome sequencing was employed to explore the downstream signaling pathways of Nesfatin-1. Additionally, brain cannula implantation was performed to facilitate targeted drug administration. Results: Our findings demonstrated that EA reduced the hypothalamic overexpression of CRH and Nesfatin-1, as well as serum levels of ACTH and CORT. Additionally, it alleviated anxiety-like behaviors resulting from surgical trauma. We observed that overexpression of Nesfatin-1 in the hypothalamic paraventricular nucleus (PVN) triggered hyperactivity of the HPA axis and anxiety. Conversely, knocking down Nesfatin-1 in the PVN reversed these effects caused by surgical trauma. Transcriptome sequencing identified the extracellular regulated protein kinases (ERK)/cAMP-response element binding protein (CREB) pathway as a key mediator in the impacts of surgical trauma and EA on the hypothalamus. Both in vivo and in vitro studies showed that overexpression of Nesfatin-1 activated the ERK/CREB pathway. Furthermore, administering ERK or CREB inhibitors into the PVN mitigated HPA axis hyperactivity and anxiety-like behaviors induced by surgical trauma. Finally, EA was observed to decrease the phosphorylation levels of ERK and CREB in the PVN. Conclusion: EA alleviates HPA axis hyperactivity and anxiety-like behaviors caused by surgical trauma through inhibition of Nesfatin-1/ERK/CREB pathway in the hypothalamus. [ABSTRACT FROM AUTHOR]
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- 2024
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36. Total tanshinones ameliorates cGAS-STING-mediated inflammatory and autoimmune diseases by affecting STING-IRF3 binding.
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Li, Chengwei, Wen, Jincai, Zhan, Xiaoyan, Shi, Wei, Ye, Xiu, Yao, Qing, Chen, Simin, Zheng, Congyang, Wang, Xianlin, Wen, Xinru, Xiao, Xiaohe, Wang, Yinghao, and Bai, Zhaofang
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ANTI-inflammatory agents , *CHINESE medicine , *ACETIC acid , *PHOSPHORYLATION , *RESEARCH funding , *POLYMERASE chain reaction , *PLASMIDS , *CELL physiology , *CELLULAR signal transduction , *FLUORESCENT antibody technique , *TREATMENT effectiveness , *IN vivo studies , *LIVER diseases , *PLANT extracts , *MICE , *INTERFERONS , *GENES , *AUTOIMMUNE diseases , *ANIMAL experimentation , *WESTERN immunoblotting , *LIPOPOLYSACCHARIDES , *NATURAL immunity , *MEMBRANE proteins , *PHARMACODYNAMICS - Abstract
Background: An important signaling pathway connecting illness and natural immunity is the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, but aberrant activation of this pathway is associated with the development of autoimmune and inflammatory diseases. Hence, targeted inhibition of the activation of the cGAS-STING pathway is potentially valuable in the treatment of disease. The primary active component of Salvia miltiorrhiza is total tanshinone (TTN). Research has indicated that TTN possesses noteworthy anti-inflammatory properties. However, the protective mechanism of TTN against acute liver injury (ALI) and autoimmune diseases is unknown. Methods: A model of aberrant activation of the cGAS-STING pathway was established in various cells and treated with TTN, and the expression of cGAS-STING pathway-related proteins, type I interferon, interferon stimulated genes and inflammatory factors was assessed by western blotting, real-time qPCR. Immunofluorescence analysis of the effect of TTN on the entry of associated proteins into the nucleus following aberrant activation of the cGAS-STING pathway. The effect of TTN on STING oligomerisation was investigated using 2'-3'-cyclic GMP-AMP (2',3'-cGAMP) to induce STING oligomerisation. Western blotting was used to examine the impact of TTN on the interactions of STING, tank-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3) after HA or Flag-labelled plasmids were transfected into HEK-293 T cells. A dimethylxanthenone-4-acetic acid (DMXAA) -induced activation model of the cGAS-STING pathway in mice was established to study the effect of TTN on aberrant activation of the cGAS-STING pathway in vivo. On the other hand, an animal model of lipopolysaccharide/D-galactosamine (LPS/D-GaIN)-induced ALI and an autoimmune disease model induced by trex1 knockout were established to study the effects of TTN on inflammatory and autoimmune diseases mediated by the cGAS-STING pathway in vivo. Results: In several models of aberrant activation of the cGAS-STING pathway, TTN significantly inhibited the phosphorylation of STING and IRF3, thereby suppressing the expression of type I interferon, interferon-stimulated genes and inflammatory factors. Additionally, TTN prevented P65 and IRF3 from entering the nucleus after the cGAS-STING signalling pathway was abnormally activated. Subsequent research indicated that TTN was not involved in the oligomerization of STING or the integration of STING-TBK1 and TBK1-IRF3. However, TTN was found to have a substantial effect on the binding process between STING and IRF3. On the other hand, DMXAA-induced STING activation and activation of downstream signalling in vivo are inhibited by TTN. Furthermore, TTN exhibits positive treatment effects on autoimmune diseases caused by deficiency of trex1 and LPS/D-GaIN-induced ALI. Conclusion: Our research indicates that TTN effectively treats ALI and autoimmune illnesses mediated by the cGAS-STING pathway by inhibiting the abnormal activation of this pathway. [ABSTRACT FROM AUTHOR]
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- 2024
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37. Post-Translational Modification of PTEN Protein: Quantity and Activity.
