Combined transcriptomics and proteomics unveil the impact of vitamin C in modulating specific protein abundance in the mouse liver.

BACKGROUND: Vitamin C (ascorbate) is a water-soluble antioxidant and an important cofactor for various biosynthetic and regulatory enzymes. Mice can synthesize vitamin C thanks to the key enzyme gulonolactone oxidase (Gulo) unlike humans. In the current investigation, we used Gulo-/- mice, which cannot synthesize their own ascorbate to determine the impact of this vitamin on both the transcriptomics and proteomics profiles in the whole liver. The study included Gulo-/- mouse groups treated with either sub-optimal or optimal ascorbate concentrations in drinking water. Liver tissues of females and males were collected at the age of four months and divided for transcriptomics and proteomics analysis. Immunoblotting, quantitative RT-PCR, and polysome profiling experiments were also conducted to complement our combined omics studies. RESULTS: Principal component analyses revealed distinctive differences in the mRNA and protein profiles as a function of sex between all the mouse cohorts. Despite such sexual dimorphism, Spearman analyses of transcriptomics data from females and males revealed correlations of hepatic ascorbate levels with transcripts encoding a wide array of biological processes involved in glucose and lipid metabolisms as well as in the acute-phase immune response. Moreover, integration of the proteomics data showed that ascorbate modulates the abundance of various enzymes involved in lipid, xenobiotic, organic acid, acetyl-CoA, and steroid metabolism mainly at the transcriptional level, especially in females. However, several proteins of the mitochondrial complex III significantly correlated with ascorbate concentrations in both males and females unlike their corresponding transcripts. Finally, poly(ribo)some profiling did not reveal significant enrichment difference for these mitochondrial complex III mRNAs between Gulo-/- mice treated with sub-optimal and optimal ascorbate levels. CONCLUSIONS: Thus, the abundance of several subunits of the mitochondrial complex III are regulated by ascorbate at the post-transcriptional levels. Our extensive omics analyses provide a novel resource of altered gene expression patterns at the transcriptional and post-transcriptional levels under ascorbate deficiency.

Specific alterations in riboproteomes composition of isonicotinic acid treated arabidopsis seedlings.

Plants have developed strategies to deal with the great variety of challenges they are exposed to. Among them, common targets are the regulation of transcription and translation to finely modulate protein levels during both biotic and abiotic stresses. Increasing evidence suggests that ribosomes are highly adaptable modular supramolecular structures which remodel to adapt to stresses. Each Arabidopsis thaliana ribosome consists of approximately 81 distinct ribosomal proteins (RPs), each of which is encoded by two to seven genes. To investigate the identity of ribosomal proteins of the small subunit (RPS) and of the large subunit (RPL) as well as ribosomes-associated proteins, we analysed by LC/MS/MS immunopurified ribosomes from A. thaliana leaves treated with isonicotinic acid (INA), an inducer of plant innate immunity. We quantified a total of 2084 proteins. 165 ribosome-associated proteins showed increased abundance while 52 were less abundant. Of the 52 identified RPS (from a possibility of 104 encoding genes), 15 were deregulated. Similarly, from the 148 possible RPL, 80 were detected and 9 were deregulated. Our results revealed potential candidates involved in innate immunity that could be interesting targets for functional genomic studies.

Vitamin C modulates the levels of several proteins of the mitochondrial complex III and its activity in the mouse liver.

Ascorbate is a crucial antioxidant and essential cofactor of biosynthetic and regulatory enzymes. Unlike humans, mice can synthesize ascorbate thanks to the key enzyme gulonolactone oxidase (Gulo). In the present study, we used the Gulo-/- mouse model, which cannot synthesize their own ascorbate to determine the impact of this vitamin on the liver proteome of specific subcellular organelles. We performed label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) global quantitative proteomic profiling to identify and quantify proteins in microsomal enriched liver extracts (MEE) from Gulo-/- mice treated with 0-0.4% (w/v) ascorbate in drinking water until the age of four months. Using a principal component analysis on normalized and imputed data of the label-free protein quantifications, a sex-based difference in MEE proteome profiles was observed for all the different ascorbate treated mice. Suboptimal hepatic ascorbate concentrations affected the levels of more proteins and hence biochemical processes in females than in males. Nevertheless, Pearson correlation analyses revealed that the MS intensities of various proteins involved in complement activation inversely correlated with liver ascorbate concentrations in both Gulo-/- males and females. Moreover, the correlation analyses also indicated that several proteins in the mitochondrial complex III of the electron transport chain positively correlated with liver ascorbate concentrations in both Gulo-/- females and males. Consequently, the mitochondrial complex III activity in Gulo-/- female and male mice treated with suboptimal hepatic concentrations of ascorbate was significantly lower than Gulo-/- mice treated with optimal ascorbate concentration. Finally, the whole liver of ascorbate-deficient Gulo-/- mice exhibited lower ATP levels and increased reactive oxygen species. These findings provide new information on how ascorbate deficiency potentially induces mitochondrial dysfunction in the liver of mice.

