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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 mck1Δ 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.

Meningiomas are associated with inactivation of NF2/Merlin, but approximately one-third of meningiomas with favorable clinical outcomes retain Merlin expression. Biochemical mechanisms underlying Merlin-intact meningioma growth are incompletely understood, and non-invasive biomarkers that may be used to guide treatment de-escalation or imaging surveillance are lacking. Here, we use single-cell RNA sequencing, proximity-labeling proteomic mass spectrometry, mechanistic and functional approaches, and magnetic resonance imaging (MRI) across meningioma xenografts and patients to define biochemical mechanisms and an imaging biomarker that underlie Merlin-intact meningiomas. We find Merlin serine 13 (S13) dephosphorylation drives meningioma Wnt signaling and tumor growth by attenuating inhibitory interactions with β-catenin and activating the Wnt pathway. MRI analyses show Merlin-intact meningiomas with S13 phosphorylation and favorable clinical outcomes are associated with high apparent diffusion coefficient (ADC). These results define mechanisms underlying a potential imaging biomarker that could be used to guide treatment de-escalation or imaging surveillance for patients with Merlin-intact meningiomas.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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/ ).

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.

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.

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.

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.

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.

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.

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.

Neuroligin-3 is a postsynaptic adhesion molecule involved in synapse development and function. It is implicated in rare, monogenic forms of autism, and its shedding is critical to the tumor microenvironment of gliomas. While other members of the neuroligin family exhibit synapse-type specificity in localization and function through distinct interactions with postsynaptic scaffold proteins, the specificity of neuroligin-3 synaptic localization remains largely unknown. We investigated the synaptic localization of neuroligin-3 across regions in mouse and human brain samples after validating antibody specificity in knockout animals. We raised a phospho-specific neuroligin antibody and used phosphoproteomics, cell-based assays, and in utero CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) knockout and gene replacement to identify mechanisms that regulate neuroligin-3 localization to distinct synapse types. Neuroligin-3 exhibits region-dependent synapse specificity, largely localizing to excitatory synapses in cortical regions and inhibitory synapses in subcortical regions of the brain in both mice and humans. We identified specific phosphorylation of cortical neuroligin-3 at a key binding site for recruitment to inhibitory synapses, while subcortical neuroligin-3 remained unphosphorylated. In vitro, phosphomimetic mutation of that site disrupted neuroligin-3 association with the inhibitory postsynaptic scaffolding protein gephyrin. In vivo, phosphomimetic mutants of neuroligin-3 localized to excitatory postsynapses, while phospho-null mutants localized to inhibitory postsynapses. These data reveal an unexpected region-specific pattern of neuroligin-3 synapse specificity, as well as a phosphorylation-dependent mechanism that regulates its recruitment to either excitatory or inhibitory synapses. These findings add to our understanding of how neuroligin-3 is involved in conditions that may affect the balance of excitation and inhibition. [ABSTRACT FROM AUTHOR]

A key strategy to improve photocatalytic reaction efficiency is to achieve effective separation and migration of photo-generated carriers. Heterojunctions and co-catalysts are essential for boosting charge separation and transfer, reducing activation energy, and providing active sites for surface redox reactions. Herein, we introduce a composite catalytic system based on NiTiO 3 , enhanced with Graphdiyne (GDY) and NiP, achieving a hydrogen production activity of 8.37 mmol g−1 h−1. This performance is attributed to the combined effects of S-scheme heterojunction formation and NiP nanoparticle generation through phosphorylation. The S-scheme heterojunction effectively separates electron-hole pairs, reducing charge transport distance. Additionally, NiP nanoparticles formed in situ promote faster charge separation from NiTiO 3 and act as active sites to accelerate the reduction half-reaction. Consequently, NiTiO 3 /GDY-P exhibits higher light absorption, faster charge separation, and superior redox capacity compared to NiTiO 3. This work presents an efficient, eco-friendly multiphase catalytic system based on an S-scheme heterojunction and metal phosphide. • GDY was prepared by a new method of ball milling HEB-TMS. • GDY and NiTiO 3 construct S-scheme heterojunction to enhance electron-hole separation. • NiP nanoparticles boost NiTiO 3 's photoinduced electron transfer, enhancing charge separation. • In-situ XPS and DFT calculations proved the charge transfer process of NiTiO 3 /GDY-P. [ABSTRACT FROM AUTHOR]

