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The phosphorylation state of 20 types of intracellular proteins in the presence of the protein phosphatase 1 (PP1)- and PP2A-specific Ser/Thr phosphatase inhibitor calyculin A or the Tyr phosphatase inhibitor pervanadate was visualized by Phos-tag SDS-PAGE followed by immunoblotting. All blots showed a Phos-tag pattern indicating increased phosphorylation in the presence of one or both phosphatase inhibitors. The increase in phosphorylation stoichiometry per protein tends to be greater for Ser/Thr phosphatase inhibition than for Tyr phosphatase inhibition. This is consistent with the fact that the number of Ser/Thr kinase genes in the human genome is greater than that of Tyr kinases and with the fact that the phospho-Ser/phospho-Thr ratio in the actual human phosphoproteome is far greater than that of phospho-Tyr ratio. This suggests that cellular proteins are routinely and randomly phosphorylated by different kinases with no biological significance, simply depending on the frequency of substrate encounters. Phosphatase is responsible for routinely removing these unwanted phosphate groups systematically and maintaining the dynamic equilibrium of physiological protein phosphorylation. Phos-tag SDS-PAGE visualized that the kinase reaction involves many incidental phosphorylation and that phosphatases play broader roles besides being strict counterparts to kinases. [ABSTRACT FROM AUTHOR]

Chronic cerebral ischemia (CCI) is a clinical syndrome characterised by brain dysfunction due to decreased chronic cerebral perfusion. CCI initiates several inflammatory pathways, including pyroptosis. RNA-binding proteins (RBPs) play important roles in CCI. This study aimed to explore whether the interaction between RBP-Cpeb4 and Dclk2 affected Ehf phosphorylation to regulate neuronal pyroptosis. HT22 cells and mice were used to construct oxygen glucose deprivation (OGD)/CCI models. We found that Cpeb4 and Dclk2 were upregulated in OGD-treated HT22 cells and CCI-induced hippocampal CA1 tissues. Cpeb4 upregulated Dclk2 expression by increasing Dclk2 mRNA stability. Knockdown of Cpeb4 or Dclk2 inhibited neuronal pyroptosis in OGD-treated HT22 cells and CCI-induced hippocampal CA1 tissues. By binding to the promoter regions of Caspase1 and Caspase3, the transcription factor Ehf reduced their promoter activities and inhibited the transcription. Dclk2 phosphorylated Ehf and changed its nucleoplasmic distribution, resulting in the exit of p-Ehf from the nucleus and decreased Ehf levels. It promoted the expression of Caspase1 and Caspase3 and stimulated neuronal pyroptosis of HT22 cells induced by OGD. Cpeb4/Dclk2/Ehf pathway plays an important role in the regulation of cerebral ischemia-induced neuronal pyroptosis. [ABSTRACT FROM AUTHOR]

Cancerous genetic mutations result in a complex and comprehensive post-translational modification (PTM) dynamics, in which protein succinylation is well known for its ability to reprogram cell metabolism and is involved in the malignant evolution. Little is known about the regulatory interactions between succinylation and other PTMs in the PTM network. Here, we developed a conjoint analysis and systematic clustering method to explore the intermodification communications between succinylome and phosphorylome from eight lung cancer patients. We found that the intermodification coorperation in both parallel and series. Besides directly participating in metabolism pathways, some phosphosites out of mitochondria were identified as an upstream regulatory modification directing succinylome dynamics in cancer metabolism reprogramming. Phosphorylated activation of histone deacetylase (HDAC) in lung cancer resulted in the removal of acetylation and favored the occurrence of succinylation modification of mitochondrial proteins. These results suggest a tandem regulation between succinylation and phosphorylation in the PTM network and provide HDAC-related targets for intervening mitochondrial succinylation and cancer metabolism reprogramming. [ABSTRACT FROM AUTHOR]

We have demonstrated that SAMHD1 (sterile alpha motif and histidineaspartic domain HD-containing protein 1) is a restriction factor for the human papillomavirus 16 (HPV16) life cycle. Here, we demonstrate that in HPV-negative cervical cancer C33a cells and human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), SAMHD1 is recruited to E1-E2 replicating DNA. Homologous recombination (HR) factors are required for HPV16 replication, and viral replication promotes phosphorylation of SAMHD1, which converts it from a dNTPase to an HR factor independent from E6/E7 expression. A SAMHD1 phospho-mimic (SAMHD1 T592D) reduces E1-E2-mediated DNA replication in C33a cells and has enhanced recruitment to the replicating DNA. In HFK+HPV16 cells, SAMHD1 T592D is recruited to the viral DNA and attenuates cellular growth, but does not attenuate growth in isogenic HFK cells immortalized by E6/E7 alone. SAMHD1 T592D also attenuates the development of viral replication foci following keratinocyte differentiation. The results indicated that enhanced SAMHD1 phosphorylation could be therapeutically beneficial in cells with HPV16 replicating genomes. Protein phosphatase 2A (PP2A) can dephosphorylate SAMHD1, and PP2A function can be inhibited by endothall. We demonstrate that endothall reduces E1-E2 replication and promotes SAMHD1 recruitment to E1-E2 replicating DNA, mimicking the SAMHD1 T592D phenotypes. Finally, we demonstrate that in head and neck cancer cell lines with HPV16 episomal genomes, endothall attenuates their growth and promotes recruitment of SAMHD1 to the viral genome. The results suggest that targeting cellular phosphatases has therapeutic potential for the treatment of HPV infections and cancers. IMPORTANCE Human papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. The development of anti-viral therapeutics depends upon an increased understanding of the viral life cycle. Here, we demonstrate that HPV16 replication converts sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) into a homologous recombination (HR) factor via phosphorylation. This phosphorylation promotes recruitment of SAMHD1 to viral DNA to assist with replication. A SAMHD1 mutant that mimics phosphorylation is hyper-recruited to viral DNA and attenuates viral replication. Expression of this mutant in HPV16-immortalized cells attenuates the growth of these cells, but not cells immortalized by the viral oncogenes E6/E7 alone. Finally, we demonstrate that the phosphatase inhibitor endothall promotes hyper-recruitment of endogenous SAMHD1 to HPV16 replicating DNA and can attenuate the growth of both HPV16-immortalized human foreskin keratinocytes (HFKs) and HPV16-positive head and neck cancer cell lines. We propose that phosphatase inhibitors represent a novel tool for combating HPV infections and disease. [ABSTRACT FROM AUTHOR]