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Xiao Li, Pu Yang, Xiaoli Hou, and Shaoping Ji
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POST-translational modification , *PTEN protein , *PROTEOLYSIS , *PROTEIN stability , *PI3K/AKT pathway - Abstract
Post-translational modifications play crucial roles in regulating protein functions and stabilities. PTEN is a critical tumor suppressor involved in regulating cellular proliferation, survival, and migration processes. However, dysregulation of PTEN is common in various human cancers. PTEN stability and activation/suppression have been extensively studied in the context of tumorigenesis through inhibition of the PI3K/AKT signaling pathway. PTEN undergoes various post-translational modifications, primarily including phosphorylation, acetylation, ubiquitination, SUMOylation, neddylation, and oxidation, which finely tune its activity and stability. Generally, phosphorylation modulates PTEN activity through its lipid phosphatase function, leading to altered power of the signaling pathways. Acetylation influences PTEN protein stability and degradation rate. SUMOylation has been implicated in PTEN localization and interactions with other proteins, affecting its overall function. Neddylation, as a novel modification of PTEN, is a key regulatory mechanism in the loss of tumor suppressor function of PTEN. Although current therapeutic approaches focus primarily on inhibiting PI3 kinase, understanding the post-translational modifications of PTEN could help provide new therapeutic strategies that can restore PTEN's role in PIP3-dependent tumors. The present review summarizes the major recent developments in the regulation of PTEN protein level and activity. We expect that these insights will contribute to better understanding of this critical tumor suppressor and its potential implications for cancer therapy in the future. [ABSTRACT FROM AUTHOR]
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- 2024
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38. TLK1>Nek1 Axis Promotes Nuclear Retention and Activation of YAP with Implications for Castration-Resistant Prostate Cancer.
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Olatunde, Damilola and De Benedetti, Arrigo
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CASTRATION-resistant prostate cancer , *YAP signaling proteins , *PROTEIN kinases , *CANCER invasiveness , *RESEARCH funding , *PHOSPHORYLATION , *PROGRAMMED death-ligand 1 , *CELLULAR signal transduction , *XENOGRAFTS , *MESSENGER RNA , *RATS , *CELL culture , *GENES , *CELL lines , *ANIMAL experimentation , *HIPPO signaling pathway - Abstract
Simple Summary: Treatment with ARSI results in an increased expression of the TLK1B splice variant and consequent activation of the TLK1>NEK1>YAP axis. We now provide important details of the significance of the NEK1-mediated phosphorylation of the YAP-Y407F residue for its nuclear localization and transcriptional output. Furthermore, we study the effect of the pharmacologic inhibition of this pathway in a xenograft model that readily converts from androgen-dependent to castration-resistant (CRPC) prostate cancer, as a first approach to translating our work toward a clinical investigation. Despite some advances in controlling the progression of prostate cancer (PCa) that is refractory to the use of ADT/ARSI, most patients eventually succumb to the disease, and there is a pressing need to understand the mechanisms that lead to the development of CRPC. A common mechanism is the ability to integrate AR signals from vanishing levels of testosterone, with the frequent participation of YAP as a co-activator, and pointing to the deregulation of the Hippo pathway as a major determinant. We have recently shown that YAP is post-transcriptionally activated via the TLK1>NEK1 axis by stabilizing phosphorylation at Y407. We are now solidifying this work by showing the following: (1) The phosphorylation of Y407 is critical for YAP retention/partition in the nuclei, and J54 (TLK1i) reverses this along with YAP-Y407 dephosphorylation. (2) The enhanced degradation of (cytoplasmic) YAP is increased by J54 