Protein Phosphorylation Profiles Research Articles

Comprehensive study of protein phosphorylation, acetylation, ubiquitination and S-nitrosylation profiles in pre- and post-rigor meat

Comprehensive study of protein phosphorylation, acetylation, ubiquitination and S-nitrosylation profiles in pre- and post-rigor meat

A Comparison of White and Yellow Seminal Plasma Phosphoproteomes Obtained from Turkey (Meleagris gallopavo) Semen.

Seminal plasma is rich in proteins originating from various male reproductive organs. The phosphorylation of these proteins can significantly impact sperm motility, capacitation, and acrosome reaction. Phosphoproteomics identifies, catalogues, and characterizes phosphorylated proteins. The phosphoproteomic profiling of seminal plasma offers valuable insights into the molecular mechanisms that influence semen quality and male fertility. Thus, the aim of this study was a phosphoproteomic analysis of white and yellow turkey seminal plasma. The experimental material consisted of 100 ejaculates from BIG-6 turkeys between 39 and 42 weeks of age. The collected white and yellow turkey seminal plasmas were analyzed for total protein content; the activity of selected enzymes, i.e., alkaline phosphatase (ALP), acid phosphatase (ACP), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT); and the content of reduced glutathione (GSH) and malondialdehyde (MDA). Phosphoproteins were isolated from white and yellow seminal fluids, and the resulting protein fractions were separated by SDS-PAGE and Western blotting. Phosphorylated residues were immunodetected, and the isolated phosphoproteins were identified (nano LC-MS/MS). Yellow seminal plasmas were characterized by higher levels of total protein, GSH, and MDA, as well as higher levels of ALP, ACP, and GPx activity. There were no significant differences in the activity of SOD and CAT. A total of 113 phosphoproteins were identified in turkey seminal fluids. The functional analysis demonstrated that these phosphoproteins were mainly involved in oocyte fertilization, organization and metabolism of the actin cytoskeleton, amplification of the intracellular signal transduction pathway, general regulation of transport, vesicular transport, proteome composition of individual cellular compartments, and the organization and localization of selected cellular components and macromolecules. Increased phosphorylation of the fractions containing proteins encoded by SPARC, PPIB, TRFE, QSOX1, PRDX1, PRDX6, and FASN genes in white plasmas and the proteins encoded by CKB, ORM2, APOA1, SSC5D, RAP1B, CDC42, FTH, and TTH genes in yellow plasmas was observed based on differences in the optical density of selected bands. The obtained results indicate that the phosphorylation profiles of turkey seminal plasma proteins vary depending on the type of ejaculate.

Abstract 4682: Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma

Abstract The B-cell Receptor (BCR), comprising membrane Ig (mIg) and CD79a/b, controls normal signaling in B-cells, but dysregulation of this signaling is the dominant driver of leukemia/lymphoma. The class-specific mIg subunit of the BCR is a rational drug target for B-cell malignancies. It plays a critical role in the functioning of these cells, and allows for restricted targeting of only B-cells expressing an Ig class, offering a more tumor-specific approach than current pan-B-cell treatments. We developed a first-in-class antibody, WBMP-4, to the mIgM-BCR, which inhibits cell growth and induces apoptosis via widespread down-regulation of tyrosine kinase activity. Given the potent inhibition of intracellular signaling induced upon WBMP-4 binding to the mIgM-BCR, we expect that translation of membrane proteins will be impacted. Here we investigate the expression and phosphorylation profile of cell surface proteins following treatment of malignant B-cells with WBMP-4. Using western blot and flow cytometry, we measure levels of CD79a/b, CD19, CD20, CD21, CD22, CD32a, CD81, and CXCR4 at 6, 24, and 48 hours after low (5ug/mL) and high (20ug/mL) dose WBMP-4 treatment, as compared to control, untreated Burkitt lymphoma (CA46) cells. We analyze phosphorylation levels of those surface proteins that serve as kinases in this system (CD79a/b, CD19, CXCR4, etc.). Using these same methods, we also examine the expression and phosphorylation of key B-cell transcription factors (e.g. NF-kB, c-MYC, STAT5, PAX5, EBF1, FOXO, and BCL-2). Preliminary analyses show a complete absence of CD79a protein (western blot), and decreased phospho-kinase activity of CD79b (Pamgene phosphokinase array) following treatment of Burkitt lymphoma cells with WBMP-4, as compared to untreated cells. While the effect of an antibody binding to mIg may be most pronounced for CD79a/b relative to other proteins, as mIg and CD79a/b form a complex, we expect that the absence of CD79a/b is a result of WBMP-4-induced mIgM inhibition modulating downstream signaling and transcription factor activity. By examining cell surface markers, we may gain insight into how WBMP-4 impacts a cell’s interaction with the tumor microenvironment, and this information is valuable for diagnostic and patient monitoring purposes, and to determine drug combination strategies. This work will be considered in the context of our ongoing research on WBMP-4’s inhibition of intracellular signaling pathways, and the resulting biologic effects, and will contribute to the continued development of WBMP-4 by providing an understanding of the effects achieved (cell growth inhibition, cell differentiation, apoptosis) at different dosages and if these effects are maintained over time. As a novel, efficacious, and low toxicity treatment strategy for B-cell leukemia and lymphoma, WBMP-4 has significant clinical potential. Citation Format: Rachel S. Welt, David Kostyal, Virginia Raymond, Jose M. Lobo, Sydney Welt. Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4682.