The production of preconditioned freeze-dried Oenococcus oeni primes its metabolism to withstand environmental stresses encountered upon inoculation into wine.

Oenococcus oeni is the most resistant lactic acid bacteria species to the environmental stresses encountered in wine, particularly the acidity, presence of ethanol and phenolic compounds. Indigenous strains develop spontaneously following the yeast-driven alcoholic fermentation and may perform the malolactic fermentation whereby improving taste, aroma, and the microbial stability of wine. However, spontaneous fermentation is sometimes delayed, prolonged or incomplete. In order to better control its timing and quality, O. oeni strains are selected and developed to be used as malolactic starters. They are prepared under proprietary manufacturing processes to survive direct inoculation and are predominantly provided as freeze-dried preparations. In this study, we have investigated the physiological and molecular alterations occurring in O. oeni cells prepared by an industrial process that consists of preconditioning protocols and freeze-drying, and compared them to the same strain grown in a grape juice medium. We found that compared to cultured cells, the industrial production process improved survival under extreme conditions, i. e. at low pH or high tannin concentrations. In contrast, cultured cells resumed active growth more quickly and strongly than freeze-dried preparations in standard pH wines. A proteomic analysis showed that during the industrial production most non-essential metabolic processes are shut down and components of the general and the stringent stress response are upregulated. The presence of major components of the stress response facilitates protein homeostasis and physiological changes that further ensure the integrity of cells.

Vitamin C Differentially Impacts the Serum Proteome Profile in Female and Male Mice.

A suboptimal blood vitamin C (ascorbate) level increases the risk of several chronic diseases. However, the detection of hypovitaminosis C is not a simple task, as ascorbate is unstable in blood samples. In this study, we examined the serum proteome of mice lacking the gulonolactone oxidase (Gulo) required for the ascorbate biosynthesis. Gulo-/- mice were supplemented with different concentrations of ascorbate in drinking water, and serum was collected to identify proteins correlating with serum ascorbate levels using an unbiased label-free liquid chromatography-tandem mass spectrometry global quantitative proteomic approach. Parallel reaction monitoring was performed to validate the correlations. We uncovered that the serum proteome profiles differ significantly between male and female mice. Also, unlike Gulo-/- males, a four-week ascorbate treatment did not entirely re-establish the serum proteome profile of ascorbate-deficient Gulo-/- females to the optimal profile exhibited by Gulo-/- females that never experienced an ascorbate deficiency. Finally, the serum proteins involved in retinoid metabolism, cholesterol, and lipid transport were similarly affected by ascorbate levels in males and females. In contrast, the proteins regulating serum peptidases and the protein of the acute phase response were different between males and females. These proteins are potential biomarkers correlating with blood ascorbate levels and require further study in standard clinical settings. The complete proteomics data set generated in this study has been deposited to the public repository ProteomeXchange with the data set identifier: PXD027019.

Lipoprotein Proteomics and Aortic Valve Transcriptomics Identify Biological Pathways Linking Lipoprotein(a) Levels to Aortic Stenosis.

