Microglial TNFα orchestrates protein phosphorylation in the cortex during the sleep period and controls homeostatic sleep.

Sleep intensity is adjusted by the length of previous awake time, and under tight homeostatic control by protein phosphorylation. Here, we establish microglia as a new cellular component of the sleep homeostasis circuit. Using quantitative phosphoproteomics of the mouse frontal cortex, we demonstrate that microglia-specific deletion of TNFα perturbs thousands of phosphorylation sites during the sleep period. Substrates of microglial TNFα comprise sleep-related kinases such as MAPKs and MARKs, and numerous synaptic proteins, including a subset whose phosphorylation status encodes sleep need and determines sleep duration. As a result, microglial TNFα loss attenuates the build-up of sleep need, as measured by electroencephalogram slow-wave activity and prevents immediate compensation for loss of sleep. Our data suggest that microglia control sleep homeostasis by releasing TNFα which acts on neuronal circuitry through dynamic control of phosphorylation.

Nuclear Phosphoproteome Reveals Prolyl Isomerase PIN1 as a Modulator of Oncogene-Induced Senescence.

Mammalian cells possess intrinsic mechanisms to prevent tumorigenesis upon deleterious mutations, including oncogene-induced senescence (OIS). The molecular mechanisms underlying OIS are, however, complex and remain to be fully characterized. In this study, we analyzed the changes in the nuclear proteome and phosphoproteome of human lung fibroblast IMR90 cells during the progression of OIS induced by oncogenic RASG12V activation. We found that most of the differentially regulated phosphosites during OIS contained prolyl isomerase PIN1 target motifs, suggesting PIN1 is a key regulator of several promyelocytic leukemia nuclear body proteins, specifically regulating several proteins upon oncogenic Ras activation. We showed that PIN1 knockdown promotes cell proliferation, while diminishing the senescence phenotype and hallmarks of senescence, including p21, p16, and p53 with concomitant accumulation of the protein PML and the dysregulation of promyelocytic leukemia nuclear body formation. Collectively, our data demonstrate that PIN1 plays an important role as a tumor suppressor in response to oncogenic ER:RasG12V activation.

Molecular Signatures of Neurodegenerative Diseases Identified by Proteomic and Phosphoproteomic Analyses in Aging Mouse Brain.

A central hallmark of neurodegenerative diseases is the irreversible accumulation of misfolded proteins in the brain by aberrant phosphorylation. Understanding the mechanisms underlying protein phosphorylation and its role in pathological protein aggregation within the context of aging is crucial for developing therapeutic strategies aimed at preventing or reversing such diseases. Here, we applied multi-protease digestion and quantitative mass spectrometry to compare and characterize dysregulated proteins and phosphosites in the mouse brain proteome using three different age groups: young-adult (3-4 months), middle-age (10 months), and old mice (19-21 months). Proteins associated with senescence, neurodegeneration, inflammation, cell cycle regulation, the p53 hallmark pathway, and cytokine signaling showed significant age-dependent changes in abundances and level of phosphorylation. Several proteins implicated in Alzheimer's disease (AD) and Parkinson's disease (PD) including tau (Mapt), Nefh, and Dpysl2 (also known as Crmp2) were hyperphosphorylated in old mice brain suggesting their susceptibility to the diseases. Cdk5 and Gsk3b, which are known to phosphorylate Dpysl2 at multiple specific sites, had also increased phosphorylation levels in old mice suggesting a potential crosstalk between them to contribute to AD. Hapln2, which promotes α-synuclein aggregation in patients with PD, was one of the proteins with highest abundance in old mice. CD9, which regulates senescence through the PI3K-AKT-mTOR-p53 signaling was upregulated in old mice and its regulation was correlated with the activation of phosphorylated AKT1. Overall, the findings identify a significant association between aging and the dysregulation of proteins involved in various pathways linked to neurodegenerative diseases with potential therapeutic implications.

