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Molecular cellular proteomics [journal]
- Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 17.
Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites and the identities of core proteins. CSPGs were enriched from human urine and cerebral spinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1 and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may influence also the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.
- Quantitative proteomics reveals dynamic interaction of c-Jun N-terminal kinase (JNK) with RNA transport granule proteins Sfpq and Nono during neuronal differentiation. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 17.
The c-Jun N-terminal kinase (JNK) is an important mediator of physiological and pathophysiological processes in the central nervous system. Importantly, JNK is not only involved in neuronal cell death but also plays a significant role in neuronal differentiation and regeneration. For example, nerve growth factor (NGF) induces JNK-dependent neuronal differentiation in several model systems. The mechanism how JNK mediates neuronal differentiation is not well understood. Here, we employ a proteomic strategy to better characterize the function of JNK during neuronal differentiation. We use SILAC-based quantitative proteomics to identify proteins that interact with JNK in PC12 cells in an NGF-dependent manner. Intriguingly, we find that JNK interacts with neuronal transport granule proteins such as Sfpq and Nono upon NGF treatment. We validate the specificity of these interactions by showing that they are disrupted by a specific peptide inhibitor that blocks the interaction of JNK with its substrates. Immunoprecipitation and western blotting experiments confirm the interaction of JNK1 with Sfpq/Nono and demonstrate that it is RNA dependent. Confocal microscopy and subcellular fractionation indicates that JNK1 associates with neuronal granule proteins in the cytosol of PC12 cells, primary cortical neurons and P19-neuronal cells. Finally, siRNA experiments confirm that Sfpq is necessary for neuronal outgrowth in PC12 cells and that it is most likely acting in the same pathway as JNK. In summary, our data indicate that the interaction of JNK1 with transport granule proteins in the cytosol of differentiating neurons plays an important role during neuronal development.
- cysTMTRAQ-an integrative method for unbiased thiol-based redox proteomics. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 14.
Protein redox regulation plays important roles in many biological processes. Protein cysteine thiols are sensitive to redox changes and may function as redox switches, which turn on or turn off signaling and metabolic pathways to ensure speedy responses to environmental stimuli or stresses. Here we report a novel integrative proteomics method called cysTMTRAQ that combines two types of isobaric tags, cysteine tandem mass tag (cysTMT) and isobaric tag for relative and absolute quantification (iTRAQ) in one experiment. The method not only enables simultaneous analysis of cysteine redox changes and total protein level changes, but also allows determination of bona fide redox modified cysteines in proteins through correction of protein turnover. Overlooking the factor of protein level changes in the course of the protein posttranslational modification (PTM) experiments could lead to misleading results. The capability to analyze protein posttranslational modification dynamics and protein level changes in one experiment will advance proteomic studies in many fields of biology and medicine.
- SLP-76 N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal TCR signaling. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 14.
SRC homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T cell receptor (TCR) mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues Tyr112, Tyr128, Tyr145 in the N-terminus of SLP-76 resulted in severely impaired phosphorylation and activation of Itk and PLCγ1, which further led to defective calcium mobilization, Erk activation and NFAT activation. To expand our knowledge of the role of N terminal phosphorylation of SLP-76 from these three tyrosine sites，we characterized nearly 1000 tyrosine phosphorylation sites by mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells, where three tyrosine residues are replaced with phenylalanines (denoted as Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76 deficient cells for the majority of tyrosine phosphorylation sites observed including feedback on proximal TCR signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr192 of Lck when comparing mutants to complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr440 of Fyn, Tyr702 of PLCγ1, Tyr204, Tyr397 and Tyr69 of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N terminal tyrosine residues.
- Quantitative Proteomics Reveals a Role for Epigenetic Reprogramming During Human Monocyte Differentiation. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 14.
The differentiation of monocytes into macrophages and dendritic cells involves mechanisms for activation of the innate immune system in response to inflammatory stimuli, such as pathogen infection, and environmental cues. Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. While highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells.
- Systematic analysis of the phosphoproteome and kinase-substrate networks in the mouse testis. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 7.
Spermatogenesis is a complex process closely associated with the phosphorylation-orchestrated cell cycle. Elucidating the phosphorylation-based regulations should advance our understanding of the underlying molecular mechanisms. Here we present an integrative study of phosphorylation events in the testis. Large-scale phosphoproteome profiling in the adult mouse testis identified 17,829 phosphorylation sites in 3,955 phosphoproteins. Although only approximately half of the phosphorylation sites enriched by IMAC were also captured by TiO2, both the phosphoprotein datasets identified by the two methods significantly enriched the functional annotation of spermatogenesis. Thus, the phosphoproteome profiled in this study is a highly useful snapshot of the phosphorylation events in spermatogenesis. To further understand phosphoregulation in the testis, the site-specific kinase-substrate relations (ssKSRs) were computationally predicted for re-constructing kinase-substrate phosphorylation networks (KSPNs). A core sub-KSPN among the spermatogenesis-related proteins was retrieved and analyzed to explore the phosphoregulation during spermatogenesis. Moreover, network-based analyses demonstrated that a number of protein kinases such as MAPKs, CDK2 and CDC2 with statistically more ssKSRs might have significantly higher activities and play an essential role in spermatogenesis, and the predictions were consistent with previous studies on the regulatory roles of these kinases. In particular, the analyses proposed that the activities of POLO-like kinases (PLKs) might be dramatically higher, while the prediction was experimentally validated by detecting and comparing the phosphorylation levels of pT210, an indicator of PLK1 activation, in testis and other tissues. Further experiments showed that the inhibition of PLKs decreases cell proliferation by inducing G2/M cell cycle arrest. Taken together, this systematic study provides a global landscape of phosphoregulation in the testis, and should prove to be of value in future studies of spermatogenesis.