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.

Nucleos(t)ide analogs for hepatitis B virus infection differentially regulate the growth factor signaling in hepatocytes.

Recent clinical studies have suggested that the risk of developing HCC might be lower in patients with chronic hepatitis B receiving tenofovir disoproxil fumarate than in patients receiving entecavir, although there is no difference in biochemical and virological remission between the 2 drugs. The effects of nucleoside analogs (NsAs; lamivudine and entecavir) or nucleotide analogs (NtAs; adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide) on cell growth and the expression of growth signaling molecules in hepatoma cell lines and PXB cells were investigated in vitro. The tumor inhibitory effects of NsAs or NtAs were evaluated using a mouse xenograft model, and protein phosphorylation profiles were investigated. The binding of NsAs or NtAs to the insulin receptor (INSR) was investigated by thermal shift assays. NtAs, but not NsAs, showed direct growth inhibitory effects on hepatoma cell lines in vitro and a mouse model in vivo. A phosphoprotein array revealed that INSR signaling was impaired and the levels of phosphorylated (p)-INSRβ and downstream molecules phosphorylated (p)-IRS1, p-AKT, p-Gab1, and p-SHP2 were substantially reduced by NtAs. In addition, p-epidermal growth factor receptor and p-AKT levels were substantially reduced by NtAs. Similar findings were also found in PXB cells and nontumor lesions of liver tissues from patients with chronic hepatitis B. Prodrug NtAs, but not their metabolites (adefovir, adefovir monophosphate, adefovir diphosphate, tenofovir, tenofovir monophosphate, and tenofovir diphosphate), had such effects. A thermal shift assay showed the binding of NtAs to INSRβ. NtAs (adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide), which are adenine derivative acyclic nucleotide analogs, potentially bind to the ATP-binding site of growth factor receptors and inhibit their autophosphorylation, which might reduce the risk of HCC in patients with chronic hepatitis B.

Endometrial protein expression and phosphorylation landscape decipher aberrant insulin and mTOR signalling in patients with recurrent pregnancy loss

What are the proteomic and phosphoproteomic differences between the endometrium of women with recurrent pregnancy loss (RPL) and the endometrium of healthy control women during the proliferative and secretory phases of the menstrual cycle? In total, 54 endometrial samples were collected during the proliferative and secretory phases from women with RPL (n = 28) and healthy controls (n = 26). Comprehensive proteomic and phosphoproteomic analyses were conducted using label-free liquid chromatography-tandem mass spectrometry (n = 44), and verified through Western blotting (n = 10). Three comparison groups were established: total RPL endometrium versus total control endometrium; RPL proliferative endometrium versus control proliferative endometrium; and RPL secretory endometrium versus control secretory endometrium. Differentially expressed proteins and differentially phosphorylated proteins were identified in the three comparison groups. Combining pathway enrichment, network analysis and soft clustering analysis, the insulin/cyclic nucleotide signalling pathway and AMPK/mTOR signalling pathway were identified as the major contributors to the aberration of RPL endometrium. Western blotting verified altered expression of four proteins: cAMP-dependent protein kinase type I-β regulatory subunit, adenylate cyclase type 3, 5'-AMP-activated protein kinase catalytic subunit α-2 and phosphatidate phosphatase LPIN2. This exploratory study provides insights into the differentiated protein expression and phosphorylation profiles of the endometrium of women with RPL in both the proliferative and sectretory phases of the menstrual cycle. The results highlight potential proteins associated with the pathogenesis of RPL that may serve as potential indicators for RPL. The findings contribute to the identification of potential targets for RPL treatment as well as its pathogenesis.