Arabidopsis hypocotyl growth in darkness requires the phosphorylation of a microtubule-associated protein.

Rapid hypocotyl elongation allows buried seedlings to emerge, where light triggers de-etiolation and inhibits hypocotyl growth mainly by photoreceptors. Phosphorylation/dephosphorylation events regulate many aspects of plant development. Only recently we have begun to uncover the earliest phospho-signaling responders to light. Here, we reported a large-scale phosphoproteomic analysis and identified 20 proteins that changed their phosphorylation pattern following a 20 min light pulse compared to darkness. Microtubule-associated proteins were highly overrepresented in this group. Among them, we studied CIP7 (COP1-INTERACTING-PROTEIN 7), which presented microtubule (MT) localization in contrast to the previous description. An isoform of CIP7 phosphorylated at Serine915 was detected in etiolated seedlings but was undetectable after a light pulse in the presence of photoreceptors, while CIP7 transcript expression decays with long light exposure. The short hypocotyl phenotype and rearrangement of MTs in etiolated cip7 mutants are complemented by CIP7-YFP and the phospho-mimetic CIP7S915D-YFP, but not the phospho-null CIP7S915A-YFP suggesting that the phosphorylated S915CIP7 isoform promotes hypocotyl elongation through MT reorganization in darkness. Our evidence on Serine915 of CIP7 unveils phospho-regulation of MT-based processes during skotomorphogenic hypocotyl growth.

Endothelium-mediated regulation of platelet activation: Involvement of multiple protein kinases.

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet-endothelium interaction. Using high-shear whole-blood microfluidics, employing coagulant or non-coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real-time monitoring of the cytosolic Ca2+ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca2+ responses by collagen-related peptide (CRP-XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label-free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP-XL, and/or thrombin. High-resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable-isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium-promoted activity of a multi-kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.

PP2ACdc55 Phosphatase Imposes Ordered Cell-Cycle Phosphorylation by Opposing Threonine Phosphorylation.

In the quantitative model of cell-cycle control, progression from G1 through S phase and into mitosis is ordered by thresholds of increasing cyclin-dependent kinase (Cdk) activity. How such thresholds are read out by substrates that respond with the correct phosphorylation timing is not known. Here, using the budding yeast model, we show that the abundant PP2ACdc55 phosphatase counteracts Cdk phosphorylation during interphase and delays phosphorylation of late Cdk substrates. PP2ACdc55 specifically counteracts phosphorylation on threonine residues, and consequently, we find that threonine-directed phosphorylation occurs late in the cell cycle. Furthermore, the late phosphorylation of a model substrate, Ndd1, depends on threonine identity of its Cdk target sites. Our results support a model in which Cdk-counteracting phosphatases contribute to cell-cycle ordering by imposing Cdk thresholds. They also unveil a regulatory principle based on the phosphoacceptor amino acid, which is likely to apply to signaling pathways beyond cell-cycle control.

The Proteome and Phosphoproteome Uncovers Candidate Proteins Associated With Vacuolar Phosphate Signal Multipled by Vacuolar Phosphate Transporter 1 (VPT1) in Arabidopsis.

Plant vacuoles serve as the primary intracellular compartments for inorganic phosphate (Pi) storage. Passage of Pi across vacuolar membranes plays a critical role in buffering the cytoplasmic Pi level against fluctuations of external Pi and metabolic activities. To gain new insights into the proteins and processes, vacuolar Pi level regulated by vacuolar phosphate transporter 1 (VPT1) in Arabidopsis, we carried out tandem mass tag labeling proteome and phosphoproteome profiling of Arabidopsis WT and vpt1 loss-of-function mutant plants. The vpt1 mutant had a marked reduced vacuolar Pi level and a slight increased cytosol Pi level. The mutant was stunted as reflected in the reduction of the fresh weight compared with WT plants and bolting earlier under normal growth conditions in soil. Over 5566 proteins and 7965 phosphopeptides were quantified. About 146 and 83 proteins were significantly changed at protein abundance or site-specific phosphorylation levels, but only six proteins were shared between them. Functional enrichment analysis revealed that the changes of Pi states in vpt1 are associated with photosynthesis, translation, RNA splicing, and defense response, consistent with similar studies in Arabidopsis. Except for PAP26, EIN2, and KIN10, which were reported to be associated with phosphate starvation signal, we also found that many differential proteins involved in abscisic acid signaling, such as CARK1, SnRK1, and AREB3, were significantly changed in vpt1. Our study illuminates several new aspects of the phosphate response and identifies important targets for further investigation and potential crop improvement.