The multifunctional tegument protein pUL21 of HSV-2 is phosphorylated in infected cells. We have identified two residues in the unstructured linker region of pUL21, serine 251 and serine 253, as phosphorylation sites. Both phosphorylation sites are absent in HSV-1 pUL21, which likely explains why phosphorylated pUL21 was not detected in cells infected with HSV-1. Cells infected with HSV-2 strain 186 viruses deficient in pUL21 phosphorylation exhibited reductions in both cell-cell spread of virus infection and virus replication. Defects in secondary envelopment of cytoplasmic nucleocapsids were also observed in cells infected with viruses deficient in pUL21 phosphorylation as well as in cells infected with multiple strains of HSV-2 and HSV-1 deleted for pUL21. These results confirm a role for HSV pUL21 in the secondary envelopment of cytoplasmic nucleocapsids and indicate that phosphorylation of HSV-2 pUL21 is required for this activity. Phosphorylation of pUL21 was substantially reduced in cells infected with HSV-2 strain 186 mutants lacking the viral serine/threonine kinase pUL13, indicating a requirement for pUL13 in pUL21 phosphorylation. IMPORTANCE It is well known that post-translational modification of proteins by phosphorylation can regulate protein function. Here, we determined that phosphorylation of the multifunctional HSV-2 tegument protein pUL21 requires the viral serine/threonine kinase pUL13. In addition, we identified serine residues within HSV-2 pUL21 that can be phosphorylated. Phenotypic analysis of mutant HSV-2 strains with deficiencies in pUL21 phosphorylation revealed reductions in both cell-cell spread of virus infection and virus replication. Deficiencies in pUL21 phosphorylation also compromised the secondary envelopment of cytoplasmic nucleocapsids, a critical final step in the maturation of all herpes virions. Unlike HSV-2 pUL21, phosphorylation of HSV-1 pUL21 was not detected. This fundamental difference between HSV-2 and HSV-1 may underlie our previous observations that the requirements for pUL21 differ between HSV species. [ABSTRACT FROM AUTHOR]

Excessive fat deposition leads to obesity and cardiovascular diseases with abnormal metabolism. Pantothenic acid (PA) is a major B vitamin required for energy metabolism. However, the effect of PA on lipid metabolism and obesity has not been explored. We investigated the effects and molecular mechanism of PA on fat accumulation as well as the influence of adipogenic marker genes in both adult male mice and primary adipocytes. First, we demonstrated that PA attenuates weight gain in mice fed high-fat diet (HFD). Besides, PA supplementation substantially improved glucose tolerance and lipid metabolic disorder in obese mice. Furthermore, PA significantly inhibited white adipose tissue (WAT) deposition as well as fat droplets visualized by magnification in both chow and HFD group. More importantly, PA obviously suppressed the mRNA levels of CD36, IL-6, and TNF-α to alleviate inflammation and reduced the levels of PPARγ, aP2, and C/EBPα genes that are related to lipid metabolism in inguinal white adipose tissue (ing-WAT) and epididymal white adipose tissue (ei-WAT). In vitro, PA supplementation showed a lower lipid droplet aggregation as well as reduced expression levels of adipogentic genes. Finally, we identified that PA inhibits the phosphorylation levels of p38 and JNK in murine primary adipocytes. Collectively, our data demonstrated for the first time that PA attenuates lipid metabolic disorder as well as fat deposition by JNK/p38 MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are produced during cellular metabolism and in response to environmental stress. While low levels of ROS play essential physiological roles, excess ROS can damage cellular components, leading to cell death or transformation. ROS can also regulate protein interactions in cancer cells, thereby affecting processes such as cell growth, migration, and angiogenesis. Dysregulated interactions occur via various mechanisms, including amino acid modifications, conformational changes, and alterations in complex stability. Understanding ROS-mediated changes in protein interactions is crucial for targeted cancer therapies. In this review, we examine the role that ROS mechanisms in regulating pathways through protein–protein interactions. [ABSTRACT FROM AUTHOR]

Among polymer wastes, poly(ethylene terephthalate) (PET) is the most important commercial thermoplastic polyester. Less than 30% of total PET production is recycled into new products. Therefore, large amounts of waste PET need to be recycled. We describe a feasible approach for the direct application of the glycolysis products of PET (GP-PET), without further purification, for the synthesis of value-added products. It was established that GP-PET is valorized via phosphorylation with phenylphosphonic dichloride (PPD), as well as with trimethyl phosphate (TMP). When PPD is used, a condensation reaction takes place with the evolution of hydrogen chloride. During the interaction between GP-PET and TMP, the following reactions take place simultaneously: a transesterification with the participation of the hydroxyl group of GP-PET and the methoxy group of TMP and an exchange reaction between the ester group of GP-PET and the methyl ester group of TMP. The occurrence of the exchange reaction was confirmed by 1H, 31P, 13C NMR, and GPC analysis. Thermogravimetric analysis (TGA) revealed that the percentage of a carbon residual (CR) implies the possibility of using the end products as flame retardant (FR) additives, especially for polyurethanes as well as thermal stabilizers of polymer materials or Li-ion cells. [ABSTRACT FROM AUTHOR]

Proteins frequently undergo covalent modification at the post‐translational level, which involves the covalent attachment of chemical groups onto amino acids. This can entail the singular or multiple addition of small groups, such as phosphorylation; long‐chain modifications, such as glycosylation; small proteins, such as ubiquitination; as well as the interconversion of chemical groups, such as the formation of pyroglutamic acid. These post‐translational modifications (PTMs) are essential for the normal functioning of cells, as they can alter the physicochemical properties of amino acids and therefore influence enzymatic activity, protein localization, protein–protein interactions and protein stability. Despite their inherent importance, accurately depicting PTMs in experimental studies of protein structures often poses a challenge. This review highlights the role of PTMs in protein structures, as well as the prevalence of PTMs in the Protein Data Bank, directing the reader to accurately built examples suitable for use as a modelling reference. [ABSTRACT FROM AUTHOR]