counteracting its Enzalutamide-induced accumulation. (3) The basis for all these effects, including YAP nuclear retention, can be explained by the stronger association of pYAP-Y407 with its transcriptional co-activators, AR and TEAD1. (4) We demonstrate that ChIP for GFP-YAP-wt, but hardly for the GFP-YAP-Y407F mutant, at the promoters of typical ARE- and TEAD1-driven genes is readily detected but becomes displaced after treatment with J54. (5) While xenografts of LNCaP cells show rapid regression following treatment with ARSI+J54, in the VCaP model, driven by the TMPRSS2-ERG oncogenic translocation, tumors initially respond well to the combination but subsequently recur, despite the continuous suppression of pNek1-T141 and pYAP-Y407. This suggests an alternative parallel pathway for CRPC progression for VCaP tumors in the long term, which may be separate from the observed ENZ-driven YAP deregulation, although clearly some YAP gene targets like PD-L1, that are found to accumulate following prolonged ENZ treatment, are still suppressed by the concomitant addition of J54. [ABSTRACT FROM AUTHOR]
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- 2024
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39. MERTK Is a Potential Therapeutic Target in Ewing Sarcoma.
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Smart, Sherri K., Yeung, Tsz Y., Santos, M. Olivia, McSwain, Leon F., Wang, Xiaodong, Frye, Stephen V., Earp III, H. Shelton, DeRyckere, Deborah, and Graham, Douglas K.
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RESEARCH funding , *PHOSPHORYLATION , *PROTEIN-tyrosine kinase inhibitors , *ANTINEOPLASTIC agents , *CELL lines , *DRUG efficacy , *EWING'S sarcoma , *PHOSPHOTRANSFERASES , *PHARMACODYNAMICS - Abstract
Simple Summary: Ewing sarcoma family tumors are the second most common bone cancer affecting adolescents and young adults. Outcomes are poor in patients with metastatic or relapsed disease, and new treatments are urgently needed. MERTK is a protein found in leukemia, melanoma, lung cancer, and other cancer types where it promotes cancer cell survival and resistance to therapies. MRX-2843 is a new drug that targets MERTK and is currently in clinical trials. We show that MERTK is present on Ewing sarcoma cells and that MRX-2843 has therapeutic activity against these cells. MRX-2843 is even more effective against Ewing sarcoma cells when combined with other drugs, venetoclax or navitoclax, that target a protein called BCL-2. These results suggest that MRX-2843 could be useful to treat Ewing sarcoma in patients. Outcomes are poor in patients with advanced or relapsed Ewing sarcoma (EWS) and current treatments have significant short- and long-term side effects. New, less toxic and more effective treatments are urgently needed. MER proto-oncogene tyrosine kinase (MERTK) promotes tumor cell survival, metastasis, and resistance to cytotoxic and targeted therapies in a variety of cancers. MERTK was ubiquitously expressed in five EWS cell lines and five patient samples. Moreover, data from CRISPR-based library screens indicated that EWS cell lines are particularly dependent on MERTK. Treatment with MRX-2843, a first-in-class, MERTK-selective tyrosine kinase inhibitor currently in clinical trials, decreased the phosphorylation of MERTK and downstream signaling in a dose-dependent manner in A673 and TC106 cells and provided potent anti-tumor activity against all five EWS cell lines, with IC50 values ranging from 178 to 297 nM. Inhibition of MERTK correlated with anti-tumor activity, suggesting MERTK inhibition as a therapeutic mechanism of MRX-2843. Combined treatment with MRX-2843 and BCL-2 inhibitors venetoclax or navitoclax provided enhanced therapeutic activity compared to single agents. These data highlight MERTK as a promising therapeutic target in EWS and provide rationale for the development of MRX-2843 for the treatment of EWS, especially in combination with BCL-2 inhibitors. [ABSTRACT FROM AUTHOR]
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- 2024
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40. Therapeutic Senolysis of Axitinib-Induced Senescent Human Lung Cancer Cells.