Lipoprotein(a) (Lp(a)) is one of the most important risk factors for the development of calcific aortic valve stenosis (CAVS). However, the mechanisms through which Lp(a) causes CAVS are currently unknown. Our objectives were to characterize the Lp(a) proteome and to identify proteins that may be differentially associated with Lp(a) in patients with versus without CAVS. Our second objective was to identify genes that may be differentially regulated by exposure to high versus low Lp(a) levels in explanted aortic valves from patients with CAVS. We isolated Lp(a) from the blood of 21 patients with CAVS and 22 volunteers and performed untargeted label-free analysis of the Lp(a) proteome. We also investigated the transcriptomic signature of calcified aortic valves from patients who underwent aortic valve replacement with high versus low Lp(a) levels (n = 118). Proteins involved in the protein activation cascade, platelet degranulation, leukocyte migration, and response to wounding may be associated with Lp(a) depending on CAVS status. The transcriptomic analysis identified genes involved in cardiac aging, chondrocyte development, and inflammation as potentially influenced by Lp(a). Our multi-omic analyses identified biological pathways through which Lp(a) may cause CAVS, as well as key molecular events that could be triggered by Lp(a) in CAVS development.

A Comparative Analysis of the Lipoprotein(a) and Low-Density Lipoprotein Proteomic Profiles Combining Mass Spectrometry and Mendelian Randomization.

BACKGROUND: Lipoprotein(a) (Lp[a]), which consists of a low-density lipoprotein (LDL) bound to apolipoprotein(a), is one of the strongest genetic risk factors for atherosclerotic cardiovascular diseases. Few studies have performed hypothesis-free direct comparisons of the Lp(a) and the LDL proteomes. Our objectives were to compare the Lp(a) and the LDL proteomic profiles and to evaluate the effect of lifelong exposure to elevated Lp(a) or LDL cholesterol levels on the plasma proteomic profile. METHODS: We performed a label-free analysis of the Lp(a) and LDL proteomic profiles of healthy volunteers in a discovery (n = 6) and a replication (n = 9) phase. We performed inverse variance weighted Mendelian randomization to document the effect of lifelong exposure to elevated Lp(a) or LDL cholesterol levels on the plasma proteomic profile of participants of the INTERVAL study. RESULTS: We identified 15 proteins that were more abundant on Lp(a) compared with LDL (serping1, pi16, itih1, itih2, itih3, pon1, podxl, cd44, cp, ptprg, vtn, pcsk9, igfals, vcam1, and ttr). We found no proteins that were more abundant on LDL compared with Lp(a). After correction for multiple testing, lifelong exposure to elevated LDL cholesterol levels was associated with the variation of 18 plasma proteins whereas Lp(a) did not appear to influence the plasma proteome. CONCLUSIONS: Results of this study highlight marked differences in the proteome of Lp(a) and LDL as well as in the effect of lifelong exposure to elevated LDL cholesterol or Lp(a) on the plasma proteomic profile.

CONTEXTE: La lipoprotéine(a) (Lp[a]), qui est constituée d'une lipoprotéine de basse densité (LDL) liée à une apolipoprotéine(a), est l'un des plus importants facteurs de risque génétiques de survenue d'une maladie cardiovasculaire athéroscléreuse. Peu d'études comparatives directes sans hypothèse ont porté sur le protéome de la Lp(a) et celui des LDL. Nos objectifs étaient de comparer les profils protéomiques de la Lp(a) et des LDL et d'évaluer l'effet d'une exposition à vie à un taux élevé de Lp(a) ou de cholestérol LDL sur le profil protéomique plasmatique. MÉTHODOLOGIE: Nous avons réalisé une analyse sans marquage des profils protéomiques de la Lp(a) et des LDL chez des volontaires en bonne santé dans le cadre d'une phase de découverte (n = 6) et d'une phase de réplication (n = 9). Pour rendre compte de l'effet d'une exposition à vie à un taux élevé de Lp(a) ou de cholestérol des LDL sur le profil protéomique plasmatique des participants de l'étude INTERVAL, nous avons utilisé une analyse de randomisation Mendélienne avec pondération par l'inverse de la variance. RÉSULTATS: Nous avons relevé 15 protéines associées en plus grande abondance à la Lp(a) qu'aux LDL (serping1, pi16, itih1, itih2, itih3, pon1, podxl, cd44, cp, ptprg, vtn, pcsk9, igfals, vcam1 et ttr). Nous n'avons noté aucune protéine associée en plus grande abondance aux LDL qu'à la Lp(a). Après correction pour tenir compte de la multiplicité des tests, l'exposition à vie à un taux élevé de cholestérol LDL a été associée à la variation de 18 protéines plasmatiques, tandis que le taux de Lp(a) ne semblait pas influencer le protéome plasmatique. CONCLUSIONS: Les résultats de notre étude font ressortir les différences marquées entre le protéome de la Lp(a) et celui des LDL, ainsi qu'entre l'effet sur le profil protéomique plasmatique de l'exposition à vie à un taux élevé de cholestérol LDL et celui de l'exposition à vie à un taux élevé de Lp(a).