Simple Summary: The YAP/TAZ signaling pathways, which is involved in tumor development and proliferation in a variety of solid tumors, inhibits hypoxia-induced cell death, increases and stabilizes angiogenesis, and activates tumor proliferation, both independently and in conjunction with HIF in hypoxic tumors. It also promotes tumor metastasis by regulating different TME environments. Therefore, understanding the activity of YAP/TAZ as a mechanism of tumor development may lead to the development of novel therapeutic strategies. In solid tumors such as hepatocellular carcinoma (HCC), hypoxia is one of the important mechanisms of cancer development that closely influences cancer development, survival, and metastasis. The development of treatments for cancer was temporarily revolutionized by immunotherapy but continues to be constrained by limited response rates and the resistance and high costs required for the development of new and innovative strategies. In particular, solid tumors, including HCC, a multi-vascular tumor type, are sensitive to hypoxia and generate many blood vessels for metastasis and development, making it difficult to treat HCC, not only with immunotherapy but also with drugs targeting blood vessels. Therefore, in order to develop a treatment strategy for hypoxic tumors, various mechanisms must be explored and analyzed to treat these impregnable solid tumors. To date, tumor growth mechanisms linked to hypoxia are known to be complex and coexist with various signal pathways, but recently, mechanisms related to the Hippo signal pathway are emerging. Interestingly, Hippo YAP/TAZ, which appear during early tumor and normal tumor growth, and YAP/TAZ, which appear during hypoxia, help tumor growth and proliferation in different directions. Peculiarly, YAP/TAZ, which have different phosphorylation directions in the hypoxic environment of tumors, are involved in cancer proliferation and metastasis in various carcinomas, including HCC. Analyzing the mechanisms that regulate the function and expression of YAP in addition to HIF in the complex hypoxic environment of tumors may lead to a variety of anti-cancer strategies and combining HIF and YAP/TAZ may develop the potential to change the landscape of cancer treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: The naturally occurring derivative of estrogen, 2-methoxyestradiol (2-ME), has been shown to have good anti-cancer properties. However, it is broken down too quickly within the blood to be clinically useful. We designed 2-ME analogs with modifications which could avoid rapid metabolism, would preferably stay in the tumor, and were more toxic to cancer cells. Here, we looked more closely at how these compounds work within cancer cells, and how they communicate within themselves and with their environment. ESE-15-one and ESE-16 interfere with the functioning of the intracellular cytoskeleton (actin and microtubules), sending messages that stop cell division, intracellular transport of proteins, and cell migration and invasion, eventually inducing cell suicide. They also inhibited the movement of cells that make blood vessels that would support tumor growth. Using eggs with chicken embryos, we could show that the compounds reduced the tumor size and diminished the spread of cancer cells in a living system. The microtubule-disrupting agent 2-methoxyestradiol (2-ME) displays anti-tumor and anti-angiogenic properties, but its clinical development is halted due to poor pharmacokinetics. We therefore designed two 2-ME analogs in silico—an ESE-15-one and an ESE-16 one—with improved pharmacological properties. We investigated the effects of these compounds on the cytoskeleton in vitro, and their anti-angiogenic and anti-metastatic properties in ovo. Time-lapse fluorescent microscopy revealed that sub-lethal doses of the compounds disrupted microtubule dynamics. Phalloidin fluorescent staining of treated cervical (HeLa), metastatic breast (MDA-MB-231) cancer, and human umbilical vein endothelial cells (HUVECs) displayed thickened, stabilized actin stress fibers after 2 h, which rearranged into a peripheral radial pattern by 24 h. Cofilin phosphorylation and phosphorylated ezrin/radixin/moesin complexes appeared to regulate this actin response. These signaling pathways overlap with anti-angiogenic, extra-cellular communication and adhesion pathways. Sub-lethal concentrations of the compounds retarded both cellular migration and invasion. Anti-angiogenic and extra-cellular matrix signaling was evident with TIMP2 and P-VEGF receptor-2 upregulation. ESE-15-one and ESE-16 exhibited anti-tumor and anti-metastatic properties in vivo, using the chick chorioallantoic membrane assay. In conclusion, the sulfamoylated 2-ME analogs displayed promising anti-tumor, anti-metastatic, and anti-angiogenic properties. Future studies will assess the compounds for myeloproliferative effects, as seen in clinical applications of other drugs in this class. [ABSTRACT FROM AUTHOR]

Simple Summary: NAD+ is a key cofactor that plays a role in various metabolic and signaling pathways. Understanding its ability to regulate cellular homeostasis also sheds light on its role in tumorigenesis and disease progression in gynecologic cancers. By reviewing the enzymes that consume NAD+ as well as the pathways and enzymes that synthesize NAD+, the vast influence NAD+ has on the survival of cancer cells becomes clear. With that information, advances have been made in the development of unique inhibitors to target NAD+ synthesizing and consuming enzymes. Nicotinamide adenine dinucleotide (NAD+) is an important cofactor for both metabolic and signaling pathways, with the dysregulation of NAD+ levels acting as a driver for diseases such as neurodegeneration, cancers, and metabolic diseases. NAD+ plays an essential role in regulating the growth and progression of cancers by controlling important cellular processes including metabolism, transcription, and translation. NAD+ regulates several metabolic pathways such as glycolysis, the citric acid (TCA) cycle, oxidative phosphorylation, and fatty acid oxidation by acting as a cofactor for redox reactions. Additionally, NAD+ acts as a cofactor for ADP-ribosyl transferases and sirtuins, as well as regulating cellular ADP-ribosylation and deacetylation levels, respectively. The cleavage of NAD+ by CD38—an NAD+ hydrolase expressed on immune cells—produces the immunosuppressive metabolite adenosine. As a result, metabolizing and maintaining NAD+ levels remain crucial for the function of various cells found in the tumor microenvironment, hence its critical role in tissue homeostasis. The NAD+ levels in cells are maintained by a balance between NAD+ biosynthesis and consumption, with synthesis being controlled by the Preiss–Handler, de novo, and NAD+ salvage pathways. The primary source of NAD+ synthesis in a variety of cell types is directed by the expression of the enzymes central to the three biosynthesis pathways. In this review, we describe the role of NAD+ metabolism and its synthesizing and consuming enzymes' control of cancer cell growth and immune responses in gynecologic cancers. Additionally, we review the ongoing efforts to therapeutically target the enzymes critical for NAD+ homeostasis in gynecologic cancers. [ABSTRACT FROM AUTHOR]

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]

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]

Objectives: The underlying mechanisms of burning mouth syndrome (BMS) remain unclear leading to challenges and unsatisfactory management. Current treatments focus primarily on symptom relief, with few consistently achieving a 50% reduction in pain. This review aims to explore animal models of BMS to gain a better understanding of the underlying mechanisms and to discuss potential and existing knowledge gaps. Methods: A comprehensive review of PubMed®, Google Scholar, and Scopus was performed to assess advances and significant gaps of existing rodent models that mimic BMS‐related symptoms. Results: Rodent models of BMS involve reproduction of dry‐tongue, chorda tympani transection, or overexpression of artemin protein. Existing preclinical models tend to highlight one specific etiopathogenesis and often overlook sex‐ and hormone‐specific factors. Conclusion: Combining aspects from various BMS models could prove beneficial in developing comprehensive experimental designs and outcomes encompassing the multifaceted nature of BMS. [ABSTRACT FROM AUTHOR]

Objectives: Odontogenesis, an intricate process initiated by epithelium‐mesenchyme interaction, is meticulously regulated by a cascade of regulatory mechanisms. Epigenetic modifications, especially histone modification, have been found to exhibit spatiotemporal specificity during tooth development. However, the expression patterns and roles of enzymes associated with histone modifications have yet to be systematically explored in odontogenesis. This review aims to summarize the histone‐modifying enzymes in odontogenesis and their regulation mechanism during tooth development and provide the potential theoretical basis for the clinical management and intervention of dental developmental diseases. Subjects and Methods: This study conducted a systematic search across PubMed and Web of Science databases, utilizing the keywords "odontogenesis," "histone modification," and "enzyme" for pertinent articles. Results: No doubt histone modification contributes extensively to odontogenesis regulation, and the disturbances in histone modifications can derange the odontogenesis process. Conclusion: Further studies are warranted to elucidate these roles and their potential downstream effects, positioning histone modifications as a pivotal focal point for unraveling the intricacies of tooth development and regeneration. [ABSTRACT FROM AUTHOR]

The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70 kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health. [ABSTRACT FROM AUTHOR]

Pathogenic variations in the NTRK1 can cause congenital insensitivity to pain with anhidrosis (CIPA), a rare autosomal recessive inherited neuropathy. The precise diagnosis of CIPA relies on the identification of pathogenic genotypes. Therefore, it is essential to expand the NTRK1 variation spectrum and improve molecular diagnosis methods. In this study, 74 probands with typical manifestations of CIPA but unknown genotypes were recruited. A comprehensive molecular genetic analysis was performed to identify variations in the NTRK1, using techniques including Sanger and next-generation sequencing, bioinformatic analysis, quantitative polymerase chain reaction (qPCR), gap-PCR, short tandem repeat (STR) genotyping, and reverse-transcription PCR. In addition, functional assays were conducted to determine the pathogenicity of variants of uncertain significance (VUS) and further characterized changes in glycosylation and phosphorylation of 14 overexpressed mutant vectors with variants at different domains in the TrkA protein, which is encoded by NTRK1. A total of 48 variations in the NTRK1 were identified, including 22 novel ones. When combined with data from another 53 CIPA patients examined in our previous work, this study establishes the largest genotypic and phenotypic spectra of CIPA worldwide, including 127 CIPA families. Moreover, functional studies indicated that the pathogenicity of VUS mainly affected insufficient glycosylation in the extracellular domain and abnormal phosphorylation in the intracellular domain. This study not only provides important evidence for precise diagnosis of CIPA but also further enriches our understanding of the pathogenesis of this disease. [ABSTRACT FROM AUTHOR]