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Kotani, Hitoshi, Han, Wei, Iida, Yuichi, Tanino, Ryosuke, Katakawa, Kazuaki, Okimoto, Tamio, Tsubata, Yukari, Isobe, Takeshi, and Harada, Mamoru
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VASCULAR endothelial growth factors , *BIOLOGICAL models , *ADENOCARCINOMA , *FLOW cytometry , *PHOSPHORYLATION , *RESEARCH funding , *CELLULAR aging , *ANTINEOPLASTIC agents , *PROTEIN-tyrosine kinase inhibitors , *APOPTOSIS , *POLYMERASE chain reaction , *XENOGRAFTS , *DESCRIPTIVE statistics , *CELL lines , *MICE , *GENE expression , *REACTIVE oxygen species , *MESSENGER RNA , *CELL death , *LUNG tumors , *ANIMAL experimentation , *MOLECULAR structure , *CELL size , *LUNG cancer , *GLYCOSIDASES , *CELL receptors , *INTERLEUKINS , *PHARMACODYNAMICS - Abstract
Simple Summary: Tyrosine kinase inhibitors (TKIs) inhibit receptor-mediated signals in cancer and vascular endothelial cells. Especially, axitinib inhibits signaling via vascular endothelial growth factor receptors (VEGFRs). In this study, we report an unforeknown effect of axitinib on human lung cancer cells. We show that axitinib increased the cell size and enhanced the expression of β-galactosidase in a panel of human cancer cell lines, irrespective of their expression of VEGFRs. Especially, axitinib-treated human lung adenocarcinoma A549 cells showed typical senescence and subsequent treatment with the senolytic drug ABT-263 induced drastic cell death (senolysis). Senolysis of senescent A549 cells by ABT-263 was attributed to caspase-dependent apoptosis and Bcl-xL inhibition. Reactive oxygen species were involved in axitinib-induced senescence, but not in senolysis, of A549 cells. In an A549-xenografted mouse model, combination therapy with axitinib and ABT-263 significantly suppressed tumor growth. Background: Tyrosine kinase inhibitors (TKIs) inhibit receptor-mediated signals in cells. Axitinib is a TKI with high specificity for vascular endothelial growth factor receptors (VEGFRs). Aim: We determined whether axitinib could induce senescence in human cancer cells and be lysed by the senolytic drug ABT-263. Methods: Human lung and breast adenocarcinoma cell lines were used. These cells were cultured with axitinib or a multi-target TKI lenvatinib. The expression of β-galactosidase, VEGFRs, Ki-67, reactive oxygen species (ROS) of cancer cells, and their BrdU uptake were evaluated by flow cytometry. The mRNA expression of p21 and IL-8 was examined by quantitative PCR. The effects of TKIs on phosphorylation of Akt and Erk1/2, as downstream molecules of VEGFR signaling, were examined by immunoblot. The in vivo anti-cancer effect was examined using a xenograft mice model. Results: Axitinib, but not lenvatinib, induced cellular senescence (increased cell size and enhanced expression of β-galactosidase) in all adenocarcinoma cell lines. Axitinib-induced senescence was unrelated to the expression of VEGFRs on cancer cells. ROS were involved in axitinib-induced senescence. Axitinib-induced senescent lung adenocarcinoma A549 cells were drastically lysed by ABT-263. In A549-xenografted mice, combination therapy with axitinib and ABT-263 significantly suppressed tumor growth with the induction of apoptotic cancer cells. [ABSTRACT FROM AUTHOR]
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- 2024
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41. The Anticancer Activity of Monosaccharides: Perspectives and Outlooks.