Secreted protein Reelin is implicated in neuropsychiatric disorders and its supplementation ameliorates neurological symptoms in mouse disease models. Recombinant human Reelin protein may be useful for the treatment of human diseases, but its properties remain uncharacterized. Here, we report that full-length human Reelin was well secreted from transfected cells and was able to induce Dab1 phosphorylation. Unexpectedly, the central fragment of human Reelin was much less secreted than that of mouse Reelin. Three residues in the sixth Reelin repeat contributed to the secretion inefficiency, and their substitutions with mouse residues increased the secretion without affecting its biological activity. Our findings help efficient production of human Reelin protein for the supplementation therapy. [ABSTRACT FROM AUTHOR]

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]

Parkinson's disease (PD) is a neurodegenerative disorder caused by the degeneration of dopaminergic neurons in the brain stem. PD is mostly sporadic, but familial PD (FPD) cases are recorded in different studies. The first gene mutation that is linked to FPD is α‐synuclein (α‐syn). It was then found that α‐syn is also accumulated in Lewy body (LB), a classical pathological hallmark in PD patients. Different studies have shown that α‐syn accumulation and aggregation can be a crucial factor contributing to the degeneration of dopaminergic neurons in PD. α‐syn has been found to be degraded by the ubiquitin proteasomal system (UPS) and autophagy–lysosomal pathway (ALP). In this study, we initially explored how α‐syn phosphorylation by GRK6, PLK2 and CK2α would facilitate its degradation in relation to the UPS or ALP. Unexpectedly, we found that the degradation of α‐syn through PLK2 phosphorylation could be modulated by UPS and ALP in a novel mechanism. Specially, attenuation of UPS could increase the amount of PLK2 and then could facilitate the phosphorylation and degradation of α‐syn through ALP. To test this further in vivo, we attenuate the proteasomal activity in a well‐established A53T α‐syn transgenic PD mouse model. We found that attenuation of proteasomal activity in the A53T α‐syn transgenic mice could reduce the accumulation of α‐syn in the striatum and midbrain. Based on our results, this study provides a new insight into how α‐syn is degraded through the UPS and ALP. [ABSTRACT FROM AUTHOR]

Summary Reactive oxygen species (ROS) are among the most important signaling molecules, playing a significant role in plant growth, development, and responses to various environmental stresses. Respiratory burst oxidase homologs (RBOHs) are key enzymes in ROS production. Plants tightly regulate the activation and deactivation of RBOHs through various post‐translational modifications (PTMs), including phosphorylation, ubiquitination, S‐nitrosylation, and persulfidation. These PTMs fine‐tune ROS production, ensuring normal plant growth and development while facilitating rapid responses to abiotic and biotic stresses. This review discusses the effects of different PTMs on RBOH function and their biological relevance. Additionally, we examine the evolutionary conservation of PTM sites and emphasize the complex interplay between multiple PTMs regulating RBOHs. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Sepsis-induced myopathy (SIM) has been recognized as a critical risk factor for the development of acquired muscle weakness among patients in the intensive care unit. These individuals frequently encounter inadequate dietary intake and malnutrition. With the aggravation of the severity of the person's condition, leading to increased skeletal muscle protein breakdown and reduced synthesis, which is an urgent problem to be solved in clinical nutritional treatment. Whole milk protein powder (WMPP) has promising bioactive nutrients and holds promising potential for enhancing skeletal muscle mass. The study was designed to delve into the potential effects and mechanisms of WMPP intervention for increaseing skeletal muscle mass on SIM mice. Our results clearly show that the intervention with WMPP can significantly improve the exercise capacity and skeletal muscle mass in SIM mice. It significantly increases the diameter and cross-sectional area (CSA) of skeletal muscle fibers, while effectively reducing the excessive aggregation of collagen fibers and the abnormal accumulation of adipose tissue in the skeletal muscle of SIM mice. Moreover, WMPP intervention also significantly alleviated the oxidative stress status of mitochondria, which subsequently enhanced the expression of mitochondrial metabolic enzymes. The mechanism may be associated with decreased AMPK phosphorylation in skeletal muscle tissue and simultaneously increased phosphorylation of mTOR, p70S6K1, and 4EBP-1 in SIM mice. In summary, the WMPP intervention significantly enhances exercise capacity and skeletal muscle mass while mitigating the oxidative stress status of mitochondria. Furthermore, it regulates skeletal muscle anabolism via the AMPK/mTOR signaling pathway in SIM mice. [ABSTRACT FROM AUTHOR]