Proteomic markers of low and high fertility bovine spermatozoa separated by Percoll gradient.

In the context of artificial insemination, male fertility is defined as the ability to produce functional spermatozoa able to withstand cryopreservation. We hypothesized that interindividual variations in fertility depend on the proportion of the fully functional sperm population contained in the insemination dose. The objective of this study was to identify protein markers of the fully functional sperm subpopulation. Insemination doses from four high-fertility (HF) and four low-fertility (LF) bulls with comparable post-thaw quality parameters were selected for proteomic analysis using iTRAQ technology. Thawed semen was centrifuged through a Percoll gradient to segregate the motile (high density [HD]) from the immotile (low density [LD]) sperm populations. Sperm proteins were extracted with sodium deoxycholate and four groups were compared: LD and HD spermatozoa from LF and HF bulls. A total of 498 unique proteins were identified and quantified. Comparison of HD spermatozoa from HF and LF bulls revealed that five proteins were significantly more abundant in the HF group (AK8, TPI1, TSPAN8, OAT, and DBIL5) whereas five proteins were more abundant in the LF group (RGS22, ATP5J, CLU, LOC616319, and CCT5). Comparison of LD spermatozoa from HF and LF bulls revealed that four proteins were significantly more abundant in the HF group (IL4I1, CYLC2, OAT, and ARMC3) whereas 15 proteins were significantly more abundant in the LF group (HADHA, HSP90AA1, DNASE1L3, SLC25A20, GPX5, TCP1, HIP1, CLU, G5E622, LOC616319, HSPA2, NUP155, DPY19L2, SPERT, and SERPINE2). DBIL5, TSPAN8, and TPI1 showed potential as putative markers of the fully functional sperm subpopulation.

Identification of protein markers for extracellular vesicle (EV) subsets in cow's milk.

Extracellular vesicles (EVs), like exosomes, are small membrane vesicles involved in cell-to-cell communications that modulate numerous biological processes. We previously discovered a new EV subset in milk (sedimenting at 35,000 g; 35 K) that protected its cargo (RNAs and proteins) during simulated digestion and was more enriched in microRNAs than exosomes (sedimenting at 100 K). Here, we used LC-MS/MS to push further the comparison between these two pellets. Commonly used EV markers were not differentially enriched between the pellets, questioning their use with cow's milk EVs. Similarly, the majority of the quantified proteins were equally enriched between the two pellets. Nevertheless, 20 proteins were specific to 35 K, while 41 were specifically enriched in 100 K (p < 0.05), suggesting their potential use as specific markers. Loaded with these proteins, the EVs in these pellets might regulate translation, proliferation and cell survival for 35 K, and metabolism, extracellular matrix turnover and immunity for 100 K. This approach also brought new insights into milk EV-associated integrins and their possible role in specifically targeting recipient cell types. These findings may help better discriminate between milk EVs, improve our understanding of milk EV-associated protein function and their possible use as therapeutic tools for the management of immunity- and metabolism-associated disorders. WEB PAGE: http://www.crchuq.ca/en/research/researchers/4691.

A type 2 diabetes disease module with a high collective influence for Cdk2 and PTPLAD1 is localized in endosomes.

Despite the identification of many susceptibility genes our knowledge of the underlying mechanisms responsible for complex disease remains limited. Here, we identified a type 2 diabetes disease module in endosomes, and validate it for functional relevance on selected nodes. Using hepatic Golgi/endosomes fractions, we established a proteome of insulin receptor-containing endosomes that allowed the study of physical protein interaction networks on a type 2 diabetes background. The resulting collated network is formed by 313 nodes and 1147 edges with a topology organized around a few major hubs with Cdk2 displaying the highest collective influence. Overall, 88% of the nodes are associated with the type 2 diabetes genetic risk, including 101 new candidates. The Type 2 diabetes module is enriched with cytoskeleton and luminal acidification-dependent processes that are shared with secretion-related mechanisms. We identified new signaling pathways driven by Cdk2 and PTPLAD1 whose expression affects the association of the insulin receptor with TUBA, TUBB, the actin component ACTB and the endosomal sorting markers Rab5c and Rab11a. Therefore, the interactome of internalized insulin receptors reveals the presence of a type 2 diabetes disease module enriched in new layers of feedback loops required for insulin signaling, clearance and islet biology.