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McCallum, Niamh and Najlah, Mohammad
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THERAPEUTIC use of antineoplastic agents , *DRUG toxicity , *PHOSPHORYLATION , *GLYCOLYSIS , *CARBOHYDRATES , *CARRIER proteins , *MONOSACCHARIDES , *BIOLOGICAL products , *SOLUBILITY , *CANCER chemotherapy , *ENERGY metabolism , *MOLECULAR structure , *CELL death , *TUMORS , *SYNTHETIC drugs - Abstract
Simple Summary: Despite recent advances in treatment options, such as chemotherapy, cancer continues to be the second-leading cause of death worldwide. Significant hurdles in the success of chemotherapy regimens include severe adverse effects, as well as drug resistance. As such, there is a strong requirement for novel pharmacological interventions in the treatment of cancer. Natural products, such as monosaccharides, are a promising potential treatment option for cancer due to their low toxicity, high solubility, and high specificity for tumour cells. Several naturally occurring and synthetically modified sugars have displayed toxicity in a variety of cancer and tumour cells. A major hallmark of cancer is the reprogramming of cellular metabolism from oxidative phosphorylation (OXPHOS) to glycolysis, a phenomenon known as the Warburg effect. To sustain high rates of glycolysis, cancer cells overexpress GLUT transporters and glycolytic enzymes, allowing for the enhanced uptake and consumption of glucose. The Warburg effect may be exploited in the treatment of cancer; certain epimers and derivatives of glucose can enter cancer cells and inhibit glycolytic enzymes, stunting metabolism and causing cell death. These include common dietary monosaccharides (ᴅ-mannose, ᴅ-galactose, ᴅ-glucosamine, ʟ-fucose), as well as some rare monosaccharides (xylitol, ᴅ-allose, ʟ-sorbose, ʟ-rhamnose). This article reviews the literature on these sugars in in vitro and in vivo models of cancer, discussing their mechanisms of cytotoxicity. In addition to this, the anticancer potential of some synthetically modified monosaccharides, such as 2-deoxy-ᴅ-glucose and its acetylated and halogenated derivatives, is reviewed. Further, this article reviews how certain monosaccharides can be used in combination with anticancer drugs to potentiate conventional chemotherapies and to help overcome chemoresistance. Finally, the limitations of administering two separate agents, a sugar and a chemotherapeutic drug, are discussed. The potential of the glycoconjugation of classical or repurposed chemotherapy drugs as a solution to these limitations is reviewed. [ABSTRACT FROM AUTHOR]
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- 2024
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42. Protein Charge Neutralization Is the Proximate Driver Dynamically Tuning Reflectin Assembly.
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Levenson, Robert, Malady, Brandon, Lee, Tyler, Al Sabeh, Yahya, Gordon, Michael J., and Morse, Daniel E.
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OSMOTIC pressure , *REFRACTIVE index , *SQUIDS , *PHOSPHORYLATION , *REFLECTANCE - Abstract
Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, the neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing their refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that the reduction in repulsive net charge of the purified, recombinant reflectin—either (for the first time) by generalized anionic screening with salt or by pH titration—drives a finely tuned, precisely calibrated increase in the size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. The precise proportionality between the assembly size and charge neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth by a continual, equilibrium tuning of the balance between the protein's Coulombic repulsion and short-range interactive forces. The resulting stability of reflectin assemblies with time ensures a reciprocally precise control of the particle number concentration, encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The charge regulation of reflectin assembly precisely fine-tunes a colligative property-based nanostructured biological machine. A physical mechanism is proposed. [ABSTRACT FROM AUTHOR]
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- 2024
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43. Bacterial Production of CDKL5 Catalytic Domain: Insights in Aggregation, Internal Translation and Phosphorylation Patterns.
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Colarusso, Andrea, Lauro, Concetta, Canè, Luisa, Cozzolino, Flora, and Tutino, Maria Luisa
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CATALYTIC domains , *BACTERIAL proteins , *PROTEIN folding , *CATALYTIC activity , *GENETIC translation , *PROTEIN stability , *POST-translational modification - Abstract
Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase involved in human brain development and functioning. Mutations in CDKL5, especially in its catalytic domain, cause a severe developmental condition named CDKL5 deficiency disorder. Nevertheless, molecular studies investigating the structural consequences of such mutations are still missing. The CDKL5 catalytic domain harbors different sites of post-translational modification, such as phosphorylations, but their role in catalytic activity, protein folding, and stability has not been entirely investigated. With this work, we describe the expression pattern of the CDKL5 catalytic domain in Escherichia coli demonstrating that it predominantly aggregates. However, the use of solubility tags, the lowering of the expression temperature, the manual codon optimization to overcome an internal translational start, and the incubation of the protein with K+ and MgATP allow the collection of a soluble catalytically active kinase. Interestingly, the resulting protein exhibits hypophosphorylation compared to its eukaryotic counterpart, proving that bacteria are a useful tool to achieve almost unmodified CDKL5. Posing questions about the CDKL5 autoactivation mechanism and the determinants for its stability, this research provides a valuable platform for comparative biophysical studies between bacterial and eukaryotic-expressed proteins, contributing to our understanding of neurodevelopmental disorders associated with CDKL5 dysfunction. [ABSTRACT FROM AUTHOR]
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- 2024
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44. Post-Translational Variants of Major Proteins in Amyotrophic Lateral Sclerosis Provide New Insights into the Pathophysiology of the Disease.