Translational and Posttranslational Dynamics in a Model Peptidergic System.

The cell bodies of hypothalamic magnocellular neurones are densely packed in the hypothalamic supraoptic nucleus, whereas their axons project to the anatomically discrete posterior pituitary gland. We have taken advantage of this unique anatomical structure to establish proteome and phosphoproteome dynamics in neuronal cell bodies and axonal terminals in response to physiological stimulation. We have found that proteome and phosphoproteome responses to neuronal stimulation are very different between somatic and axonal neuronal compartments, indicating the need of each cell domain to differentially adapt. In particular, changes in the phosphoproteome in the cell body are involved in the reorganization of the cytoskeleton and in axonal terminals the regulation of synaptic and secretory processes. We have identified that prohormone precursors including vasopressin and oxytocin are phosphorylated in axonal terminals and are hyperphosphorylated following stimulation. By multiomic integration of transcriptome and proteomic data, we identify changes to proteins present in afferent inputs to this nucleus.

Characterization of p38α Signaling Networks in Cancer Cells Using Quantitative Proteomics and Phosphoproteomics.

p38α (encoded by MAPK14) is a protein kinase that regulates cellular responses to almost all types of environmental and intracellular stresses. Upon activation, p38α phosphorylates many substrates both in the cytoplasm and nucleus, allowing this pathway to regulate a wide variety of cellular processes. While the role of p38α in the stress response has been widely investigated, its implication in cell homeostasis is less understood. To investigate the signaling networks regulated by p38α in proliferating cancer cells, we performed quantitative proteomic and phosphoproteomic analyses in breast cancer cells in which this pathway had been either genetically targeted or chemically inhibited. Our study identified with high confidence 35 proteins and 82 phosphoproteins (114 phosphosites) that are modulated by p38α and highlighted the implication of various protein kinases, including MK2 and mTOR, in the p38α-regulated signaling networks. Moreover, functional analyses revealed an important contribution of p38α to the regulation of cell adhesion, DNA replication, and RNA metabolism. Indeed, we provide experimental evidence supporting that p38α facilitates cancer cell adhesion and showed that this p38α function is likely mediated by the modulation of the adaptor protein ArgBP2. Collectively, our results illustrate the complexity of the p38α-regulated signaling networks, provide valuable information on p38α-dependent phosphorylation events in cancer cells, and document a mechanism by which p38α can regulate cell adhesion.

Proteomic and Phosphoproteomic Analysis of Right Ventricular Hypertrophy in the Pulmonary Hypertension Rat Model.