Direct Phosphorylation of SRC Homology 3 Domains by Tyrosine Kinase Receptors Disassembles Ligand-Induced Signaling Networks.

Phosphotyrosine (pTyr) signaling has evolved into a key cell-to-cell communication system. Activated receptor tyrosine kinases (RTKs) initiate several pTyr-dependent signaling networks by creating the docking sites required for the assembly of protein complexes. However, the mechanisms leading to network disassembly and its consequence on signal transduction remain essentially unknown. We show that activated RTKs terminate downstream signaling via the direct phosphorylation of an evolutionarily conserved Tyr present in most SRC homology (SH) 3 domains, which are often part of key hub proteins for RTK-dependent signaling. We demonstrate that the direct EPHA4 RTK phosphorylation of adaptor protein NCK SH3s at these sites results in the collapse of signaling networks and abrogates their function. We also reveal that this negative regulation mechanism is shared by other RTKs. Our findings uncover a conserved mechanism through which RTKs rapidly and reversibly terminate downstream signaling while remaining in a catalytically active state on the plasma membrane.

Proteomic Markers of Functional Sperm Population in Bovines: Comparison of Low- and High-Density Spermatozoa Following Cryopreservation.

Mammalian semen contains a heterogeneous population of sperm cells. This heterogeneity results from variability in the complex processes of cell differentiation in the testis, biochemical modifications undergone by spermatozoa during transit along the male reproductive tract, interactions with secretions from accessory sex glands at ejaculation, and, in the context of reproductive technologies, in the ability of ejaculated spermatozoa to resist damage associated with freeze-thaw procedures. When submitted to density gradient centrifugation, ejaculated spermatozoa distribute themselves into two distinct populations: a low-density population characterized by low motility parameters, and a high-density population with high motility characteristics. To understand the origin of ejaculated spermatozoa heterogeneity, cryopreserved semen samples from bulls used by the artificial insemination (A.I.) industry were submitted to Percoll gradient centrifugation. Proteins from low and high density spermatozoa were then extracted with sodium deoxycholate and submitted to proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) methodologies. Quantification of selected sperm proteins was confirmed by multiple reaction monitoring (MRM). Overall, 31 different proteins were more abundant in low-density spermatozoa, while 80 different proteins were more abundant in the high-density subpopulation. Proteins enriched in high-density spermatozoa were markers of sperm functionality such as the glycolytic process, binding to the egg zona pellucida, and motility. Low-density spermatozoa were not solely characterized by loss of proteins and their associated functions. Chaperonin-containing TCP1s and chaperones are hallmarks of the low-density subpopulation. iTRAQ analysis revealed that other proteins such as binder of sperm proteins, histone, GPX5, ELSPBP1, and clusterin are overexpressed in low-density spermatozoa suggesting that these proteins represent defects occurring at different steps during the sperm journey. These differences contribute to the sperm cell heterogeneity present in mammalian semen.

Daylily protein constituents of the pollen and stigma a proteomics approach.

This study was aimed at the identification and quantification of the protein components of the pollen grains in parallel with the distal stigmatic tissue of tetraploid cultivars. Proteomes were analyzed using iTRAQ 4plex labeling, peptides separation by online RP-nano-LC and analysis by ESI-MS/MS. Protein identification and quantification were made using the Asparagales database as a reference. A total of 524,037 MS/MS spectra were produced from pollen and stigma samples. From these, a total of 8368 peptides wereidentified corresponding to 994 unique peptides and 432 protein groups. Among them, 128 differentially expressed proteins were retained for further analysis. In absence of the daylily genome availability, we exploited numerous databases and bioinformatics resources to exploring the putative biological functions of these proteins. The profile of differentially expressed proteins suggests an important representation of functions associated to the signalling and response against endogenous and environmental stresses, including several enzymes implicated in the biosynthesis of antibiotics. The abundance in stigma of several structural proteins of the ribosomal sub-units as well as of the core histones suggest that the translation processes and the regulation of gene expression in stigma is a more active mechanism than in pollen. In addition, pollen prioritizes the synthesis of fructose and glucose as opposed to sucrose in stigma as a source of energy. Finally, the modulated proteins in Hemerocallis point to several pathways that give potential clues concerning the molecular mechanisms underlying the functions of the pollen and the stigmatic fluid in daylily reproduction.

iTRAQ-based quantitative proteomics of stratum corneum of dandruff scalp reveals new insights into its aetiology and similarities with atopic dermatitis.