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Bedja-Iacona, Léa, Richard, Elodie, Marouillat, Sylviane, Brulard, Céline, Alouane, Tarek, Beltran, Stéphane, Andres, Christian R., Blasco, Hélène, Corcia, Philippe, Veyrat-Durebex, Charlotte, and Vourc'h, Patrick
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AMYOTROPHIC lateral sclerosis , *UBIQUITINATION , *PATHOLOGICAL physiology , *PHOSPHORYLATION , *ACETYLATION , *POST-translational modification - Abstract
Post-translational modifications (PTMs) affecting proteins during or after their synthesis play a crucial role in their localization and function. The modification of these PTMs under pathophysiological conditions, i.e., their appearance, disappearance, or variation in quantity caused by a pathological environment or a mutation, corresponds to post-translational variants (PTVs). These PTVs can be directly or indirectly involved in the pathophysiology of diseases. Here, we present the PTMs and PTVs of four major amyotrophic lateral sclerosis (ALS) proteins, SOD1, TDP-43, FUS, and TBK1. These modifications involve acetylation, phosphorylation, methylation, ubiquitination, SUMOylation, and enzymatic cleavage. We list the PTM positions known to be mutated in ALS patients and discuss the roles of PTVs in the pathophysiological processes of ALS. In-depth knowledge of the PTMs and PTVs of ALS proteins is needed to better understand their role in the disease. We believe it is also crucial for developing new therapies that may be more effective in ALS. [ABSTRACT FROM AUTHOR]
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- 2024
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45. MNK-driven eIF4E phosphorylation regulates the fibrogenic transformation of mesenchymal cells and chronic lung allograft dysfunction.
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Walker, Natalie M., Yuta Ibuki, McLinden, A. Patrick, Keizo Misumi, Mitchell, Dylan C., Kleer, Gabriel G., Lock, Alison M., Vittal, Ragini, Sonenberg, Nahum, Garner, Amanda L., and Lama, Vibha N.
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CELL transformation , *HOMOGRAFTS , *PHOSPHORYLATION , *MITOGEN-activated protein kinases , *LUNGS , *KINASES , *THREONINE - Abstract
Tissue fibrosis remains unamenable to meaningful therapeutic interventions and is the primary cause of chronic graft failure after organ transplantation. Eukaryotic translation initiation factor (eIF4E), a key translational regulator, serves as convergent target of multiple upstream profibrotic signaling pathways that contribute to mesenchymal cell (MC) activation. Here, we investigate the role of MAP kinase--interacting serine/threonine kinase--induced (MNK-induced) direct phosphorylation of eIF4E at serine 209 (Ser209) in maintaining fibrotic transformation of MCs and determine the contribution of the MNK/ eIF4E pathway to the pathogenesis of chronic lung allograft dysfunction (CLAD). MCs from patients with CLAD demonstrated constitutively higher eIF4E phosphorylation at Ser209, and eIF4E phospho-Ser209 was found to be critical in regulating key fibrogenic protein autotaxin, leading to sustained β-catenin activation and profibrotic functions of CLAD MCs. MNK1 signaling was upregulated in CLAD MCs, and genetic or pharmacologic targeting of MNK1 activity inhibited eIF4E phospho-Ser209 and profibrotic functions of CLAD MCs in vitro. Treatment with an MNK1/2 inhibitor (eFT-508) abrogated allograft fibrosis in an orthotopic murine lung-transplant model. Together these studies identify what we believe is a previously unrecognized MNK/eIF4E/ATX/β-catenin signaling pathway of fibrotic transformation of MCs and present the first evidence, to our knowledge, for the utility of MNK inhibitors in fibrosis. [ABSTRACT FROM AUTHOR]
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- 2024
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46. 丙酮酸激酶翻译后修饰影响肉品质研究进展.