SUMMARY The sophisticated regulation of state transition is required to maintain optimal photosynthetic performance under fluctuating light condition, through balancing the absorbed light energy between photosystem II and photosystem I. This exquisite process incorporates phosphorylation and dephosphorylation of light‐harvesting complexes and PSII core subunits, accomplished by thylakoid membrane‐localized kinases and phosphatases that have not been fully identified. In this study, one Chlamydomonas high light response gene, THYLAKOID ENRICHED FRACTION 8 (TEF8), was characterized. The Chlamydomonas tef8 mutant showed high light sensitivity and defective state transition. The enzymatic activity assays showed that TEF8 is a bona fide phosphatase localized in thylakoid membranes. Biochemical assays, including BN‐PAGE, pull‐down, and phosphopeptide mass spectrometry, proved that TEF8 associates with photosystem II and is involved in the dephosphorylation of D2 and CP29 subunits during state 2 to state 1 transition. Taken together, our results identified TEF8 as a thylakoid phosphatase with multiple dephosphorylation targets on photosystem II, and provide new insight into the regulatory mechanism of state transition and high light resistance in Chlamydomonas. [ABSTRACT FROM AUTHOR]

Psilocybin, the natural hallucinogen from Psilocybe (magic) mushrooms, is a highly promising drug candidate for the treatment of depression and several other mental health conditions. Biosynthesis of psilocybin from the amino acid l‐tryptophan involves four strictly sequential modifications. The third of these, ATP‐dependent phosphorylation of the intermediate 4‐hydroxytryptamine, is catalysed by PsiK. Here we present a crystallographic analysis and a structure‐based mutagenesis study of this kinase, providing insight into its mode of substrate recognition. The results of our work will support future bioengineering efforts aimed at generating variants of psilocybin with enhanced therapeutic properties. [ABSTRACT FROM AUTHOR]

Kinesin-5 motors play an essential role during mitotic spindle assembly in many organisms 1,2,3,4,5,6,7,8,9,10,11 : they crosslink antiparallel spindle microtubules, step toward plus ends, and slide the microtubules apart. 12,13,14,15,16,17 This activity separates the spindle poles and chromosomes. Kinesin-5s are not only plus-end-directed but can walk or be carried toward MT minus ends, 18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 where they show enhanced localization. 3,5,7,27,29,32 The kinesin-5 C-terminal tail interacts with and regulates the motor, affecting structure, motility, and sliding force of purified kinesin-5 35,36,37 along with motility and spindle assembly in cells. 27,38,39 The tail contains phosphorylation sites, particularly in the conserved BimC box. 6,7,40,41,42,43,44 Nine mitotic tail phosphorylation sites were identified in the kinesin-5 motor of the fission yeast Schizosaccharomyces pombe , 45,46,47,48 suggesting that multi-site phosphorylation may regulate kinesin-5s. Here, we show that mutating all nine sites to either alanine or glutamate causes temperature-sensitive lethality due to a failure of bipolar spindle assembly. We characterize kinesin-5 localization and sliding force in the spindle based on Cut7-dependent microtubule minus-end protrusions in cells lacking kinesin-14 motors. 39,49,50,51,52 Imaging and computational modeling show that Cut7p simultaneously moves toward the minus ends of protrusion MTs and the plus ends of spindle midzone MTs. Phosphorylation mutants show dramatic decreases in protrusions and sliding force. Comparison to a model of force to create protrusions suggests that tail truncation and phosphorylation mutants decrease Cut7p sliding force similarly to tail-truncated human Eg5. 36 Our results show that C-terminal tail phosphorylation is required for kinesin-5/Cut7 sliding force and bipolar spindle assembly in fission yeast. [Display omitted] • Phosphorylation site mutations in the kinesin-5 tail affect motor function • Phosphorylation site mutants show impaired mitotic spindle assembly and elongation • Kinesin-5 sliding force is reduced in phosphorylation mutants • Computational modeling reproduces kinesin-5 accumulation in protrusions Jones et al. show that phosphorylation of the kinesin-5 C-terminal tail is required for proper spindle assembly in fission yeast. Mutations in multiple tail phosphorylation sites impair both spindle function and motor sliding force, similarly to full tail truncations of fission yeast and (by comparison to a model of force) human kinesin-5s. [ABSTRACT FROM AUTHOR]