Stomata are micropores on the leaf epidermis that allow carbon dioxide (CO2) uptake for photosynthesis at the expense of water loss through transpiration. Stomata coordinate the plant gas exchange of carbon and water with the atmosphere through their opening and closing dynamics. In the context of global climate change, it is essential to better understand the mechanism of stomatal movements under different environmental stimuli. Aquaporins (AQPs) are considered important regulators of stomatal movements by contributing to membrane diffusion of water, CO2 and hydrogen peroxide. This review compiles the most recent findings and discusses future directions to update our knowledge of the role of AQPs in stomatal movements. After highlighting the role of subsidiary cells (SCs), which contribute to the high water use efficiency of grass stomata, we explore the expression of AQP genes in guard cells and SCs. We then focus on the cellular regulation of AQP activity at the protein level in stomata. After introducing their post‐translational modifications, we detail their trafficking as well as their physical interaction with various partners that regulate AQP subcellular dynamics towards and within specific regions of the cell membranes, such as microdomains and membrane contact sites. Summary statement: Stomatal movements require the activity of aquaporins in guard cells and, when present, in subsidiary cells. This review explores several regulation mechanisms that control aquaporin expression, trafficking and activity and, consequently stomatal dynamics. [ABSTRACT FROM AUTHOR]

Cancer is a mysterious disease. Among other alterations, tumor cells, importantly, have metabolic modifications. A well-known metabolic modification commonly observed in cancer cells has been termed the Warburg effect. This phenomenon is defined as a high preference for glucose uptake, and increased lactate production from that glucose, even when oxygen is readily available. Some anti-cancer drugs target the proposed Warburg effect, and some dietary regimens can function similarly. However, the most suitable dietary strategies for treating particular cancers are not yet well understood. The aim of this review was to describe findings regarding the impact of various proposed dietary regimens targeting the Warburg effect. The evidence suggests that combining routine cancer therapies with diet-based strategies may improve the outcome in treating cancer. However, designing individualized therapies must be our ultimate goal. [ABSTRACT FROM AUTHOR]

Protein kinase B (AKT1) is a serine/threonine kinase that regulates fundamental cellular processes, including cell survival, proliferation, and metabolism. AKT1 activity is controlled by two regulatory phosphorylation sites (Thr308, Ser473) that stimulate a downstream signaling cascade through phosphorylation of many target proteins. At either or both regulatory sites, hyperphosphorylation is associated with poor survival outcomes in many human cancers. Our previous biochemical and chemoproteomic studies showed that the phosphorylated forms of AKT1 have differential selectivity toward peptide substrates. Here, we investigated AKT1‐dependent activity in human cells, using a cell‐penetrating peptide (transactivator of transcription, TAT) to deliver inactive AKT1 or active phospho‐variants to cells. We used enzyme engineering and genetic code expansion relying on a phosphoseryl‐transfer RNA (tRNA) synthetase (SepRS) and tRNASep pair to produce TAT‐tagged AKT1 with programmed phosphorylation at one or both key regulatory sites. We found that all TAT‐tagged AKT1 variants were efficiently delivered into human embryonic kidney (HEK 293T) cells and that only the phosphorylated AKT1 (pAKT1) variants stimulated downstream signaling. All TAT‐pAKT1 variants induced glycogen synthase kinase (GSK)‐3α phosphorylation, as well as phosphorylation of ribosomal protein S6 at Ser240/244, demonstrating stimulation of downstream AKT1 signaling. Fascinatingly, only the AKT1 variants phosphorylated at S473 (TAT‐pAKT1S473 or TAT‐pAKT1T308,S473) were able to increase phospho‐GSK‐3β levels. Although each TAT‐pAKT1 variant significantly stimulated cell proliferation, cells transduced with TAT‐pAKT1T308 grew significantly faster than with the other pAKT1 variants. The data demonstrate differential activity of the AKT1 phospho‐forms in modulating downstream signaling and proliferation in human cells. [ABSTRACT FROM AUTHOR]

Type 1 diabetes arises from the selective destruction of pancreatic β-cells by autoimmune mechanisms, and intracellular pathways driven by Janus kinase (JAK)–mediated phosphorylation of STAT isoforms (especially STAT1 and STAT2) are implicated as mediators of β-cell demise. Despite this, the molecular mechanisms that regulate JAK-STAT signaling in β-cells during the autoimmune attack remain only partially disclosed, and the factors acting to antagonize proinflammatory STAT1 signaling are uncertain. We have recently implicated signal regulatory protein α (SIRPα) in promoting β-cell viability in the face of ongoing islet autoimmunity and have now revealed that this protein controls the availability of a cytosolic lysine deacetylase, HDAC6, whose activity regulates the phosphorylation and activation of STAT1. We provide evidence that STAT1 serves as a substrate for HDAC6 in β-cells and that sequestration of HDAC6 by SIRPα in response to anti-inflammatory cytokines (e.g., IL-13) leads to increased STAT1 acetylation. This then impairs the ability of STAT1 to promote gene transcription in response to proinflammatory cytokines, including interferon-γ. We further found that SIRPα is lost from the β-cells of subjects with recent-onset type 1 diabetes under conditions when HDAC6 is retained and STAT1 levels are increased. On this basis, we report a previously unrecognized role for cytokine-induced regulation of STAT1 acetylation in the control of β-cell viability and propose that targeted inhibition of HDAC6 activity may represent a novel therapeutic modality to promote β-cell viability in the face of active islet autoimmunity. Article Highlights: Signal regulatory protein α (SIRPα) is present in human islet cells, but its levels decline in β-cells in type 1 diabetes. Decreases in SIRPα expression are associated with a reduction in the viability of cultured β-cells. Immunoprecipitation of β-cell SIRPα reveals a direct interaction with the lysine deacetylase HDAC6. Sequestration of HDAC6 by SIRPα results in increased acetylation, reduced phosphorylation, and impaired activation of STAT1 during exposure of β-cells to interferon-γ. Pharmacological targeting of HDAC6 might yield improvements in β-cell viability during progression to type 1 diabetes. [ABSTRACT FROM AUTHOR]