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黄小兰, 陈丽, 任驰, 摆玉蔷, 侯成立, 李欣, 罗瑞明, and 张德权
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POST-translational modification ,POWER resources ,GLYCOLYSIS ,PHOSPHORYLATION ,ACETYLATION ,MEAT quality - Abstract
Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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- 2024
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47. Network analysis and experimental verification of Salvia miltiorrhiza Bunge-Reynoutria japonica Houtt. drug pair in the treatment of non-alcoholic fatty liver disease.
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Chen, Huafeng, Yan, Shengzhe, Xiang, Qianru, Liang, Jiamin, Deng, Xuejian, He, Wanqin, Cheng, Yanzhen, and Yang, Li
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PHYTOTHERAPY ,PROTEIN metabolism ,NON-alcoholic fatty liver disease ,IN vitro studies ,RESEARCH funding ,PHOSPHORYLATION ,AUTOPHAGY ,LIPIDS ,CELLULAR signal transduction ,PLANT extracts ,METABOLITES ,FLAVONES ,TRIGLYCERIDES ,PHOSPHOTRANSFERASES - Abstract
Context: There are currently no approved specific clinical drugs for non-alcoholic fatty liver disease (NAFLD). Salvia miltiorrhiza Bunge-Reynoutria japonica Houtt. drug pair (SRDP) has been widely used in the treatment of chronic liver diseases. However, the mechanism of SRDP treating NAFLD remains unclear. Objective: Based on network analysis and in vitro experimental verification, we investigated the effect of SRDP on lipid deposition and explored its possible mechanism for the treatment of NAFLD. Methods: The TCMSP platform was used to screen the active metabolites of SRDP and corresponding targets. The GeneCards and OMIM databases were used to screen the NAFLD targets. The drug-disease intersecting targets were extracted to obtain the potential targets. Then the protein–protein interaction (PPI) and drug-active metabolites-target-disease network map was constructed. The DAVID database was performed to GO and KEGG pathway enrichment analysis for the intersecting targets. The core active metabolite and signaling pathway were verified by in vitro experiments. Results: Network analysis predicted 59 active metabolites and 89 targets of SRDP for the treatment of NAFLD. 112 signaling pathways were enriched for KEGG pathways, including PI3K-AKT signaling pathway,etc. It was confirmed that luteolin, the core active metabolite of SRDP, effectively reduced fat accumulation and intracellular triglyceride content in HepG2 fatty liver cell model. Luteolin could inhibit mTOR pathway by inhibiting PI3K-AKT signaling pathway phosphorylation, thereby activating autophagy to alleviate NAFLD. Discussion and conclusion: The results of this study validate and predict the possible role of various active metabolites of SRDP in the treatment of NAFLD through multiple targets and signaling pathways. The core active metabolite of SRDP, luteolin can alleviate NAFLD by acting on the PI3K-AKT-mTOR signaling pathway to induce autophagy. [ABSTRACT FROM AUTHOR]
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- 2024
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48. Nemo‐like kinase blocks myeloid differentiation by targeting tumor suppressor C/EBPα in AML.
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Singh, Anil Kumar, Thacker, Gatha, Upadhyay, Vishal, Mishra, Mukul, Sharma, Akshay, Sethi, Arppita, Chowdhury, Sangita, Siddiqui, Shumaila, Verma, Shailendra Prasad, Pandey, Amita, Bhatt, Madan L. B., and Trivedi, Arun Kumar
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GRANULOCYTE colony stimulating factor receptor , *MONONUCLEAR leukocytes , *TRANSCRIPTION factors , *ACUTE myeloid leukemia , *PHOSPHORYLATION - Abstract
CCAAT/enhancer‐binding protein α (C/EBPα), a key myeloid transcription factor, drives myeloid differentiation from blast cells by regulating the expression of granulocyte colony stimulating factor receptor and C/EBPε as required for promoting granulocyte differentiation. Here, we show that serine/threonine‐protein kinase NLK, also known as Nemo‐like kinase, physically associates with C/EBPα and phosphorylates it at multiple sites, including Ser21, Thr226, Thr230 and S234, leading to its ubiquitin‐mediated degradation. Individual phospho‐point mutants of C/EBPα could be phosphorylated by NLK, but a mutant with all phosphorylatable residues replaced by alanine resisted phosphorylation and degradation by NLK, as did the single point mutants. Furthermore, although ectopic expression of NLK enhanced phosphorylation of C/EBPα levels, it markedly inhibited total C/EBPα protein levels. Conversely, NLK depletion inhibited endogenous C/EBPα phosphorylation but enhanced its total protein levels in several acute myeloid leukemia (AML) cell lines and in peripheral blood mononuclear cells isolated from number of AML patient samples. Importantly, NLK depletion in peripheral blood mononuclear cells from primary AML patients not only restored C/EBPα protein levels, but also induced myeloid differentiation, suggesting that NLK could be therapeutically targeted to restore C/EBPα to resolve differentiation arrest in AML. [ABSTRACT FROM AUTHOR]
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- 2024
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49. GhMPK9‐GhRAF39_1‐GhWRKY40a Regulates the GhERF1b‐ and GhABF2‐Mediated Pathways to Increase Cotton Disease Resistance.