This article reviews the latest research progress on the role of mitochondrial autophagy receptor FUN14 domain containing 1 (FUNDC1) in mitochondrial events and kidney disease. FUNDC1 is a protein located in the outer membrane of mitochondria, which maintains the function and quality of mitochondria by regulating mitochondrial autophagy, that is, the selective degradation process of mitochondria. The structural characteristics of FUNDC1 enable it to respond to intracellular signal changes and regulate the activity of mitochondrial autophagy through phosphorylation and dephosphorylation. During phosphorylation, unc-51-like kinase 1 (ULK1) promotes the activation of mitophagy by phosphorylating Ser17 of FUNDC1. In contrast, Src and CK2 kinases inhibit the interaction between FUNDC1 and LC3 by phosphorylating Tyr18 and Ser13, thereby inhibiting mitophagy. During dephosphorylation, PGAM5 phosphatase enhances the interaction between FUNDC1 and LC3 by dephosphorylating Ser13, thereby activating mitophagy. BCL2L1 inhibits the activity of PGAM5 by interacting with PGAM5, thereby preventing the dephosphorylation of FUNDC1 and inhibiting mitophagy. FUNDC1 plays an important role in mitochondrial events, participating in mitochondrial fission, maintaining the homeostasis of iron and proteins in mitochondrial matrix, and mediating crosstalk between mitochondria, endoplasmic reticulum and lysosomes, which have important effects on cell energy metabolism and programmed death. In the aspect of kidney disease, the abnormal function of FUNDC1 is closely related to the occurrence and development of many diseases. In acute kidney injury (AKI), cardiorenal syndrome (CRS), diabetic nephropathy (DN), chronic kidney disease (CKD), renal fibrosis (RF) and renal anemia, FUNDC1-mediated imbalance of mitophagy may be one of the key factors in disease progression. Therefore, in-depth study of the regulatory mechanism and function of FUNDC1 is of great significance for understanding the pathogenesis of renal disease and developing new treatment strategies. [ABSTRACT FROM AUTHOR]

Background: Epigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage. Methods: KDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay. Results: RSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation. Conclusions: Overall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy. [ABSTRACT FROM AUTHOR]

The insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) is a conserved RNA-binding protein that regulates RNA stability, localization and translation. IGF2BP1 is part of various ribonucleoprotein (RNP) condensates. However, the mechanism that regulates its assembly into condensates remains unknown. By using proteomics, we demonstrate that phosphorylation of IGF2BP1 at S181 in a disordered linker is regulated in a stress-dependent manner. Phosphomimetic mutations in two disordered linkers, S181E and Y396E, modulate RNP condensate formation by IGF2BP1 without impacting its binding affinity for RNA. Intriguingly, the S181E mutant, which lies in linker 1, impairs IGF2BP1 condensate formation in vitro and in cells, whereas a Y396E mutant in the second linker increases condensate size and dynamics. Structural approaches show that the first linker binds RNAs nonspecifically through its RGG/RG motif, an interaction weakened in the S181E mutant. Notably, linker 2 interacts with IGF2BP1's folded domains and these interactions are partially impaired in the Y396E mutant. Importantly, the phosphomimetic mutants impact IGF2BP1's interaction with RNAs and remodel the transcriptome in cells. Our data reveal how phosphorylation modulates low-affinity interaction networks in disordered linkers to regulate RNP condensate formation and RNA metabolism. How phosphorylation regulates the RNA-binding protein IGF2BP1 remained largely unclear. Here, the authors discovered that phosphorylation of IGF2BP1 in disordered linkers regulates its assembly into ribonucleoprotein granules and impacts metabolism of its target RNAs. [ABSTRACT FROM AUTHOR]

p57Kip2 is a member of the cyclin-dependent kinase (CDK) Interacting Protein/Kinase Inhibitory Protein (CIP/Kip) family that also includes p21Cip1/WAF1 and p27Kip1. Different from its siblings, few data are available about the p57Kip2 protein, especially in humans. Structurally, p57Kip2 is an intrinsically unstructured protein, a characteristic that confers functional flexibility with multiple transient interactions influencing the metabolism and roles of the protein. Being an IUP, its localization, stability, and binding to functional partners might be strongly modulated by post-translational modifications, especially phosphorylation. In this work, we investigated by two-dimensional analysis the phosphorylation pattern of p57Kip2 in different cellular models, revealing how the human protein appears to be extensively phosphorylated, compared to p21Cip1/WAF1 and p27Kip1. We further observed clear differences in the phosphoisoforms distributed in the cytosolic and nuclear compartments in asynchronous and synchronized cells. Particularly, the unmodified form is detectable only in the nucleus, while the more acidic forms are present in the cytoplasm. Most importantly, we found that the phosphorylation state of p57Kip2 influences the binding with some p57Kip2 partners, such as CDKs, LIMK1 and CRM1. Thus, it is necessary to completely identify the phosphorylated residues of the protein to fully unravel the roles of this CIP/Kip protein, which are still partially identified. [ABSTRACT FROM AUTHOR]