Phosphoproteomics Reveal New Candidates in Abnormal Spermatogenesis of Pseudomales in Cynoglossus semilaevis.

Phosphorylation is a post-translational modification that contributes to versatile protein functions in spermatogenesis, and the variations they generate usually results in abnormal spermatogenesis or sperm dysfunction. The sex-reversal phenomenon exists in Chinese tongue sole under certain conditions such that individuals with a ZW genotype can acquire a male phenotype and are thus called pseudomales. Pseudomale tongue sole can reach sexual maturity but produce only Z-type sperm, and the Z sperm carries paternal epigenetic information. Whether phosphorylation plays a role in the sperm abnormality of pseudomales is unknown. In this study, a phosphoproteomic analysis was performed to compare protein phosphorylation profiles between pseudomale and male testes. Altogether, we identified 14,253 phosphopeptides matching with 4843 proteins, with 1329 differentially phosphorylated peptides corresponding to 1045 differentially phosphorylated proteins (DPPs). Phosphorylation at 781 sites was upregulated and at 548 sites was downregulated. Four motifs were identified among differentially phosphorylated peptides, which were "SP", "SD", "RxxS", and "TP". Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that the cell cycle and DNA/RNA processing were significantly enriched with the genes encoding DPPs. To analyze DPP function in depth, a protein-protein interaction network was constructed, and Ran-binding protein 2 was found to play a central role in spermatogenesis by regulating several processes such as the cell cycle, eukaryotic translation, ubiquitination, and minichromosome maintenance. In kinase-associated network analyses, two "mitogen-activated protein kinase (Mapk)-centered" clusters were identified that may account for abnormal spermatogenesis in pseudomales. One cluster was centered on Mapk6, which predominantly regulated the cell cycle by interacting with several cyclin-dependent kinases, and the other was centered on the "testis-expressed kinase 1-like (Tesk1l)/Pim1l-Mapk4l- testis-expressed 14 (Tex14)" kinase cascade, which might contribute to spermatogenesis by regulating β-catenin. Taken together, these data suggested the new candidates involved in pseudomale sperm abnormalities and provided clues to discover the phosphorylated regulatory mechanism underlying tongue sole spermatogenesis.

Salt-inducible kinase inhibition promotes the adipocyte thermogenic program and adipose tissue browning

ObjectiveNorepinephrine stimulates the adipose tissue thermogenic program through a β-adrenergic receptor (βAR)–cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA) signaling cascade. We discovered that a noncanonical activation of the mechanistic target of rapamycin complex 1 (mTORC1) by PKA is required for the βAR-stimulation of adipose tissue browning. However, the downstream events triggered by PKA-phosphorylated mTORC1 activation that drive this thermogenic response are not well understood. MethodsWe used a proteomic approach of Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) to characterize the global protein phosphorylation profile in brown adipocytes treated with the βAR agonist. We identified salt-inducible kinase 3 (SIK3) as a candidate mTORC1 substrate and further tested the effect of SIK3 deficiency or SIK inhibition on the thermogenic gene expression program in brown adipocytes and in mouse adipose tissue. ResultsSIK3 interacts with RAPTOR, the defining component of the mTORC1 complex, and is phosphorylated at Ser884 in a rapamycin-sensitive manner. Pharmacological SIK inhibition by a pan-SIK inhibitor (HG-9-91-01) in brown adipocytes increases basal Ucp1 gene expression and restores its expression upon blockade of either mTORC1 or PKA. Short-hairpin RNA (shRNA) knockdown of Sik3 augments, while overexpression of SIK3 suppresses, Ucp1 gene expression in brown adipocytes. The regulatory PKA phosphorylation domain of SIK3 is essential for its inhibition. CRISPR-mediated Sik3 deletion in brown adipocytes increases type IIa histone deacetylase (HDAC) activity and enhances the expression of genes involved in thermogenesis such as Ucp1, Pgc1α, and mitochondrial OXPHOS complex protein. We further show that HDAC4 interacts with PGC1α after βAR stimulation and reduces lysine acetylation in PGC1α. Finally, a SIK inhibitor well-tolerated in vivo (YKL-05-099) can stimulate the expression of thermogenesis-related genes and browning of mouse subcutaneous adipose tissue. ConclusionsTaken together, our data reveal that SIK3, with the possible contribution of other SIKs, functions as a phosphorylation switch for β-adrenergic activation to drive the adipose tissue thermogenic program and indicates that more work to understand the role of the SIKs is warranted. Our findings also suggest that maneuvers targeting SIKs could be beneficial for obesity and related cardiometabolic disease.