The study aimed at detecting differentially expressed proteins in the stratum corneum of dandruff versus non-dandruff scalps to better understand dandruff aetiology. iTRAQ-based quantitative proteomic analysis revealed a total of 68 differentially expressed biomarkers. A detailed analysis of their known physiological functions provided new insights into the affected metabolic pathways of a dandruff scalp. Dandruff scalp showed (1) profound changes in the expression and maturation of structural and epidermal differentiation related proteins, that are responsible for the integrity of the skin, (2) altered relevant factors that regulate skin hydration, and (3) an imbalanced physiological protease-protease inhibitor ratio. Stratum corneum proteins with antimicrobial activity, mainly those derived from sweat and sebaceous glands were also found modified. Comparing our data with those reported for atopic dermatitis revealed that about 50 % of the differentially expressed proteins in the superficial layers of the stratum corneum from dandruff and atopic dermatitis are identical.

Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.

This article contains raw and processed data related to research published in "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation" [1]. The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s. LC-MS/MS mass spectrometry analysis was then performed and the processed data related to the identified glycoproteins show that several hundred proteins are differentially expressed between control and GALNT3 KD A2780s cells. The obtained data also uncover numerous novel glycoproteins; some of which could represent new potential EOC biomarkers and/or therapeutic targets.

A metabolic labeling approach for glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.

UNLABELLED: Epithelial ovarian cancer (EOC) is a disease responsible for more deaths among women in the Western world than all other gynecologic malignancies. There is urgent need for new therapeutic targets and a better understanding of EOC initiation and progression. We have previously identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene, a member of the GalNAc-transferases (GalNAc-Ts) gene family, as hypomethylated and overexpressed in high-grade serous EOC tumors, compared to low malignant potential EOC tumors and normal ovarian tissues. This data also suggested for a role of GALNT3 in aberrant EOC glycosylation, possibly implicated in disease progression. To evaluate differential glycosylation in EOC caused by modulations in GALNT3 expression, we used a metabolic labeling strategy for enrichment and mass spectrometry-based characterization of glycoproteins following GALNT3 gene knockdown (KD) in A2780s EOC cells. A total of 589 differentially expressed glycoproteins were identified upon GALNT3 KD. Most identified proteins were involved in mechanisms of cellular metabolic functions, post-translational modifications, and some have been reported to be implicated in EOC etiology. The GALNT3-dependent glycoproteins identified by this metabolic labeling approach support the oncogenic role of GALNT3 in EOC dissemination and may be pursued as novel EOC biomarkers and/or therapeutic targets. BIOLOGICAL SIGNIFICANCE: Knowledge of the O-glycoproteome has been relatively elusive, and the functions of the individual polypeptide GalNAc-Ts have been poorly characterized. Alterations in GalNAc-Ts expression were shown to provide huge variability in the O-glycoproteome in various pathologies, including cancer. The application of a chemical biology approach for the metabolic labeling and subsequent characterization of O-glycoproteins in EOC using the Ac4GalNAz metabolite has provided a strategy allowing for proteomic discovery of GalNAc-Ts specific functions. Our study supports an essential role of one of the GalNAc-Ts - GALNT3, in EOC dissemination, including its implication in modulating PTMs and EOC metabolism. Our approach validates the use of the applied metabolic strategy to identify important functions of GalNAc-Ts in normal and pathological conditions.

STAR syndrome-associated CDK10/Cyclin M regulates actin network architecture and ciliogenesis.