- Proteogenomics of Gammarus fossarum to document the reproductive system of amphipods. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 7.
Because of their ecological importance, amphipod crustacea are employed worldwide as test species in environmental risk assessment. While proteomics allows new insights into the molecular mechanisms related to the stress response, such investigations are rare for these organisms due to the lack of comprehensive protein sequence databases. Here, we propose a proteogenomic approach for identifying specific proteins of the freshwater amphipod, Gammarus fossarum, a keystone species in European freshwater ecosystems. After deep RNA sequencing, we created a comprehensive ORF database. We identified and annotated the most relevant proteins detected, through a shotgun tandem mass spectrometry analysis carried out on the proteomes from three major tissues involved in the organism reproductive function: the male and female reproductive systems, and the cephalon, where different neuroendocrine glands are present. The 1,873 mass-spectrometry-certified proteins represent the largest crustacean proteomic resource to date, 218 proteins being lineage specific. Comparative proteomics between the male and female reproductive systems indicated key proteins with strong sexual dimorphism. Protein expression profiles during spermatogenesis at seven different stages highlighted the major gammarid proteins involved in the different facets of reproduction.
- The functional landscape of Hsp27 reveals new cellular processes such as DNA repair and alternative splicing and proposes novel anticancer targets. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 2.
Previously, we identified the stress-induced chaperone, Hsp27, as highly overexpressed in castration-resistant prostate cancer and developed an Hsp27 inhibitor (OGX-427) currently tested in phase I/II clinical trials as a chemosensitizing agent in different cancers. To better understand the Hsp27 poorly-defined cytoprotective functions in cancers and increase the OGX-427 pharmacological safety, we established the Hsp27-protein interaction network using a yeast two-hybrid approach and identified 226 interaction partners. As an example, we showed that targeting Hsp27 interaction with TCTP, a partner protein identified in our screen increases therapy sensitivity, opening a new promising field of research for therapeutic approaches that could decrease or abolish toxicity for normal cells. Results of an in-depth bioinformatics network analysis allying the Hsp27 interaction map into the human interactome underlined the multifunctional character of this protein. We identified interactions of Hsp27 with proteins involved in 8 well known functions previously related to Hsp27 and uncovered 17 potential new ones, such as DNA repair and RNA splicing. Validation of Hsp27 involvement in both processes in human prostate cancer cells supports our system biology-predicted functions and provides new insights into Hsp27 roles in cancer cells.
- Analysis of differential expression patterns of mRNA and protein during cold- and de-acclimation in Arabidopsis. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 2.
Overwintering plants are capable of exhibiting high levels of cold tolerance, which is acquired through the process of cold acclimation (CA). In contrast to CA, the acquired freezing tolerance is rapidly reduced during cold de-acclimation (DA) and plants resume growth after sensing warm temperatures. In order to better understand plant growth and development, and to aid in the breeding of cold-tolerant plants, it is important to decipher the functional mechanisms of the DA process. In this study, we performed comparative transcriptomic and proteomic analyses during CA and DA. As revealed by shotgun proteomics, we identified 3,987 peptides originating from 1,569 unique proteins and the corresponding mRNAs were analyzed. Among the 1,569 genes, 658 genes were specifically induced at the transcriptional level during the process of cold acclimation. In order to investigate the relationship between mRNA and the corresponding protein expression pattern, a Pearson correlation was analyzed. Interestingly, 199 genes showed a positive correlation of mRNA and protein expression pattern, indicating that both their transcription and translation occurred during CA. However, 226 genes showed a negative correlation of mRNA and protein expression pattern, indicating that their mRNAs were transcribed during CA and were stored for the subsequent DA step. Under this scenario, those proteins were specifically increased during DA without additional transcription of mRNA. In order to confirm the negative correlation of mRNA and protein expression patterns, qRT-PCR and western blot analyses were performed. Mitochondrial malate dehydrogenase1 (mMDH1) exhibited a negative correlation of mRNA and protein levels, which was characterized by CA-specific mRNA induction and protein accumulation specifically during DA. These data indicate that the expression of specific mRNAs and subsequent accumulation of corresponding proteins are not always in accordance under low temperature stress conditions in plants.
- Integrated omic analysis of oropharyngeal carcinomas reveals HPV-dependent regulation of the AP-1 pathway. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Sep 30.
While HPV-positive oropharyngeal carcinoma (OPC) patients have superior outcome relative to HPV-negative patients, the underlying mechanisms remain poorly understood. We conducted a proteomic investigation of HPV-positive (n=27) and HPV-negative (n=26) formalin-fixed paraffin-embedded OPC biopsies to acquire insights into the biological pathways that correlate with clinical behavior. Among the 2,633 proteins identified, 174 were differentially abundant. These were enriched for proteins related to cell cycle, DNA replication, apoptosis and immune response. The differential abundances of cortactin (CTTN) and methylthioadenosine phosphorylase (MTAP) were validated by immunohistochemistry in an independent cohort of 29 OPC samples (p=0.023 and p=0.009, respectively). An additional 1,124 proteins were independently corroborated by comparison to a published proteomic dataset of OPC. Furthermore, utilizing The Cancer Genome Atlas (TCGA), we conducted an integrated investigation of OPC, attributing mechanisms underlying differential protein abundance to alterations in mutation, copy number, methylation, and mRNA profiles. A key finding of this integration was the identification of elevated cortactin oncoprotein levels in HPV-negative OPCs, potentially contributing to reduced survival in these patients via its established role in radiation resistance. Through interrogation of TCGA data, we demonstrated that activation of the β1-integrin/FAK/cortactin/JNK1 signaling axis and associated differential regulation of AP-1 transcription factor target genes is a plausible consequence of elevated cortactin protein levels.