Synergistic effect of cold gas plasma and experimental drug exposure exhibits skin cancer toxicity in vitro and in vivo

IntroductionSkin cancer is often fatal, which motivates new therapy avenues. Recent advances in cancer treatment are indicative of the importance of combination treatments in oncology. Previous studies have identified small molecule-based therapies and redox-based technologies, including photodynamic therapy or medical gas plasma, as promising candidates to target skin cancer. ObjectiveWe aimed to identify effective combinations of experimental small molecules with cold gas plasma for therapy in dermato-oncology. MethodsPromising drug candidates were identified after screening an in-house 155-compound library using 3D skin cancer spheroids and high content imaging. Combination effects of selected drugs and cold gas plasma were investigated with respect to oxidative stress, invasion, and viability. Drugs that had combined well with cold gas plasma were further investigated in vascularized tumor organoids in ovo and a xenograft mouse melanoma model in vivo. ResultsThe two chromone derivatives Sm837 and IS112 enhanced cold gas plasma-induced oxidative stress, including histone 2A.X phosphorylation, and further reduced proliferation and skin cancer cell viability. Combination treatments of tumor organoids grown in ovo confirmed the principal anti-cancer effect of the selected drugs. While one of the two compounds exerted severe toxicity in vivo, the other (Sm837) resulted in a significant synergistic anti-tumor toxicity at good tolerability. Principal component analysis of protein phosphorylation profiles confirmed profound combination treatment effects in contrast to the monotherapies. ConclusionWe identified a novel compound that, combined with topical cold gas plasma-induced oxidative stress, represents a novel and promising treatment approach to target skin cancer.

Discrepancy of synaptic and microtubular protein phosphorylation in the hippocampus of APP/PS1 and MAPT×P301S transgenic mice at the early stage of Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, and is caused by multiple pathological factors, such as the overproduction of β-amyloid (Aβ) and the hyperphosphorylation of tau. However, there is limited knowledge of the mechanisms underlying AD pathogenesis and no effective biomarker for the early diagnosis of this disorder. Thus in this study, a quantitative phosphoproteomics analysis was performed to evaluate global protein phosphorylation in the hippocampus of Aβ overexpressing APP/PS1 transgenic mice and tau overexpressing MAPT×P301S transgenic mice, two in vivo AD model systems. These animals, up to ten weeks old, do not exhibit cognitive dysfunctions and are widely used to simulate early-stage AD patients. The number of differentially phosphorylated proteins (DPPs) was greater for APP/PS1 transgenic mice than for MAPT×P301S transgenic mice. The function of the DPPs in APP/PS1 transgenic mice was mainly related to synapses, while the function of the DPPs in MAPT×P301S transgenic mice was mainly related to microtubules. In addition, an AD core network was established including seven phosphoproteins differentially expressed in both animal models, and the function of this core network was related to synapses and oxidative stress. The results of this study suggest that Aβ and tau induce different protein phosphorylation profiles in the early stage of AD, leading to the dysfunctions in synapses and microtubule, respectively. And the detection of same DPPs in these animal models might be used for early AD diagnosis.