SUMMARY: The casein kinase II (CK2) complex consists of catalytic (α) and regulatory (β) subunits and is highly conserved throughout eukaryotes. Plant CK2 plays critical roles in multiple physiological processes; however, its function in plant immunity remains obscure. In this study, we demonstrated that the unique chloroplast‐localized CK2 α subunit (CPCK2) is a negative regulator of Arabidopsis thaliana innate immunity. cpck2 mutants displayed enhanced resistance against the fungal pathogen powdery mildew, Golovinomyces cichoracearum and the virulent bacterial pathogen, Pseudomonas syringae pv. tomato (Pto) DC3000. Moreover, the cpck2‐1 mutant accumulated higher salicylic acid (SA) levels and mutations that disabled SA biosynthesis or signaling inhibited cpck2‐1‐mediated disease resistance. CPCK2 interacted with the chloroplast‐localized carbonic anhydrase (CA), SA‐binding protein 3 (SABP3), which was required for cpck2‐mediated immunity. Significantly, CPCK2 phosphorylated SABP3, which promoted S‐nitrosylation of this enzyme. It has previously been established that S‐nitrosylation of SABP3 reduces both its SA binding function and its CA activity, which compromises the immune‐related function of SABP3. Taken together, our results establish CPCK2 as a negative regulator of SA accumulation and associated immunity. Importantly, our findings unveil a mechanism by which CPCK2 negatively regulates plant immunity by promoting S‐nitrosylation of SABP3 through phosphorylation, which provides the first example in plants of S‐nitrosylation being promoted by cognate phosphorylation. Significance Statement: CPCK2, a chloroplast‐localized casein kinase II α subunit, plays a negative role in plant innate immunity. CPCK2 directly interacted with and phosphorylated SA‐binding protein 3 (SABP3) to enhance the S‐nitrosylation status of SABP3, implying a potential mechanism by which CPCK2 negatively regulates plant immunity by promoting S‐nitrosylation of SABP3 through phosphorylation, which is the first example in plants of S‐nitrosylation being promoted by cognate phosphorylation. [ABSTRACT FROM AUTHOR]

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]

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]

Phosphorylation is a key post-translational modification regulating protein function and biological outcomes. However, the phosphorylation dynamics orchestrating mammalian oocyte development remains poorly understood. In the present study, we apply high-resolution mass spectrometry-based phosphoproteomics to obtain the first global in vivo quantification of mouse oocyte phosphorylation. Of more than 8000 phosphosites, 75% significantly oscillate and 64% exhibit marked upregulation during meiotic maturation, indicative of the dominant regulatory role. Moreover, we identify numerous novel phosphosites on oocyte proteins and a few highly conserved phosphosites in oocytes from different species. Through functional perturbations, we demonstrate that phosphorylation status of specific sites participates in modulating critical events including metabolism, translation, and RNA processing during meiosis. Finally, we combine inhibitor screening and enzyme-substrate network prediction to discover previously unexplored kinases and phosphatases that are essential for oocyte maturation. In sum, our data define landscape of the oocyte phosphoproteome, enabling in-depth mechanistic insights into developmental control of germ cells. Synopsis: Oocytes plays a central role in the success of reproduction, yet the phosphorylation dynamics during mammalian oocyte development remain poorly understood. This resource article describes the comprehensive stage-resolved phosphoproteome and proteome landscape of mouse oocytes, and reveals novel molecular mechanisms controlling meiotic maturation. The phosphorylation landscape of oocytes during in vivo maturation is delineated. Novel phosphosites in oocytes and conserved phosphosites across multiple species are identified. Key kinases orchestrating the regulation of oocyte meiosis are pinpointed Phosphatase dynamics during meiotic maturation are deciphered. This resource article provides the most comprehensive stage-resolved phosphoproteome and proteome landscape of mouse oocytes to date, revealing novel molecular control mechanisms. [ABSTRACT FROM AUTHOR]

ABSTRACT Actin depolymerizing factors (ADFs), like other actin‐binding proteins (ABPs), are modified by phosphorylation to regulate the dynamics of the actin filaments, thereby functioning in various processes throughout the plant lifecycle. In this study, we found that the Arabidopsis thaliana cytoplasmic kinase AGC1.7 interacts with ADF7 in vitro and in vivo. AGC1.7 phosphorylates ADF7 at its Ser‐6, Ser‐103 and Ser‐104 residues in vitro, while replacing these residues with alanine promotes ADF7‐mediated actin depolymerization in vitro. Expression of the phosphorylation‐mimetic mutant protein ADF7S6/103/104D driven by the pollen‐specific LAT52 promoter fully rescues the defects in germination rate, silique length and seeds per silique in both adf7‐2 and agc1.5 agc1.7 (agcdm) mutants. Our data establish a model whereby AGC1.7‐mediated ADF7 phosphorylation plays an important role in pollen germination and pollen tube growth. [ABSTRACT FROM AUTHOR]