Pulmonary arterial hypertension (PAH) is a progressive disease that affects both the lungs and heart. Right ventricle (RV) hypertrophy is a primary pathological feature of PAH; however, its underlying molecular mechanisms remain insufficiently studied. In this study, we employed tandem mass tag (TMT)-based quantitative proteomics for the integrative analysis of the proteome and phosphoproteome of the RV derived from monocrotaline-induced PAH model rats. Compared with control samples, 564 significantly upregulated proteins, 616 downregulated proteins, 622 downregulated phosphopeptides, and 683 upregulated phosphopeptides were identified (P < 0.05, abs (log2 (fold change)) > log2 1.2) in the MCT samples. The quantitative real-time polymerase chain reaction (qRT-PCR) validated the expression levels of top 20 significantly altered proteins, including Nppa (natriuretic peptides A), latent TGF-ß binding protein 2 (Ltbp2), periostin, connective tissue growth factor 2 (Ccn2), Ncam1 (neural cell adhesion molecule), quinone reductase 2 (Nqo2), and tropomodulin 4 (Tmod4). Western blotting confirmed the upregulation of Ncam1 and downregulation of Nqo2 and Tmod4 in both MCT-induced and hypoxia-induced PH rat models. Pathway enrichment analyses indicated that the altered proteins are associated with pathways, such as vesicle-mediated transport, actin cytoskeleton organization, TCA cycle, and respiratory electron transport. These significantly changed phosphoproteins were enriched in pathways such as diabetic cardiomyopathy, hypertrophic cardiomyopathy, glycolysis/gluconeogenesis, and cardiac muscle contraction. In summary, this study provides an initial analysis of the RV proteome and phosphoproteome in the progression of PAH, highlighting several RV dysfunction-associated proteins and pathways.

Multiomics Reveals Induction of Neuroblastoma SK-N-BE(2)C Cell Death by Mitochondrial Division Inhibitor 1 through Multiple Effects.

Mitochondrial division inhibitor 1 (Mdivi-1) is a well-known synthetic compound aimed at inhibiting dynamin-related protein 1 (Drp1) to suppress mitochondrial fission, making it a valuable tool for studying mitochondrial dynamics. However, its specific effects beyond Drp1 inhibition remain to be confirmed. In this study, we employed integrative proteomics and phosphoproteomics to delve into the molecular responses induced by Mdivi-1 in SK-N-BE(2)C cells. A total of 3070 proteins and 1945 phosphorylation sites were identified, with 880 of them represented as phosphoproteins. Among these, 266 proteins and 97 phosphorylation sites were found to be sensitive to the Mdivi-1 treatment. Functional enrichment analysis unveiled their involvement in serine biosynthesis and extrinsic apoptotic signaling pathways. Through targeted metabolomics, we observed that Mdivi-1 enhanced intracellular serine biosynthesis while reducing the production of C24:1-ceramide. Within these regulated phosphoproteins, dynamic dephosphorylation of proteasome subunit alpha type 3 serine 250 (PSMA3-S250) occurred after Mdivi-1 treatment. Further site-directed mutagenesis experiments revealed that the dephosphorylation-deficient mutant PSMA3-S250A exhibited a decreased cell survival. This research confirms that Mdivi-1's inhibition of mitochondrial division leads to various side effects, ultimately influencing cell survival, rather than solely targeting Drp1 inhibition.

High-Throughput Proteomics and Phosphoproteomics of Rat Tissues Using Microflow Zeno SWATH.

High-throughput tissue proteomics has great potential in the advancement of precision medicine. Here, we investigated the combined sensitivity of trap-elute microflow liquid chromatography with a ZenoTOF for DIA proteomics and phosphoproteomics. Method optimization was conducted on HEK293T cell lines to determine the optimal variable window size, MS2 accumulation time and gradient length. The ZenoTOF 7600 was then compared to the previous generation TripleTOF 6600 using eight rat organs, finding up to 23% more proteins using a fifth of the sample load and a third of the instrument time. Spectral reference libraries generated from Zeno SWATH data in FragPipe (MSFragger-DIA/DIA-NN) contained 4 times more fragment ions than the DIA-NN only library and quantified more proteins. Replicate single-shot phosphopeptide enrichments of 50-100 µg of rat tryptic peptide were analyzed by microflow HPLC using Zeno SWATH without fractionation. Using Spectronaut we quantified a shallow phosphoproteome containing 1000-3000 phosphoprecursors per organ. Promisingly, clear hierarchical clustering of organs was observed with high Pearson correlation coefficients >0.95 between replicate enrichments and median CV of 20%. The combined sensitivity of microflow HPLC with Zeno SWATH allows for the high-throughput quantitation of an extensive proteome and shallow phosphoproteome from small tissue samples.