Alterations in cytoskeletal and Ca2+ cycling regulators in atria lacking the obscurin Ig58/59 module.

Obscurin (720-870 kDa) is a giant cytoskeletal and signaling protein that possesses both structural and regulatory functions in striated muscles. Immunoglobulin domains 58/59 (Ig58/59) of obscurin bind to a diverse set of proteins that are essential for the proper structure and function of the heart, including giant titin, novex-3, and phospholamban (PLN). Importantly, the pathophysiological significance of the Ig58/59 module has been further underscored by the discovery of several mutations within Ig58/59 that are linked to various forms of myopathy in humans. We previously generated a constitutive deletion mouse model, Obscn-ΔIg58/59, that expresses obscurin lacking Ig58/59, and characterized the effects of this deletion on cardiac morphology and function through aging. Our findings demonstrated that Obscn-ΔIg58/59 male animals develop severe arrhythmia, primarily manifesting as episodes of junctional escape and spontaneous loss of regular p-waves, reminiscent of human atrial fibrillation, accompanied by significant atrial enlargement that progresses in severity with aging. To comprehensively characterize the molecular alterations responsible for these pathologies, we performed proteomic and phospho-proteomic analyses in aging Obscn-ΔIg58/59 atria. Our studies revealed extensive and novel alterations in the expression and phosphorylation profile of major cytoskeletal proteins, Ca2+ regulators, and Z-disk associated protein complexes in the Obscn-ΔIg58/59 atria through aging. These studies implicate obscurin, particularly the Ig58/59 module, as an essential regulator of the Z-disk associated cytoskeleton and Ca2+ cycling in the atria and provide new molecular insights into the development of atrial fibrillation and remodeling.

HIV-1 Transcription Inhibitor 1E7-03 Decreases Nucleophosmin Phosphorylation

Transcription activation of latent human immunodeficiency virus-1 (HIV-1) occurs due to HIV-1 rebound, the interruption of combination antiretroviral therapy, or development of drug resistance. Thus, novel HIV-1 inhibitors, targeting HIV-1 transcription are needed. We previously developed an HIV-1 transcription inhibitor, 1E7-03, that binds to the noncatalytic RVxF-accommodating site of protein phosphatase 1 and inhibits HIV-1 replication in cultured cells and HIV-1–infected humanized mice by impeding protein phosphatase 1 interaction with HIV-1 Tat protein. However, host proteins and regulatory pathways targeted by 1E7-03 that contribute to its overall HIV-1 inhibitory activity remain to be identified. To address this issue, we performed label-free quantitative proteome and phosphoproteome analyses of noninfected and HIV-1–infected CEM T cells that were untreated or treated with 1E7-03. 1E7-03 significantly reprogramed the phosphorylation profile of proteins including PPARα/RXRα, TGF-β, and PKR pathways. Phosphorylation of nucleophosmin (NPM1) at Ser-125 residue in PPARα/RXRα pathway was significantly reduced (>20-fold, p = 1.37 × 10−9), followed by the reduced phosphorylation of transforming growth factor-beta 2 at Ser-46 (TGF-β2, >12-fold, p = 1.37 × 10−3). Downregulation of NPM1’s Ser-125 phosphorylation was further confirmed using Western blot. Phosphorylation mimicking NPM1 S125D mutant activated Tat-induced HIV-1 transcription and exhibited enhanced NPM1–Tat interaction compared to NPM1 S125A mutant. Inhibition of Aurora A or Aurora B kinases that phosphorylate NPM1 on Ser-125 residue inhibited HIV-1, further supporting the role of NPM1 in HIV-1 infection. Taken together, 1E7-03 reprogrammed PPARα/RXRα and TGF-β pathways that contribute to the inhibition of HIV-1 transcription. Our findings suggest that NPM1 phosphorylation is a plausible target for HIV-1 transcription inhibition.