Increasing cyclic GMP activates 26S proteasomes via phosphorylation by Protein Kinase G and stimulates the intracellular degradation of misfolded proteins. Therefore, agents that raise cGMP may be useful therapeutics against neurodegenerative diseases and other diseases in which protein degradation is reduced and misfolded proteins accumulate, including Charcot Marie Tooth 1A and 1B peripheral neuropathies, for which there are no treatments. Here we increased cGMP in the S63del mouse model of CMT1B by treating for three weeks with either the phosphodiesterase 5 inhibitor tadalafil, or the brain-penetrant soluble guanylyl cyclase stimulator CYR119. Both molecules activated proteasomes in the affected peripheral nerves, reduced polyubiquitinated proteins, and improved myelin thickness and nerve conduction. CYR119 increased cGMP more than tadalafil in the peripheral nerves of S63del mice and elicited greater biochemical and functional improvements. To determine whether raising cGMP could be beneficial in other neuropathies, we first showed that polyubiquitinated proteins and the disease-causing protein accumulate in the sciatic nerves of the C3 mouse model of CMT1A. Treatment of these mice with CYR119 reduced the levels of polyubiquitinated proteins and the disease-causing protein, presumably by increasing their degradation, and improved myelination, nerve conduction, and motor coordination. Thus, pharmacological agents that increase cGMP are promising treatments for CMT1 neuropathies and may be useful against other proteotoxic and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

During the embryonic developmental stage in vertebrates, internal organs are arranged along the left–right axis. Disruptions in this process can result in congenital diseases or laterality disorders. The molecular mechanisms of left–right asymmetry in vertebrate development remain largely unclear. Due to its straightforward structure, zebrafish has become a favoured model for studying early laterality events. Here, we demonstrate that growth and development factor 11 (Gdf11) is essential for left–right development via TGF‐β signalling. Morphological analysis showed that gdf11 morphants and mutants displayed clear heart and liver laterality disorders in a Nodal signal‐dependent manner. Additionally, we found that Kupffer's vesicle formation and ciliogenesis were impaired following gdf11 deletion. We also observed that Gdf11 may form a heterodimer with Spaw, which promotes Smad2/3 phosphorylation and activates TGF‐β signalling. Subsequently, Gdf11 promotes left–right laterality by stimulating Foxj1a and its target gene expression. In summary, we reveal a critical role of Gdf11 in left–right patterning, providing fundamental insights into the developmental process of left–right asymmetry. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Alzheimer's disease (AD) is a debilitating progressively neurodegenerative disease. The best-characterized hallmark of AD, which is marked by behavioral alterations and cognitive deficits, is the aggregation of deposition of amyloid-beta (Aβ) and hyper-phosphorylated microtubule-associated protein Tau. Despite decades of experimental progress, the control rate of AD remains poor, and more precise deciphering is needed for potential therapeutic targets and signaling pathways involved. In recent years, phosphoinositide 3-kinase (PI3K) and Akt have been recognized for their role in the neuroprotective effect of various agents, and glycogen synthase kinase 3 (GSK3), a downstream enzyme, is also crucial in the tau phosphorylation and Aβ deposition. An overview of the function of PI3K/Akt pathway in the pathophysiology of AD is provided in this review, along with a discussion of recent developments in the pharmaceuticals and herbal remedies that target the PI3K/Akt signaling pathway. In conclusion, despite the challenges and hurdles, cumulative findings of novel targets and agents in the PI3K/Akt signaling axis are expected to hold promise for advancing AD prevention and treatment. [ABSTRACT FROM AUTHOR]