Retinoic acid induced specific changes in the phosphoproteome of C17.2 neural stem cells.

Retinoic acid (RA), a vitamin A derivative, is an effective cell differentiating factor which plays critical roles in neuronal differentiation induction and the production of neurotransmitters in neurons. However, the specific changes in phosphorylation levels and downstream signalling pathways associated with RA remain unclear. This study employed qualitative and quantitative phosphoproteomics approaches based on mass spectrometry to investigate the phosphorylation changes induced by RA in C17.2 neural stem cells (NSCs). Dimethyl labelling, in conjunction with TiO2 phosphopeptide enrichment, was utilized to profile the phosphoproteome of self-renewing and RA-induced differentiated cells in C17.2 NSCs. The results of our study revealed that, qualitatively, 230 and 14 phosphoproteins were exclusively identified in the self-renewal and RA-induced groups respectively. Quantitatively, we successfully identified and quantified 177 unique phosphoproteins, among which 70 exhibited differential phosphorylation levels. Analysis of conserved phosphorylation motifs demonstrated enrichment of motifs corresponding to cyclin-dependent kinase and MAPK in the RA-induced group. Additionally, through a comprehensive literature and database survey, we found that the differentially expressed proteins were associated with the Wnt/ß-catenin and Hippo signalling pathways. This work sheds light on the changes in phosphorylation levels induced by RA in C17.2 NSCs, thereby expanding our understanding of the molecular mechanisms underlying RA-induced neuronal differentiation.

A dynamic phosphoproteomic analysis provides insight into the C4 plant maize (Zea mays L.) response to natural diurnal changes.

As sessile organisms, plants need to respond to rapid changes in numerous environmental factors, mainly diurnal changes of light, temperature, and humidity. Maize is the world's most grown crop, and as a C4 plant it exhibits high photosynthesis capacity, reaching the highest rate of net photosynthesis at midday; that is, there is no "midday depression." Revealing the physiological responses to diurnal changes and underlying mechanisms will be of great significance for guiding maize improvement efforts. In this study, we collected maize leaf samples and analyzed the proteome and phosphoproteome at nine time points during a single day/night cycle, quantifying 7424 proteins and 5361 phosphosites. The new phosphosites identified in our study increased the total maize phosphoproteome coverage by 8.5%. Kinase-substrate network analysis indicated that 997 potential substrates were phosphorylated by 20 activated kinases. Through analysis of proteins with significant changes in abundance and phosphorylation, we found that the response to a heat stimulus involves a change in the abundance of numerous proteins. By contrast, the high light at noon and rapidly changing light conditions induced changes in the phosphorylation level of proteins involved in processes such as chloroplast movement, photosynthesis, and C4 pathways. Phosphorylation is involved in regulating the activity of large number of enzymes; for example, phosphorylation of S55 significantly enhanced the activity of maize phosphoenolpyruvate carboxykinase1 (ZmPEPCK1). Overall, the database of dynamic protein abundance and phosphorylation we have generated provides a resource for the improvement of C4 crop plants.

Pf-Phospho: a machine learning-based phosphorylation sites prediction tool for Plasmodium proteins.