CSIG-20. PROTEOGENOMICS PROFILING REVEALS ENRICHED PROTEIN TRANSLATION REGULATORS AS NEW THERAPEUTIC TARGETS IN DIPG

Abstract Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor arising in the brainstems of children. Current therapies are ineffective resulting in a median survival rate of less than one year and it is the leading cause of brain tumor-related death in children. A novel mutation in histone H3 protein (H3K27M) was recently identified as a genetic initiation event and affects global K27 trimethylation on histone H3 proteins and DNA methylation. The epigenetic changes caused by H3K27M mutation suggest the existence of an H3K27M-specific transcriptome and proteome. We investigated DIPG tissues at the multi-omics level including total proteome, phosphoproteome, methylproteome and metabolome by mass spectrometry. A total of 30 patient tumours were profiled to identify differentially expressed proteins, differentially phosphorylated proteins, and differentially methylated proteins in tumour tissues compared to normal brains. We identified 2995 proteins that are differentially regulated, suggesting changes in key oncogenic pathways including negative regulation of apoptosis, translation, and metabolic pathways such as methionine salvage and TCA cycle in DIPG. The deregulation of these metabolic pathways due to the differentially expressed proteins in DIPG cells was confirmed by metabolomics studies. Protein phosphorylation and protein methylation profiling of DIPG implicated that translation-related proteins were the most highly modified (post-translationally) proteins in DIPG tissues. Furthermore, protein translation measured by CyTOF showed higher translation rates in DIPG and immortalized astrocytes carrying H3K27M than their WT counterparts. We investigated the functional consequence of knockdown of the highest methylated translation regulator EEF1A1 and its methyltransferase METTL13. ShRNA knockdown of both, EEF1A and METTL13 in DIPG cells significantly reduced the cell growth. Multi-omics analysis of DIPG highlighted regulation of protein translation as a potential therapeutic target.

Protein phosphorylation profile of Atlantic cod (Gadus morhua) in response to pre-slaughter pumping stress and postmortem time

This study aimed to evaluate the effect of pumping stress (pumping and pumping-resting) and postmortem time (before and after rigor mortis) on phosphorylation profiles of myofibrillar protein (MP) and sarcoplasmic protein (SP) of Atlantic cod (Gadus morhua) fillets. The result showed that MP had higher global phosphorylation levels than SP regardless of stress condition and postmortem time. The pumping process resulted in significant changes in phosphorylation of structural proteins including myosin heavy and light chains. Pumping also affected the phosphorylation status of heat shock proteins and metabolic enzymes involved in the glycolytic pathways, indicating the possible role of phosphorylation in regulating energy hemostasis of fish under stressful conditions. The pumping-induced phosphorylation changes mainly occurred before rigor mortis, and postmortem time affected the phosphorylation status to a less extent. This work contributes to a deeper understanding on protein phosphorylation affected by pre-slaughter stress and postmortem time of fish.

Comprehensive proteomic analysis reveals dynamic phospho-profiling in human early erythropoiesis.

Normal early erythropoiesis depends on the precise regulation of protein expression and phosphorylation modification. Dysregulation of protein levels or modification contributes to erythroid disorders. To date, the dynamics of protein phosphorylation profiling across human erythroid development is not fully understood. Here, we characterized quantitative proteomic and phosphoproteomic profiling by tandem mass-tagging technology. We systemically built phospho-expression profiling and expression clusters of 11 414 phosphopeptides for human early erythropoiesis. The standardization methods for multitier integrative analyses revealed multiple functional modules of phosphoproteins (e.g., regulation of the G2/M transition) and active phosphorylated signalling (e.g., cell cycle-related pathways). Our further analysis revealed that CDK family members were the main kinases that phosphorylate substrates in erythroid progenitors and identified that CDK9 played an important role in the proliferation of erythroid progenitors. Collectively, our phosphoproteomic profiling, integrative network analysis and functional studies define landscapes of the phosphoproteome and reveal signalling pathways that are involved in human early erythropoiesis. This study will serve as a valuable resource for further investigations of phosphatase and kinase functions in human erythropoiesis and erythroid-related diseases.