Background: Neuroinflammation is widely recognized as a key factor in the pathogenesis of Alzheimer's disease (AD), alongside ß-amyloid deposition and the formation of neurofibrillary tangles. The NLR family pyrin domain containing 3 (NLRP3) inflammasome, part of the innate immune system, has been implicated in the neuropathology of both preclinical amyloid and tau transgenic models. Activation of the NLRP3 pathway involves an initial priming step, which increases the expression of Nlrp3 and interleukin (IL)-1ß, followed by the assembly of the NLRP3 inflammasome complex, comprising NLRP3, ASC, and caspase-1. This assembly leads to the proteolytic maturation of the pro-inflammatory cytokines IL-1ß and IL-18. Additionally, the NLRP3 inflammasome induces Gasdermin D (GSDMD) cleavage, forming membrane pores through which IL-1ß and IL-18 are secreted. Inhibition of NLRP3 has been shown to enhance plaque clearance by modulating microglial activation. Furthermore, blocking NLRP3 in tau transgenic mice has been found to reduce tau phosphorylation by affecting the activity of certain tau kinases and phosphatases. Methods: In this study, organotypic brain slice cultures from P301S transgenic mice were treated with lipopolysaccharide (LPS) plus nigericin as a positive control or exposed to tau seeds (K18) to evaluate NLRP3 inflammasome activation. The effect of tau seeding on NLRP3 activity was further examined using Meso Scale Discovery (MSD) assays to measure IL1ß secretion levels in the presence and absence of NLRP3 inhibitors. The role of NLRP3 activity was investigated in fullbody Nlrp3 knockout mice crossbred with the tau transgenic P301S model. Additionally, full-body and microglia-selective Gsdmd knockout mice were crossbred with P301S mice, and tau pathology and neurodegeneration were evaluated at early and late stages of the disease using immunohistochemistry and biochemical assays. Results: Activation of the NLRP3 pathway was observed in the mouse organotypic slice culture (OSC) model following stimulation with LPS and nigericin or exposure to tau seeds. However, Nlrp3 deficiency did not mitigate tauopathy or neurodegeneration in P301S mice in vivo, showing only a minor effect on plasma neurofilament (NF-L) levels. Consistently, Gsdmd deficiency did not alter tau pathology in P301S mice. Furthermore, neither full-body nor microglia-selective Gsdmd deletion had an impact on neuronal pathology or the release of pro-inflammatory cytokines. Conclusion: The absence of key components of the NLRP3 inflammasome pathway did not yield a beneficial effect on tau pathology or neurodegeneration in the preclinical Tau-P301S mouse model of AD. Nonetheless, organotypic slice cultures could serve as a valuable ex vivo mechanistic model for evaluating NLRP3 pathway activation and pharmacological inhibitors. [ABSTRACT FROM AUTHOR]

Casein kinase 1δ (CK1δ) controls essential biological processes including circadian rhythms and wingless-related integration site (Wnt) signaling, but how its activity is regulated is not well understood. CK1δ is inhibited by autophosphorylation of its intrinsically disordered C-terminal tail. Two CK1 splice variants, δ1 and δ2, are known to have very different effects on circadian rhythms. These variants differ only in the last 16 residues of the tail, referred to as the extreme C termini (XCT), but with marked changes in potential phosphorylation sites. Here, we test whether the XCT of these variants have different effects in autoinhibition of the kinase. Using NMR and hydrogen/deuterium exchange mass spectrometry, we show that the δ1 XCT is preferentially phosphorylated by the kinase and the δ1 tail makes more extensive interactions across the kinase domain. Mutation of δ1-specific XCT phosphorylation sites increases kinase activity both in vitro and in cells and leads to changes in the circadian period, similar to what is reported in vivo. Mechanistically, loss of the phosphorylation sites in XCT disrupts tail interaction with the kinase domain. δ1 autoinhibition relies on conserved anion-binding sites around the CK1 active site, demonstrating a common mode of product inhibition of CK1δ. These findings demonstrate how a phosphorylation cycle controls the activity of this essential kinase. [ABSTRACT FROM AUTHOR]

Background: Chronic migraine is a severe and common neurological disorder, yet its precise physiological mechanisms remain unclear. The IGF1/IGF1r signaling pathway plays a crucial role in pain modulation. Studies have shown that IGF1, by binding to its receptor IGF1r, activates a series of downstream signaling cascades involved in neuronal survival, proliferation, autophagy and functional regulation. The activation of these pathways can influence nociceptive transmission. Furthermore, alterations in IGF1/IGF1r signaling are closely associated with the development of various chronic pain conditions. Therefore, understanding the specific mechanisms by which this pathway contributes to pain is of significant importance for the development of novel pain treatment strategies. In this study, we investigated the role of IGF1/IGF1r and its potential mechanisms in a mouse model of chronic migraine. Methods: Chronic migraine was induced in mice by repeated intraperitoneal injections of nitroglycerin. Mechanical and thermal hypersensitivity responses were assessed using Von Frey filaments and radiant heat, respectively. To determine the role of IGF1/IGF1r in chronic migraine (CM), we examined the effects of the IGF1 receptor antagonist ppp (Picropodophyllin) on pain behaviors and the expression of calcitonin gene-related peptide (CGRP) and c-Fos. Result: In the nitroglycerin-induced chronic migraine model in mice, neuronal secretion of IGF1 is elevated within the trigeminal nucleus caudalis (TNC). Increased phosphorylation of the IGF1 receptor occurs, predominantly co-localizing with neurons. Treatment with ppp alleviated basal mechanical hypersensitivity and acute mechanical allodynia. Furthermore, ppp ameliorated autophagic dysfunction and reduced the expression of CGRP and c-Fos. Conclusion: Our findings demonstrate that in the chronic migraine (CM) model in mice, there is a significant increase in IGF1 expression in the TNC region. This upregulation of IGF1 leads to enhanced phosphorylation of IGF1 receptors on neurons. Targeting and inhibiting this signaling pathway may offer potential preventive strategies for mitigating the progression of chronic migraine. [ABSTRACT FROM AUTHOR]