Even though several in silico tools are available for prediction of the phosphorylation sites for mammalian, yeast or plant proteins, currently no software is available for predicting phosphosites for Plasmodium proteins. However, the availability of significant amount of phospho-proteomics data during the last decade and advances in machine learning (ML) algorithms have opened up the opportunities for deciphering phosphorylation patterns of plasmodial system and developing ML-based phosphosite prediction tools for Plasmodium. We have developed Pf-Phospho, an ML-based method for prediction of phosphosites by training Random Forest classifiers using a large data set of 12 096 phosphosites of Plasmodium falciparum and Plasmodium bergei. Of the 12 096 known phosphosites, 75% of sites have been used for training/validation of the classifier, while remaining 25% have been used as completely unseen test data for blind testing. It is encouraging to note that Pf-Phospho can predict the kinase-independent phosphosites with 84% sensitivity, 75% specificity and 78% precision. In addition, it can also predict kinase-specific phosphosites for five plasmodial kinases-PfPKG, Plasmodium falciparum, PfPKA, PfPK7 and PbCDPK4 with high accuracy. Pf-Phospho (http://www.nii.ac.in/pfphospho.html) outperforms other widely used phosphosite prediction tools, which have been trained using mammalian phosphoproteome data. It also has been integrated with other widely used resources such as PlasmoDB, MPMP, Pfam and recently available ML-based predicted structures by AlphaFold2. Currently, Pf-phospho is the only bioinformatics resource available for ML-based prediction of phospho-signaling networks of Plasmodium and is a user-friendly platform for integrative analysis of phospho-signaling along with metabolic and protein-protein interaction networks.

Kinome and phosphoproteome reprogramming underlies the aberrant immune responses in critically ill COVID-19 patients.

SARS-CoV-2 infection triggers extensive host immune reactions, leading to severe diseases in certain individuals. However, the molecular basis underlying the excessive yet non-productive immune responses in severe COVID-19 remains incompletely understood. In this study, we conducted a comprehensive analysis of the peripheral blood mononuclear cell (PBMC) proteome and phosphoproteome in sepsis patients positive or negative for SARS-CoV-2 infection, as well as healthy subjects, using quantitative mass spectrometry. Our findings demonstrate dynamic changes in the COVID-19 PBMC proteome and phosphoproteome during disease progression, with distinctive protein or phosphoprotein signatures capable of distinguishing longitudinal disease states. Furthermore, SARS-CoV-2 infection induces a global reprogramming of the kinome and phosphoproteome, resulting in defective adaptive immune response mediated by the B and T lymphocytes, compromised innate immune responses involving the SIGLEC and SLAM family of immunoreceptors, and excessive cytokine-JAK-STAT signaling. In addition to uncovering host proteome and phosphoproteome aberrations caused by SARS-CoV-2, our work recapitulates several reported therapeutic targets for COVID-19 and identified numerous new candidates, including the kinases PKG1, CK2, ROCK1/2, GRK2, SYK, JAK2/3, TYK2, DNA-PK, PKCδ, and the cytokine IL-12.

A multi-omics approach to elucidate okadaic acid-induced changes in human HepaRG hepatocarcinoma cells.

Okadaic acid (OA), a prevalent marine biotoxin found in shellfish, is known for causing acute gastrointestinal symptoms. Despite its potential to reach the bloodstream and the liver, the hepatic effects of OA are not well understood, highlighting a significant research gap. This study aims to comprehensively elucidate the impact of OA on the liver by examining the transcriptome, proteome, and phosphoproteome alterations in human HepaRG liver cells exposed to non-cytotoxic OA concentrations. We employed an integrative multi-omics approach, encompassing RNA sequencing, shotgun proteomics, phosphoproteomics, and targeted DigiWest analysis. This enabled a detailed exploration of gene and protein expression changes, alongside phosphorylation patterns under OA treatment. The study reveals concentration- and time-dependent deregulation in gene and protein expression, with a significant down-regulation of xenobiotic and lipid metabolism pathways. Up-regulated pathways include actin crosslink formation and a deregulation of apoptotic pathways. Notably, our results revealed that OA, as a potent phosphatase inhibitor, induces alterations in actin filament organization. Phosphoproteomics data highlighted the importance of phosphorylation in enzyme activity regulation, particularly affecting proteins involved in the regulation of the cytoskeleton. OA's inhibition of PP2A further leads to various downstream effects, including alterations in protein translation and energy metabolism. This research expands the understanding of OA's systemic impact, emphasizing its role in modulating the phosphorylation landscape, which influences crucial cellular processes. The results underscore OA's multifaceted effects on the liver, particularly through PP2A inhibition, impacting xenobiotic metabolism, cytoskeletal dynamics, and energy homeostasis. These insights enhance our comprehension of OA's biological significance and potential health risks.