• LGB-IPs is a new LGB-based optimal feature fusion model for the accurate prediction of STY phosphorylation sites. • LGB-IPs explores ten different feature descriptors and assesses its discriminative capability using five different classifiers. • Extensive cross-validation and independent assessment shows that LGB-IPs outperformed the single feature models consistently. SARS-CoV-2′s global spread has instigated a critical health and economic emergency, impacting countless individuals. Understanding the virus's phosphorylation sites is vital to unravel the molecular intricacies of the infection and subsequent changes in host cellular processes. Several computational methods have been proposed to identify phosphorylation sites, typically focusing on specific residue (S/T) or Y phosphorylation sites. Unfortunately, current predictive tools perform best on these specific residues and may not extend their efficacy to other residues, emphasizing the urgent need for enhanced methodologies. In this study, we developed a novel predictor that integrated all the residues (STY) phosphorylation sites information. We extracted ten different feature descriptors, primarily derived from composition, evolutionary, and position-specific information, and assessed their discriminative power through five classifiers. Our results indicated that Light Gradient Boosting (LGB) showed superior performance, and five descriptors displayed excellent discriminative capabilities. Subsequently, we identified the top two integrated features have high discriminative capability and trained with LGB to develop the final prediction model, LGB-IPs. The proposed approach shows an excellent performance on 10-fold cross-validation with an ACC, MCC, and AUC values of 0.831, 0.662, 0.907, respectively. Notably, these performances are replicated in the independent evaluation. Consequently, our approach may provide valuable insights into the phosphorylation mechanisms in SARS-CoV-2 infection for biomedical researchers. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects of serine/arginine-rich splicing factor 7 (SRSF7) on proliferation, migration and invasion of hepatocellular carcinoma (HCC) HepG2 cells and the possible mechanisms. Methods: Differential expression of SRSF7 between HCC and adjacent non-tumor tissues and its relationship with patient prognosis were analyzed online using The Cancer Genome Atlas (TCGA) and Kaplan Meier Plotter. HepG2 cells were cultured routinely and transfected with SRSF7 RNA knockdown sequences (siSRSF7#1 and siSRSF7#2), control sequences (NC), SRSF7 overexpression vector (hSRSF7-oe), and control vector (hSRSF7-nc) using transfection reagents. Accordingly, the cells were divided into NC group, siSRSF7#1 group, siSRSF7#2 group, NC + hSRSF7-nc group, siSRSF7 + hSRSF7-nc group, and siSRSF7 + hSRSF7-oe group. The mRNA and protein expression levels of SRSF7 in each group of cells were detected by qPCR and WB assay. The proliferation, migration, and invasion abilities of each group of cells were assessed by MTS assay, plate clone formation assay, scratch assay, and Transwell invasion assay. WB assay was used to detect the expression of JAK1/STAT3 signaling pathway related proteins in HepG2 cells of each group. Results: Database analysis showed that SRSF7 mRNA is highly expressed in HCC tissues (P < 0.001), and its high expression is associated with poor prognosis in HCC patients (P < 0.05). Knockdown of SRSF7 significantly reduced the proliferation, migration, and invasion abilities of HepG2 cells (all P < 0.01). The phosphorylation levels of JAK1 and STAT3 in the SRSF7 knockdown cells were significantly reduced (both P < 0.05), while overexpression of SRSF7 resulted in a significant increase in JAK1 and STAT3 phosphorylation levels (both P < 0.05). Conclusion: SRSF7 is highly expressed in HCC tissues and may promote the proliferation, migration, and invasion of HepG2 cells by regulating the JAK1/STAT3 signaling pathway. [ABSTRACT FROM AUTHOR]

Objective: To investigate whether Buthus martensii karsch (Scorpiones), Scolopendra subspinipes mutilans L. Koch (Scolopendra) and Gekko gecko Linnaeus (Gekko) could ameliorate the hypoxic tumor microenvironment and inhibit lung cancer growth and metastasis by regulating phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin/hypoxia-inducible factor-1α (PI3K/AKT/mTOR/HIF-1α) signaling pathway. Methods: Male C57BL/6J mice were inoculated with luciferase labeled LL/2-luc-M38 cell suspension to develop lung cancer models, with rapamycin and cyclophosphamide as positive controls. Carboxy methyl cellulose solutions of Scorpiones, Scolopendra and Gekko were administered intragastrically as 0.33, 0.33, and 0.83 g/kg, respectively once daily for 21 days. Fluorescent expression were detected every 7 days after inoculation, and tumor growth curves were plotted. Immunohistochemistry was performed to determine CD31 and HIF-1α expressions in tumor tissue and microvessel density (MVD) was analyzed. Western blot was performed to detect the expression of PI3K/AKT/mTOR/HIF-1α signaling pathway-related proteins. Enzyme-linked immunosorbent assay was performed to detect serum basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) in mice. Results: Scorpiones, Scolopendra and Gekko prolonged the survival time and inhibited lung cancer metastasis and expression of HIF-1α (all P<0.01). Moreover, Scorpiones, Scolopendra and Gekko inhibited the phosphorylation of AKT and ribosomal protein S6 kinase (p70S6K) (P<0.05 or P<0.01). In addition, they also decreased the expression of CD31, MVD, bFGF, TGF-β1 and VEGF compared with the model group (P<0.05 or P<0.01). Conclusion: Scorpiones, Scolopendra and Gekko all showed beneficial effects on lung cancer by ameliorating the hypoxic tumor microenvironment via PI3K/AKT/mTOR/HIF-1α signaling pathway. [ABSTRACT FROM AUTHOR]

Protein post‐translational modifications (PTMs) play an intricate role in a diverse range of cellular processes creating a complex PTM code that governs cell homeostasis. Understanding the molecular build‐up and the critical factors regulating this PTM code is essential for targeted therapeutic design whereby PTM mis‐regulation is prevalent. Here, we focus on Pin1, a peptidyl‐prolyl cis‐trans isomerase whose regulatory function is altered by a diverse range of PTMs. Through employing advanced mass spectrometry techniques in combination with fluorescence polarization and enzyme activity assays, we elucidate the impact of combinatorial phosphorylation on Pin1 function. Moreover, two phosphorylation sites were identified whereby Ser71 phosphorylation preceded Ser16 phosphorylation, leading to the deactivation of Pin1's prolyl isomerase activity before affecting substrate binding. Together, these findings shed light on the regulatory mechanisms underlying Pin1 function and emphasize the importance of understanding PTM landscapes in health and disease. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) poses a significant challenge because of the difficulty in identifying aggressive tumors, leading to overtreatment and missed personalized therapies. Although only 8% of cases progress beyond the prostate, the accurate prediction of aggressiveness remains crucial. Thus, this study focused on studying retinoblastoma phosphorylated at Serine 249 (Phospho-Rb S249), N-cadherin, β-catenin, and E-cadherin as biomarkers for identifying aggressive PCa using a logistic regression model and a classification and regression tree (CART). Using immunohistochemistry (IHC), we targeted the expression of these biomarkers in PCa tissues and correlated their expression with clinicopathological data of the tumor. The results showed a negative correlation between E-cadherin and β-catenin with aggressive tumor behavior, whereas Phospho-Rb S249 and N-cadherin positively correlated with increased tumor aggressiveness. Furthermore, patients were stratified based on Gleason scores and E-cadherin staining patterns to evaluate their capability for early identification of aggressive PCa. Our findings suggest that the classification tree is the most effective method for measuring the utility of these biomarkers in clinical practice, incorporating β-catenin, tumor grade, and Gleason grade as relevant determinants for identifying patients with Gleason scores ≥ 4 + 3. This study could potentially benefit patients with aggressive PCa by enabling early disease detection and closer monitoring. [ABSTRACT FROM AUTHOR]

The serine-threonine kinase Akt plays a fundamental role in cell survival, metabolism, proliferation, and migration. To keep these essential processes under control, Akt activity and stability must be tightly regulated; otherwise, life-threatening conditions might prevail. Although it is well understood that phosphorylation regulates Akt activity, much remains to be known about how its stability is maintained. Here, we characterize BAG5, a chaperone regulator, as a novel Akt-interactor and substrate that attenuates Akt stability together with Hsp70. BAG5 switches monoubiquitination to polyubiquitination of Akt and increases its degradation caused by Hsp90 inhibition and Hsp70 overexpression. Akt interacts with BAG5 at the linker region that joins the first and second BAG domains and phosphorylates the first BAG domain. The Akt-BAG5 complex is formed in serum-starved conditions and dissociates in response to HGF, coincident with BAG5 phosphorylation. BAG5 knockdown attenuated Akt degradation and facilitated its activation, whereas the opposite effect was caused by BAG5 overexpression. Altogether, our results indicate that Akt stability and signaling are dynamically regulated by BAG5, depending on growth factor availability.