• PD-O-α-Syn and MSA-O-α-Syn are differentially phosphorylated at serine 129. • PD-O-α-Syn and MSA-O-α-Syn have different cytotoxicity and seeding activity. • PD-O-α-Syn and MSA-O-α-Syn cause distinct dopaminergic damage and motor disorders. Previous studies have shown that α-synuclein (α-Syn) aggregates derived from the brains of patients with Parkinson's disease (PD) and multiple system atrophy (MSA) exhibit different phosphorylation, cytotoxicity, and seeding activity. However, the mechanism underlying the differences remains poorly understood. Here, recombinant human α-Syn was incubated in the plasma of patients with PD and MSA, and the oligomers formed in the plasma (PD-O-α-Syn and MSA-O-α-Syn) were purified and analyzed for their phosphorylation, cytotoxicity and seeding activity. In vitro assays revealed that both PD-O-α-Syn and MSA-O-α-Syn were phosphorylated at serine 129. However, the phosphorylation degree of MSA-O-α-Syn was significantly higher than that of PD-O-α-Syn. In addition, MSA-O-α-Syn exhibited stronger cytotoxicity and seeding activity compared with PD-O-α-Syn. In vivo experiments showed that mice receiving intrastriatal inoculation of MSA-O-α-Syn developed more severe motor dysfunction and dopaminergic degeneration than mice receiving intrastriatal inoculation of PD-O-α-Syn. Compared with the mice inoculated with PD-O-α-Syn, the mice inoculated with MSA-O-α-Syn accumulated more phosphorylated and oligomerized α-Syn in the striatum and brain regions (substantia nigra, hippocampus and prefrontal cortex) away from the inoculated site. The results obtained suggest that α-Syn oligomers formed in PD and MSA plasma are different in phosphorylation, cytotoxicity, and seeding activity. [ABSTRACT FROM AUTHOR]

Mcm10 plays an essential role in the activation of replicative helicase CMG through the cell cycle-regulated interaction with the prototype MCM double hexamer in Saccharomyces cerevisiae. In this study, we reported that Mcm10 is phosphorylated by S-phase cyclin-dependent kinases (S-CDKs) at S66, which enhances Mcm10--MCM association during the S phase. S66A single mutation or even deletion of whole N-terminus (a.a. 1-128) only causes mild growth defects. Nevertheless, S66 becomes indispensable in the absence of the Mcm10 C-terminus ((a.a. 463-571), the major MCM-binding domain. Using a two-degron strategy to efficiently deplete Mcm10, we show that mcm10-S66AΔC has a severe defect in proceeding into the S phase. Notably, both lethality and S-phase deficiency can be rescued by artificially tethering mcm10-S66AΔC to MCM. These findings illustrate how the Mcm10-MCM association is regulated as a crucial event in DNA replication initiation. [ABSTRACT FROM AUTHOR]

Simple Summary: Phosphorylation is a crucial process that regulates various cellular activities. Detecting phosphorylation sites, especially in cells infected by the SARS-CoV-2 virus, is challenging due to technical limitations. To address this, we developed GBMPhos, an advanced tool combining deep learning techniques, to accurately identify these sites. GBMPhos outperformed traditional methods and current state-of-the-art approaches in identifying phosphorylation sites. We have developed a free web server, which helps researchers gain a better understanding of protein modifications during a SARS-CoV-2 infection, potentially aiding in the development of therapeutic strategies and contributing to the fight against COVID-19. Phosphorylation, a reversible and widespread post-translational modification of proteins, is essential for numerous cellular processes. However, due to technical limitations, large-scale detection of phosphorylation sites, especially those infected by SARS-CoV-2, remains a challenging task. To address this gap, we propose a method called GBMPhos, a novel method that combines convolutional neural networks (CNNs) for extracting local features, gating mechanisms to selectively focus on relevant information, and a bi-directional gated recurrent unit (Bi-GRU) to capture long-range dependencies within protein sequences. GBMPhos leverages a comprehensive set of features, including sequence encoding, physicochemical properties, and structural information, to provide an in-depth analysis of phosphorylation sites. We conducted an extensive comparison of GBMPhos with traditional machine learning algorithms and state-of-the-art methods. Experimental results demonstrate the superiority of GBMPhos over existing methods. The visualization analysis further highlights its effectiveness and efficiency. Additionally, we have established a free web server platform to help researchers explore phosphorylation in SARS-CoV-2 infections. The source code of GBMPhos is publicly available on GitHub. [ABSTRACT FROM AUTHOR]