CDK10/CycM is a protein kinase deficient in STAR (toe Syndactyly, Telecanthus and Anogenital and Renal malformations) syndrome, which results from mutations in the X-linked FAM58A gene encoding Cyclin M. The biological functions of CDK10/CycM and etiology of STAR syndrome are poorly understood. Here, we report that deficiency of CDK10/Cyclin M promotes assembly and elongation of primary cilia. We establish that this reflects a key role for CDK10/Cyclin M in regulation of actin network organization, which is known to govern ciliogenesis. In an unbiased screen, we identified the RhoA-associated kinase PKN2 as a CDK10/CycM phosphorylation substrate. We establish that PKN2 is a bone fide regulator of ciliogenesis, acting in a similar manner to CDK10/CycM. We discovered that CDK10/Cyclin M binds and phosphorylates PKN2 on threonines 121 and 124, within PKN2's core RhoA-binding domain. Furthermore, we demonstrate that deficiencies in CDK10/CycM or PKN2, or expression of a non-phosphorylatable version of PKN2, destabilize both the RhoA protein and the actin network architecture. Importantly, we established that ectopic expression of RhoA is sufficient to override the induction of ciliogenesis resulting from CDK10/CycM knockdown, indicating that RhoA regulation is critical for CDK10/CycM's negative effect on ciliogenesis. Finally, we show that kidney sections from a STAR patient display dilated renal tubules and abnormal, elongated cilia. Altogether, these results reveal CDK10/CycM as a key regulator of actin dynamics and a suppressor of ciliogenesis through phosphorylation of PKN2 and promotion of RhoA signaling. Moreover, they suggest that STAR syndrome is a ciliopathy.

Evaluation of iTRAQ and SWATH-MS for the Quantification of Proteins Associated with Insulin Resistance in Human Duodenal Biopsy Samples.

Insulin resistance (IR) is associated with increased production of triglyceride-rich lipoproteins of intestinal origin. In order to assess whether insulin resistance affects the proteins involved in lipid metabolism, we used two mass spectrometry based quantitative proteomics techniques to compare the intestinal proteome of 14 IR patients to that of 15 insulin sensitive (IS) control patients matched for age and waist circumference. A total of 3886 proteins were identified by the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry approach and 2290 by the SWATH-MS strategy (Serial Window Acquisition of Theoretical Spectra). Using these two methods, 208 common proteins were identified with a confidence corresponding to FDR < 1%, and quantified with p-value < 0.05. The quantification of those 208 proteins has a Pearson correlation coefficient (r2) of 0.728 across the two techniques. Gene Ontology analyses of the differentially expressed proteins revealed that annotations related to lipid metabolic process and oxidation reduction process are overly represented in the set of under-expressed proteins in IR subjects. Furthermore, both methods quantified proteins of relevance to IR. These data also showed that SWATH-MS is a promising and compelling alternative to iTRAQ for protein quantitation of complex mixtures.

Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs.

An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADP-ribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process.

Skeletal muscle proteomic signature and metabolic impairment in pulmonary hypertension.

UNLABELLED: Exercise limitation comes from a close interaction between cardiovascular and skeletal muscle impairments. To better understand the implication of possible peripheral oxidative metabolism dysfunction, we studied the proteomic signature of skeletal muscle in pulmonary arterial hypertension (PAH). Eight idiopathic PAH patients and eight matched healthy sedentary subjects were evaluated for exercise capacity, skeletal muscle proteomic profile, metabolism, and mitochondrial function. Skeletal muscle proteins were extracted, and fractioned peptides were tagged using an iTRAQ protocol. Proteomic analyses have documented a total of 9 downregulated proteins in PAH skeletal muscles and 10 upregulated proteins compared to healthy subjects. Most of the downregulated proteins were related to mitochondrial structure and function. Focusing on skeletal muscle metabolism and mitochondrial health, PAH patients presented a decreased expression of oxidative enzymes (pyruvate dehydrogenase, p < 0.01) and an increased expression of glycolytic enzymes (lactate dehydrogenase activity, p < 0.05). These findings were supported by abnormal mitochondrial morphology on electronic microscopy, lower citrate synthase activity (p < 0.01) and lower expression of the transcription factor A of the mitochondria (p < 0.05), confirming a more glycolytic metabolism in PAH skeletal muscles. We provide evidences that impaired mitochondrial and metabolic functions found in the lungs and the right ventricle are also present in skeletal muscles of patients. KEY MESSAGE: • Proteomic and metabolic analysis show abnormal oxidative metabolism in PAH skeletal muscle. • EM of PAH patients reveals abnormal mitochondrial structure and distribution. • Abnormal mitochondrial health and function contribute to exercise impairments of PAH. • PAH may be considered a vascular affliction of heart and lungs with major impact on peripheral muscles.