Signaling by the extracellular signal-regulated kinase (ERK) pathway controls many cellular processes, including cell division, death, and differentiation. In this second installment of a two-part review, we address the question of how the ERK pathway exerts distinct and context-specific effects on multiple processes. We discuss how the dynamics of ERK activity induce selective changes in gene expression programs, with insights from both experiments and computational models. With a focus on single-cell biosensor-based studies, we summarize four major functional modes for ERK signaling in tissues: adjusting the size of cell populations, gradient-based patterning, wave propagation of morphological changes, and diversification of cellular gene expression states. These modes of operation are disrupted in cancer and other related diseases and represent potential targets for therapeutic intervention. By understanding the dynamic mechanisms involved in ERK signaling, there is potential for pharmacological strategies that not only simply inhibit ERK, but also restore functional activity patterns and improve disease outcomes.

Extracellular signal-regulated kinase (ERK) has long been studied as a key driver of both essential cellular processes and disease. A persistent question has been how this single pathway is able to direct multiple cell behaviors, including growth, proliferation, and death. Modern biosensor studies have revealed that the temporal pattern of ERK activity is highly variable and heterogeneous, and critically, that these dynamic differences modulate cell fate. This two-part review discusses the current understanding of dynamic activity in the ERK pathway, how it regulates cellular decisions, and how these cell fates lead to tissue regulation and pathology. In part 1, we cover the optogenetic and live-cell imaging technologies that first revealed the dynamic nature of ERK, as well as current challenges in biosensor data analysis. We also discuss advances in mathematical models for the mechanisms of ERK dynamics, including receptor-level regulation, negative feedback, cooperativity, and paracrine signaling. While hurdles still remain, it is clear that higher temporal and spatial resolution provide mechanistic insights into pathway circuitry. Exciting new algorithms and advanced computational tools enable quantitative measurements of single-cell ERK activation, which in turn inform better models of pathway behavior. However, the fact that current models still cannot fully recapitulate the diversity of ERK responses calls for a deeper understanding of network structure and signal transduction in general.

BackgroundNitric oxide (NO) has been identified as a signaling molecule generated during β-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during β-adrenergic receptor stimulation.MethodsWe measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 μM), sodium nitroprusside (200 μM), and β-adrenergic agonist isoproterenol (100 nmol/L).ResultsBoth WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events.ConclusionsWe conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent β-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain β-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Focal adhesion kinase (FAK) relays integrin signaling from outside to inside cells and contributes to cell adhesion and motility. However, the spatiotemporal dynamics of FAK activity in single FAs is unclear due to the lack of a robust FAK reporter, which limits our understanding of these essential biological processes. Here we have engineered a genetically encoded FAK activity sensor, dubbed FAK-separation of phases-based activity reporter of kinase (SPARK), which visualizes endogenous FAK activity in living cells and vertebrates. Our work reveals temporal dynamics of FAK activity during FA turnover. Most importantly, our study unveils polarized FAK activity at the distal tip of newly formed single FAs in the leading edge of a migrating cell. By combining FAK-SPARK with DNA tension probes, we show that tensions applied to FAs precede FAK activation and that FAK activity is proportional to the strength of tension. These results suggest tension-induced polarized FAK activity in single FAs, advancing the mechanistic understanding of cell migration.

Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells before cell lysate preparation significantly simplifies lysate preparation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.

G protein-coupled receptors (GPCRs) are highly druggable and implicated in numerous diseases, including vascular inflammation. GPCR signals are transduced from the plasma membrane as well as from endosomes and controlled by posttranslational modifications. The thrombin-activated GPCR protease-activated receptor-1 is modified by ubiquitin. Ubiquitination of protease-activated receptor-1 drives recruitment of transforming growth factor-β-activated kinase-1-binding protein 2 (TAB2) and coassociation of TAB1 on endosomes, which triggers p38 mitogen-activated protein kinase-dependent inflammatory responses in endothelial cells. Other endothelial GPCRs also induce p38 activation via a noncanonical TAB1-TAB2-dependent pathway. However, the regulatory processes that control GPCR ubiquitin-driven p38 inflammatory signaling remains poorly understood. We discovered mechanisms that turn on GPCR ubiquitin-dependent p38 signaling, however, the mechanisms that turn off the pathway are not known. We hypothesize that deubiquitination is an important step in regulating ubiquitin-driven p38 signaling. To identify specific deubiquitinating enzymes (DUBs) that control GPCR-p38 mitogen-activated protein kinase signaling, we conducted a siRNA library screen targeting 96 DUBs in endothelial cells and HeLa cells. We identified nine DUBs and validated the function two DUBs including cylindromatosis and ubiquitin-specific protease-34 that specifically regulate thrombin-induced p38 phosphorylation. Depletion of cylindromatosis expression by siRNA enhanced thrombin-stimulated p38 signaling, endothelial barrier permeability, and increased interleukin-6 cytokine expression. Conversely, siRNA knockdown of ubiquitin-specific protease-34 expression decreased thrombin-promoted interleukin-6 expression and had no effect on thrombin-induced endothelial barrier permeability. These studies suggest that specific DUBs distinctly regulate GPCR-induced p38-mediated inflammatory responses.

Abstract Background Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a fatal congenital lung disorder strongly associated with genomic alterations in the Forkhead box F1 (FOXF1) gene and its regulatory region. However, little is known about how FOXF1 genomic alterations cause ACD/MPV and what molecular mechanisms are affected by these mutations. Therefore, the effect of ACD/MPV patient-specific mutations in the FOXF1 gene on the molecular function of FOXF1 was studied. Methods Epitope-tagged FOXF1 constructs containing one of the ACD/MPV-associated mutations were expressed in mammalian cell lines to study the effect of FOXF1 mutations on protein function. EMSA binding assays and luciferase assays were performed to study the effect on target gene binding and activation. Immunoprecipitation followed by SDS‒PAGE and western blotting were used to study protein‒protein interactions. Protein phosphorylation was studied using phos-tag western blotting. Results An overview of the localization of ACD/MPV-associated FOXF1 mutations revealed that the G91-S101 region was frequently mutated. A three-dimensional model of the forkhead DNA-binding domain of FOXF1 showed that the G91-S101 region consists of an α-helix and is predicted to be important for DNA binding. We showed that FOXF1 missense mutations in this region differentially affect the DNA binding of the FOXF1 protein and influence the transcriptional regulation of target genes depending on the location of the mutation. Furthermore, we showed that some of these mutations can affect the FOXF1 protein at the posttranscriptional level, as shown by altered phosphorylation by MST1 and MST2 kinases. Conclusion Missense mutations in the coding region of the FOXF1 gene alter the molecular function of the FOXF1 protein at multiple levels, such as phosphorylation, DNA binding and target gene activation. These results indicate that FOXF1 molecular pathways may be differentially affected in ACD/MPV patients carrying missense mutations in the DNA-binding domain and may explain the phenotypic heterogeneity of ACD/MPV.