Phosphoproteome Analysis Identifies a Synaptotagmin-1-Associated Complex Involved in Ischemic Neuron Injury.

Cerebral stroke is one of the leading causes of death in adults worldwide. However, the molecular mechanisms of stroke-induced neuron injury are not fully understood. Here, we obtained phosphoproteomic and proteomic profiles of the acute ischemic hippocampus by LC-MS/MS analysis. Quantitative phosphoproteomic analyses revealed that the dysregulated phosphoproteins were involved in synaptic components and neurotransmission. We further demonstrated that phosphorylation of Synaptotagmin-1 (Syt1) at the Thr112 site in cultured hippocampal neurons aggravated oxygen-glucose deprivation-induced neuronal injury. Immature neurons with low expression of Syt1 exhibit slight neuronal injury in a cerebral ischemia model. Administration of the Tat-Syt1T112A peptide protects neurons against cerebral ischemia-induced injury in vitro and in vivo. Surprisingly, potassium voltage-gated channel subfamily KQT member 2 (Kcnq2) interacted with Syt1 and Annexin A6 (Anxa6) and alleviated Syt1-mediated neuronal injury upon oxygen-glucose deprivation treatment. These results reveal a mechanism underlying neuronal injury and may provide new targets for neuroprotection after acute cerebral ischemia onset.

Spatiotemporally resolved subcellular phosphoproteomics.

Proteome-wide profiling of protein phosphorylation has been widely used to reveal the underlying mechanism of diverse cellular signaling events. Yet, characterizing subcellular phosphoproteome with high spatial-temporal resolution has remained challenging. Herein, we developed a subcellular-specific uncaging-assisted biotinylation and mapping of phosphoproteome (SubMAPP) strategy to monitor the phosphorylation dynamics of subcellular proteome in living cells and animals. Our method capitalizes on the genetically encoded bioorthogonal decaging strategy, which enables the rapid activation of subcellular localized proximity labeling biotin ligase through either light illumination or small-molecule triggers. By further adopting an integrated orthogonal pull-down strategy with quantitative mass spectrometry, SubMAPP allowed for the investigation of subcellular phosphoproteome dynamics, revealing the altered phosphorylation patterns of endoplasmic reticulum (ER) luminal proteins under ER stress. Finally, we further expanded the scope of the SubMAPP strategy to primary neuron culture and living mice.

Phosphoproteome Microarray Analysis of Extracellular Particles as a Tool to Explore Novel Biomarker Candidates for Alzheimer's Disease.

Phosphorylation plays a key role in Alzheimer's disease (AD) pathogenesis, impacting distinct processes such as amyloid-beta (Aß) peptide production and tau phosphorylation. Impaired phosphorylation events contribute to senile plaques and neurofibrillary tangles' formation, two major histopathological hallmarks of AD. Blood-derived extracellular particles (bdEP) can represent a disease-related source of phosphobiomarker candidates, and hence, in this pilot study, bdEP of Control and AD cases were analyzed by a targeted phosphoproteomics approach using a high-density microarray that featured at least 1145 pan-specific and 913 phosphosite-specific antibodies. This approach, innovatively applied to bdEP, allowed the identification of 150 proteins whose expression levels and/or phosphorylation patterns were significantly altered across AD cases. Gene Ontology enrichment and Reactome pathway analysis unraveled potentially relevant molecular targets and disease-associated pathways, and protein-protein interaction networks were constructed to highlight key targets. The discriminatory value of both the total proteome and the phosphoproteome was evaluated by univariate and multivariate approaches. This pilot experiment supports that bdEP are enriched in phosphotargets relevant in an AD context, holding value as peripheral biomarker candidates for disease diagnosis.