Decoupling PER phosphorylation, stability and rhythmic expression from circadian clock function by abolishing PER-CK1 interaction

Robust rhythms of abundances and phosphorylation profiles of PERIOD proteins were thought be the master rhythms that drive mammalian circadian clock functions. PER stability was proposed to be a major determinant of period length. In mammals, CK1 forms stable complexes with PER. Here we identify the PER residues essential for PER-CK1 interaction. In cells and in mice, their mutation abolishes PER phosphorylation and CLOCK hyperphosphorylation, resulting in PER stabilization, arrhythmic PER abundance and impaired negative feedback process, indicating that PER acts as the CK1 scaffold in circadian feedback mechanism. Surprisingly, the mutant mice exhibit robust short period locomotor activity and other physiological rhythms but low amplitude molecular rhythms. PER-CK1 interaction has two opposing roles in regulating CLOCK-BMAL1 activity. These results indicate that the circadian clock can function independently of PER phosphorylation and abundance rhythms due to another PER-CRY-dependent feedback mechanism and that period length can be uncoupled from PER stability.

Dysregulated Brain Protein Phosphorylation Linked to Increased Human Tau Expression in the hTau Transgenic Mouse Model.

Altered protein phosphorylation is a major pathologic modification in tauopathies and Alzheimer’s disease (AD) linked to abnormal tau fibrillar deposits in neurofibrillary tangles (NFTs) and pre-tangles and β-amyloid deposits in AD. hTau transgenic mice, which express 3R and less 4R human tau with no mutations in a murine knock-out background, show increased tau deposition in neurons but not NFTs and pre-tangles at the age of nine months. Label-free (phospho)proteomics and SWATH-MS identified 2065 proteins in hTau and wild-type (WT) mice. Only six proteins showed increased levels in hTau; no proteins were down-regulated. Increased tau phosphorylation in hTau was detected at Ser199, Ser202, Ser214, Ser396, Ser400, Thr403, Ser404, Ser413, Ser416, Ser422, Ser491, and Ser494, in addition to Thr181, Thr231, Ser396/Ser404, but not at Ser202/Thr205. In addition, 4578 phosphopeptides (corresponding to 1622 phosphoproteins) were identified in hTau and WT mice; 64 proteins were differentially phosphorylated in hTau. Sixty proteins were grouped into components of membranes, membrane signaling, synapses, vesicles, cytoskeleton, DNA/RNA/protein metabolism, ubiquitin/proteasome system, cholesterol and lipid metabolism, and cell signaling. These results showed that over-expression of human tau without pre-tangle and NFT formation preferentially triggers an imbalance in the phosphorylation profile of specific proteins involved in the cytoskeletal–membrane-signaling axis.

Elucidating the Bitter Taste Signaling Paradox in Airway Smooth Muscle Relaxation

RATIONALEAirway smooth muscle (ASM) hypersensitivity and hypercontractility play a pivotal role in the pathophysiology of obstructive airway diseases such as asthma. Furthermore, over half of affected individuals are unable to reliably control these disorders using the current standard of care, β‐(adrenergic) agonists. Previously, our group discovered the expression of a novel group of G protein‐coupled receptors (GPCRs), type 2 taste receptors (TAS2Rs), in the human airway which evoke ASM relaxation. Previous work in our lab suggests that TAS2R agonists evoke relaxation in a G protein beta‐gamma subunit‐, phospholipase C beta‐ and calcium‐dependent manner. We hypothesized that the downstream molecular targets of contractile‐related signaling components are differentially regulated by TAS2Rs compared to other GPCRs. The goal of this study was to establish the molecular mechanism by which TAS2Rs induce ASM relaxation.METHODSWe employed a phosphoproteomic approach, involving dual‐mass spectrometry, to determine differences in the phosphorylation status of contractile‐related proteins in ASM following the stimulation of TAS2Rs and other GPCRs. Healthy human ASM cells were treated with the TAS2R agonist chloroquine or β‐agonist isoproterenol alone or in combination with the bronchoconstrictive agonist histamine to study the ability of each relaxant compound to attenuate the histamine‐mediated phosphorylation profile of contractile‐related proteins. These phosphoproteomic profiles were compared and a select set of targets were validated using western blot analysis.RESULTSOur phosphoproteomic data demonstrated that histamine stimulation results in increased phosphorylation of Ras homolog family member A (RhoA) guanine nucleotide exchange factor (ARHGEF) 12 and A kinase anchoring proteins (AKAP) 2 and 12, as well as decreased phosphorylation of RhoA GTPase‐activating protein (ARHGAP) 6. Furthermore, chloroquine treatment attenuated histamine‐induced phosphorylation of ARHGEF12 but not AKAP2 or AKAP12. Interestingly, isoproterenol attenuated histamine‐induced phosphorylation of AKAP2 and AKAP12 but not ARHGEF12. Validation studies using western blot analysis revealed chloroquine mediated inhibition of histamine‐induced RhoA activation and myosin phosphatase target subunit 1 (MYPT1) phosphorylation at the T853 residue, which was not observed with isoproterenol.CONCLUSIONOur findings were able to show that contractile‐related proteins are phosphorylated differentially by chloroquine and isoproterenol to elicit ASM relaxation. Furthermore, our studies strongly suggest the importance of RhoA and the T853 residue on MYPT1 as points of mechanistic divergence between TAS2R and other GPCR pathways. These findings provide further insight into the mechanism by which TAS2R signaling reduces airway contractility and its potential as a novel therapy for obstructive airway disease.