Abstract Circadian clocks rely on transcriptional/translational feedback loops involving clock genes and their corresponding proteins. While the primary oscillations originate from gene expression, the precise control of clock protein stability plays a pivotal role in establishing the 24-hour circadian rhythms. Most clock proteins are degraded through the ubiquitin/26S proteasome pathway, yet the enzymes responsible for ubiquitination and deubiquitination remain poorly characterised. We identified a missense allele (ubp12-3, S327F) of the UBP12 gene/protein in Arabidopsis. Despite ubp12-3 exhibited a short period phenotype similar to that of a loss-of-function allele, molecular analysis indicated elevated protease activity in ubp12-3. We demonstrated that early flowering of ubp12 mutants is a result of the shortened circadian period rather than a direct alteration of UBP12 function. Analysis of protease activity of non-phosphorylatable (S327A, S327F) and phosphomimetic (S327D) derivatives in bacteria suggested that phosphorylation of serine 327 inhibits UBP12 enzymatic activity, which could explain the over-functioning of S327F in vivo. We showed that phosphomimetic mutations of the conserved serine in the Neurospora and human orthologues reduced ubiquitin cleavage activity suggesting that not only the primary structures of UBP12-like enzymes are phylogenetically conserved across a wide range of species, but also the molecular mechanisms governing their enzymatic activity.

Abstract The complex network governing self-renewal in epidermal stem cells (EPSCs) is only partially defined. FOXM1 is one of the main players in this network, but the upstream signals regulating its activity remain to be elucidated. In this study, we identify cyclin-dependent kinase 1 (CDK1) as the principal kinase controlling FOXM1 activity in human primary keratinocytes. Mass spectrometry identified CDK1 as a key hub in a stem cell-associated protein network, showing its upregulation and interaction with essential self renewal-related markers. CDK1 phosphorylates FOXM1 at specific residues, stabilizing the protein and enhancing its nuclear localization and transcriptional activity, promoting self-renewal. Additionally, FOXM1 binds to the CDK1 promoter, inducing its expression. We identify the CDK1-FOXM1 feedforward loop as a critical axis sustaining EPSCs during in vitro cultivation. Understanding the upstream regulators of FOXM1 activity offers new insights into the biochemical mechanisms underlying self-renewal and differentiation in human primary keratinocytes.

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 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.

Mutations in protein phosphatase 2A (PP2A) are connected to intellectual disability and cancer. It has been hypothesized that these mutations might disrupt the autoinhibition and phosphorylation-induced activation of PP2A. Since they are located far from both the active and substrate binding sites, it is unclear how they exert their effect. We performed allosteric pathway analysis based on molecular dynamics simulations and combined it with biochemical experiments to investigate the autoinhibition of PP2A. In the wild type (WT), the C-arm of the regulatory subunit B56δ obstructs the active and substrate binding sites exerting a dual autoinhibition effect. We find that the disease mutant, E198K, severely weakens the allosteric pathways that stabilize the C-arm in the WT. Instead, the strongest allosteric pathways in E198K take a different route that promotes exposure of the substrate binding site. To facilitate the allosteric pathway analysis, we introduce a path clustering algorithm for lumping pathways into channels. We reveal remarkable similarities between the allosteric channels of E198K and those in phosphorylation-activated WT, suggesting that the autoinhibition can be alleviated through a conserved mechanism. In contrast, we find that another disease mutant, E200K, which is in spatial proximity of E198, does not repartition the allosteric pathways leading to the substrate binding site; however, it may still induce exposure of the active site. This finding agrees with our biochemical data, allowing us to predict the activity of PP2A with the phosphorylated B56δ and provide insight into how disease mutations in spatial proximity alter the enzymatic activity in surprisingly different mechanisms. [ABSTRACT FROM AUTHOR]

In the nucleus, transcriptionally silent genes are sequestered into heterochromatin compartments comprising nucleosomes decorated with histone H3 Lys9 trimethylation and a protein called HP1α. This protein can form liquid-liquid droplets in vitro and potentially organize heterochromatin through a phase separation mechanism that is promoted by phosphorylation. Elucidating the molecular interactions that drive HP1α phase separation and its consequences on nucleosome structure and dynamics has been challenging due to the viscous and heterogeneous nature of such assemblies. Here, we tackle this problem by a combination of solution and solid-state NMR spectroscopy, which allows us to dissect the interactions of phosphorylated HP1α with nucleosomes in the context of phase separation. Our experiments indicate that phosphorylated human HP1α does not cause any major rearrangements to the nucleosome core, in contrast to the yeast homologue Swi6. Instead, HP1α interacts specifically with the methylated H3 tails and slows the dynamics of the H4 tails. Our results shed light on how phosphorylated HP1α proteins may regulate the heterochromatin landscape, while our approach provides an atomic resolution view of a heterogeneous and dynamic biological system regulated by a complex network of interactions and post-translational modifications.

Intrinsically disordered proteins (IDPs) have been closely studied during the past decade due to their importance in many biological processes. The disordered nature of this group of proteins makes it difficult to observe its full span of the conformational space using either experimental or computational studies. In this article, we explored the conformational space of the C-terminal domain (CTD) of RNA polymerase II (Pol II), which is also an intrinsically disordered low complexity domain, using enhanced sampling methods. We provided a detailed conformational analysis of model systems of CTD with different lengths; first with the last 44 residues of the human CTD sequence and finally the CTD model with 2-heptapeptide repeating units. We then investigated the effects of phosphorylation on CTD conformations by performing simulations at different phosphorylated states. We obtained broad conformational spaces in nonphosphorylated CTD models, and phosphorylation has complex effects on the conformations of the CTD. These complex effects depend on the length of the CTD, spacing between the multiple phosphorylation sites, ion coordination, and interactions with the nearby residues.