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Mi, Xinyue, Li, Weixi, Chen, Chuan, Xu, Huijuan, Wang, Guilin, Jin, Xuanxiang, Zhang, Dayong, and Guo, Wangzhen
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TRANSCRIPTION factors , *VERTICILLIUM wilt diseases , *PROTEIN kinases , *PLANT diseases , *VERTICILLIUM dahliae - Abstract
Mitogen‐activated protein kinase (MAPK) cascade is the center of plant signal transduction system that amplify immune signals into cellular responses by phosphorylating diverse substrates. The MAPK cascade consisting of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs is well characterized in plants, in which Raf‐like kinases are generally regarded as MAPKKKs. However, it is rarely reported that Raf‐like MAPKKKs function as middle regulators to link MAPK and its downstream transcription factors in plant immunity. Verticillium wilt, caused by the soil‐borne vascular fungus Verticillium dahliae, is a serious disease in many plants, including cotton. The previous studies showed that GhMPK9 (a MAPK) is involved in the response to Verticillium wilt. Here, the Raf‐like kinase GhRAF39_1 is reported as helper regulates the phosphorylation of WRKY transcription factor GhWRKY40a by GhMPK9. The phosphorylated GhWRKY40a can further activate the transcription of GhERF1b to up‐regulate defense‐related genes while inhibit the transcription of GhABF2 to regulate the stomatal opening, thus improving the resistance to Verticillium wilt in cotton. This study reveals a new signaling module of GhMPK9‐GhRAF39_1‐GhWRKY40a to regulate GhERF1b‐ and GhABF2‐mediated defense responses, which triggers plant defense against Verticillium wilt. [ABSTRACT FROM AUTHOR]
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- 2024
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50. Analysis of homodimer formation in 12-oxophytodienoate reductase 3 in solutio and crystallo challenges the physiological role of the dimer.
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Kerschbaumer, Bianca, Macheroux, Peter, and Bijelic, Aleksandar
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JASMONIC acid , *PLANT defenses , *LIGHT scattering , *PHOSPHORYLATION , *ABIOTIC stress - Abstract
12-oxophytodienoate reductase 3 (OPR3) is a key enzyme in the biosynthesis of jasmonoyl-L-isoleucine, the receptor-active form of jasmonic acid and crucial signaling molecule in plant defense. OPR3 was initially crystallized as a self-inhibitory dimer, implying that homodimerization regulates enzymatic activity in response to biotic and abiotic stresses. Since a sulfate ion is bound to Y364, mimicking a phosphorylated tyrosine, it was suggested that dimer formation might be controlled by reversible phosphorylation of Y364 in vivo. To investigate OPR3 homodimerization and its potential physiological role in more detail, we performed analytical gel filtration and dynamic light scattering on wild-type OPR3 and three variants (R283D, R283E, and Y364P). The experiments revealed a rapid and highly sensitive monomer–dimer equilibrium for all OPR3 constructs. We crystallized all constructs with and without sulfate to examine its effect on the dimerization process and whether reversible phosphorylation of Y364 triggers homodimerization in vivo. All OPR3 constructs crystallized in their monomeric and dimeric forms independent of the presence of sulfate. Even variant Y364P, lacking the putative phosphorylation site, was crystallized as a self-inhibitory homodimer, indicating that Y364 is not required for dimerization. Generally, the homodimer is relatively weak, and our results raise doubts about its physiological role in regulating jasmonate biosynthesis. [ABSTRACT FROM AUTHOR]
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- 2024
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