The Role of Akt in Acquired Cetuximab Resistant Head and Neck Squamous Cell Carcinoma: An In Vitro Study on a Novel Combination Strategy.

The epidermal growth factor receptor (EGFR) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). Resistance to EGFR-targeted therapies, such as cetuximab, poses a challenging problem. This study aims to characterize acquired cetuximab resistance mechanisms in HNSCC cell lines by protein phosphorylation profiling. Through this, promising combination treatments can be identified to possibly overcome acquired cetuximab resistance in HNSCC. Protein phosphorylation profiling showed increased phosphorylation of Akt1/2/3 after cetuximab treatment in acquired cetuximab resistant cells compared to cetuximab sensitive cells, which was confirmed by western blotting. Based on this protein phosphorylation profile, a novel combination treatment with cetuximab and the Akt1/2/3 inhibitor MK2206 was designed. Synergy between cetuximab and MK2206 was observed in two cetuximab sensitive HNSCC cell lines and one acquired cetuximab resistant variant in simultaneous treatment schedules. In conclusion, this study demonstrates that increased Akt1/2/3 phosphorylation seems to be characteristic for acquired cetuximab resistance in HNSCC cell lines. Our results also show an additive to synergistic interaction between cetuximab and MK2206 in simultaneous treatment schedules. These data support the hypothesis that the combination of cetuximab with PI3K/Akt pathway inhibition might be a promising novel therapeutic strategy to overcome acquired cetuximab resistance in HNSCC patients.

CCCP‐induced mitochondrial dysfunction – characterization and analysis of integrated stress response to cellular signaling and homeostasis

Mitochondrial dysfunction mediated by CCCP (carbonyl cyanide m-chlorophenyl hydrazone), an inhibitor of mitochondrial oxidative phosphorylation, evokes the integrated stress response (ISR), which is analyzed here by eIF2α phosphorylation and expression profiles of ATF4 and CHOP proteins. Our findings suggest that the CCCP-induced ISR pathway is mediated by activation of HRI kinase, but not by GCN2, PERK, or PKR. Also, CCCP activates AMPK, a cellular energy sensor, and AKT, a regulator implicated in cell survival, and suppresses phosphorylation of mTORC1 substrates eIF4E-BP1 and S6K. CCCP also downregulates translation and promotes autophagy, leading to noncaspase-mediated cell death in HepG2 cells. All these events are neutralized by NAC, an anti-ROS, suggesting that CCCP-induced mitochondrial dysfunction promotes oxidative stress. ISRIB, an inhibitor of the ISR pathway, mitigates CCCP-induced expression of ATF4 and CHOP, activation of AKT, and autophagy, similar to NAC. However, it fails to reverse CCCP-induced AMPK activation, suggesting that CCCP-induced autophagy is dependent on ISR and independent of AMPK activation. ISRIB restores partly, inhibition in eIF4E-BP1 phosphorylation, promotes eIF2α phosphorylation, albeit slowly, and mitigates suppression of translation accordingly, in CCCP-treated cells. These findings are consistent with the idea that CCCP-induced oxidative stress leading to eIF2α phosphorylation and ATF4 expression, which is known to stimulate genes involved in autophagy, play a pro-survival role together with AKT activation and regulate mTOR-mediated eIF4E-BP1 phosphorylation.