β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2-adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and β-arrestins in controlling gene expression. We found that Gαs is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

In arterial myocytes, the canonical function of voltage-gated CaV1.2 and KV2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, KV2.1 also plays a sex-specific role by promoting the clustering and activity of CaV1.2 channels. However, the impact of KV2.1 protein organization on CaV1.2 function remains poorly understood. We discovered that KV2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of KV2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the KV2.1 clustering site (KV2.1S590A) eliminated KV2.1 macro-clustering and sex-specific differences in CaV1.2 cluster size and activity. We propose that the degree of KV2.1 clustering tunes CaV1.2 channel function in a sex-specific manner in arterial myocytes.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compacts the RNA genome into viral ribonucleoprotein (vRNP) complexes within virions. Assembly of vRNPs is inhibited by phosphorylation of the N protein serine/arginine (SR) region. Several SARS-CoV-2 variants of concern carry N protein mutations that reduce phosphorylation and enhance the efficiency of viral packaging. Variants of the dominant B.1.1 viral lineage also encode a truncated N protein, termed N∗ or Δ(1-209), that mediates genome packaging despite lacking the N-terminal RNA-binding domain and SR region. Here, we use mass photometry and negative stain electron microscopy to show that purified Δ(1-209) and viral RNA assemble into vRNPs that are remarkably similar in size and shape to those formed with full-length N protein. We show that assembly of Δ(1-209) vRNPs requires the leucine-rich helix of the central disordered region and that this helix promotes N protein oligomerization. We also find that fusion of a phosphomimetic SR region to Δ(1-209) inhibits RNA binding and vRNP assembly. Our results provide new insights into the mechanisms by which RNA binding promotes N protein self-association and vRNP assembly, and how this process is modulated by phosphorylation.

BackgroundAtrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility.MethodsRight atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies.ResultsIn human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (P=2.0×10-2; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (P=1.4×10-6; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=2.9×10-2 and 6.7×10-3, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (P=5.0×10-6; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (P=1.8×10-2 and 4.1×10-2, respectively; n=6, 6, and 5).ConclusionsPatients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Atypical chemokine receptor 3 (ACKR3) is an arrestin-biased receptor that regulates extracellular chemokine levels through scavenging. The scavenging process restricts the availability of the chemokine agonist CXCL12 for the G protein-coupled receptor (GPCR) CXCR4 and requires phosphorylation of the ACKR3 C-terminus by GPCR kinases (GRKs). ACKR3 is phosphorylated by GRK2 and GRK5, but the mechanisms by which these kinases regulate the receptor are unresolved. Here we determined that GRK5 phosphorylation of ACKR3 results in more efficient chemokine scavenging and β-arrestin recruitment than phosphorylation by GRK2 in HEK293 cells. However, co-activation of CXCR4-enhanced ACKR3 phosphorylation by GRK2 through the liberation of Gβγ, an accessory protein required for efficient GRK2 activity. The results suggest that ACKR3 senses CXCR4 activation through a GRK2-dependent crosstalk mechanism, which enables CXCR4 to influence the efficiency of CXCL12 scavenging and β-arrestin recruitment to ACKR3. Surprisingly, we also found that despite the requirement for phosphorylation and the fact that most ligands promote β-arrestin recruitment, β-arrestins are dispensable for ACKR3 internalization and scavenging, suggesting a yet-to-be-determined function for these adapter proteins. Since ACKR3 is also a receptor for CXCL11 and opioid peptides, these data suggest that such crosstalk may also be operative in cells with CXCR3 and opioid receptor co-expression. Additionally, kinase-mediated receptor cross-regulation may be relevant to other atypical and G protein-coupled receptors that share common ligands. SIGNIFICANCE STATEMENT: The atypical receptor ACKR3 indirectly regulates CXCR4-mediated cell migration by scavenging their shared agonist CXCL12. Here, we show that scavenging and β-arrestin recruitment by ACKR3 are primarily dependent on phosphorylation by GRK5. However, we also show that CXCR4 co-activation enhances the contribution of GRK2 by liberating Gβγ. This phosphorylation crosstalk may represent a common feedback mechanism between atypical and G protein-coupled receptors with shared ligands for regulating the efficiency of scavenging or other atypical receptor functions.

Exercise changes the tumor microenvironment by remodeling blood vessels and increasing infiltration by cytotoxic immune cells. The mechanisms driving these changes remain unclear. Herein, we demonstrate that exercise normalizes tumor vasculature and upregulates endothelial expression of VCAM1 in YUMMER 1.7 and B16F10 murine models of melanoma but differentially regulates tumor growth, hypoxia, and the immune response. We found that exercise suppressed tumor growth and increased CD8+ T-cell infiltration in YUMMER but not in B16F10 tumors. Single-cell RNA sequencing and flow cytometry revealed exercise modulated the number and phenotype of tumor-infiltrating CD8+ T cells and myeloid cells. Specifically, exercise caused a phenotypic shift in the tumor-associated macrophage population and increased the expression of MHC class II transcripts. We further demonstrated that ERK5 S496A knock-in mice, which are phosphorylation deficient at the S496 residue, mimicked the exercise effect when unexercised, yet when exercised, these mice displayed a reversal in the effect of exercise on tumor growth and macrophage polarization compared with wild-type mice. Taken together, our results reveal tumor-specific differences in the immune response to exercise and show that ERK5 signaling via the S496 residue plays a crucial role in exercise-induced tumor microenvironment changes. See related Spotlight by Betof Warner, p. 1158.

G protein-coupled receptor kinases (GRKs) are essential regulators of signaling pathways mediated by G protein-coupled receptors. Recent research suggests that GRK-mediated phosphorylation patterns dictate functional selectivity, leading to biased cellular responses. However, a comprehensive understanding of the structural mechanisms at the single-residue level remains elusive. This study aims to define the general conformational dynamics of GRKs with a particular focus on quantifying the transitions between the closed and open states. Specifically, we examined these transitions, classified based on the ionic lock between the regulatory G protein signaling homology domain and kinase domain. To facilitate a precise structural comparison, we assigned common labels to topologically identical positions across the 47 GRK structures retrieved from the Protein Data Bank. Our analysis identified both general and subfamily-specific dynamic movements within the networks and measured the conformational change scores between the two states. Elucidating these structural dynamics could provide significant insights into the regulatory mechanisms of GRK.

Genome-wide association studies (GWASs) have identified genetic susceptibility loci associated with juvenile dermatomyositis (JDM). Single nucleotide polymorphisms related to phosphorylation (phosSNPs) are critical nonsynonymous mutations exerting substantial influence on gene expression regulation. The aim of this study was to identify JDM susceptibility genes in the GWAS loci by the use of phosSNPs. We explored quantitative trait loci (QTLs) among the phosSNPs associated with JDM using data from eQTL (bulk tissues and single-cell) and pQTL studies. For gene expression and protein levels significantly influenced by JDM-associated phosSNPs, we assessed their associations with JDM through MR analyses. Additionally, we conducted differential expression gene analyses, incorporating single-cell transcriptomic profiling of 6 JDM cases and 11 juvenile controls (99,396 cells). We identified 31 phosSNPs situated in the 6p21 locus that were associated with JDM. Half of these phosSNPs showed effects on gene expression in various cells and circulating protein levels. In MR analyses, we established associations between the expression levels of pivotal JDM-associated genes, including MICB, C4A, HLA-DRB1, HLA-DRB5, and PSMB9, in skin, muscle, or blood cells and circulating levels of C4A, with JDM. Utilizing single-cell eQTL data, we identified a total of 276 association signals across 14 distinct immune cell types for 28 phosSNPs. Further insights were gained through single-cell differential expression analysis, revealing differential expression of PSMB9, HLA-A, HLA-B, HLA-C, HLA-DPB1, HLA-DQA1, HLA-DQB1, and HLA-DRB1 in immune cells. The present study pinpointed phosSNPs within susceptibility genes for JDM and unraveled the intricate relationships among these SNPs, gene expression levels, and JDM. [ABSTRACT FROM AUTHOR]