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Molecular cellular proteomics [journal]
- Proteomics of skin proteins in psoriasis: discovery and verification in a mouse model to confirmation in humans. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 28.
Herein we demonstrate the efficacy of an unbiased proteomics screening approach for studying protein expression changes in the KC-Tie2 psoriasis mouse model, identifying multiple protein expression changes in the mouse and validating these changes in human psoriasis. KC-Tie2 mouse skin samples (n=3) were compared with littermate controls (n=3) using gel-based fractionation followed by label-free protein expression analysis. 5482 peptides mapping to 1281 proteins were identified and quantitated: 105 proteins exhibited fold-changes ≥2.0 including: stefin A1 (average fold change of 342.4; average P=0.0082; cystatin A, human orthologue); slc25a5 (average fold change of 46.2; average P=0.0318); serpinb3b (average fold change of 35.6 and an average P = 0.0345; serpinB1, human orthologue); and kallikrein related peptidase 6 (average fold change of 4.7; average P=0.2474; KLK6). We independently confirmed mouse gene expression-based increases of selected genes including serpinb3b (17.4-fold, P<0.0001), KLK6 (9.0-fold, P=0.002), stefin A1 (7.3-fold; P<0.001) and slc25A5 (1.5-fold; P=0.05) using qRT-PCR on a second cohort of animals (n=8). Parallel LC/MS/MS analyses on these same samples verified protein-level increases of 1.3-fold (slc25a5; P<0.05), 29,000-fold (stefinA1; P<0.01), 322-fold (KLK6; P<0.0001) between KC-Tie2 and control mice. To underscore the utility and translatability of our combined approach, we analyzed gene and protein expression levels in psoriasis patient skin and primary keratinocytes vs. healthy controls. Increases in gene expression for slc25a5 (1.8-fold), cystatin A (3.0-fold), KLK6 (5.8-fold) and serpinB1 (76-fold; all P < 0.05) were observed between healthy controls and involved lesional psoriasis skin and primary psoriasis keratinocytes. Moreover slc25a5, cystatin A, KLK6 and serpinB1 protein were all increased in lesional psoriasis skin compared to normal skin. These results highlight the usefulness of preclinical disease models using readily-available mouse skin and demonstrate the utility of proteomic approaches for identifying novel peptides/proteins that are differentially regulated in psoriasis that could serve as sources of auto-antigens or provide novel therapeutic targets for the development of new anti-psoriatic treatments.
- Proteomes of host cell membranes modified by intracellular activities of Salmonella enterica. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 27.
Intracellular pathogens need to establish a growth-stimulating host niche for survival and replication. A unique feature of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium is the creation of extensive membrane network within its host. Understanding the origin and function of these membranes is crucial for the development of new treatment strategies. However, the characterization of this compartment is very challenging and only fragmentary knowledge of its composition and biogenesis exists. Here, we describe a new proteome-based approach to enrich and characterize Salmonella-modified membranes (SMM). Using a Salmonella mutant strain that does not form this unique membrane network as reference we identified a high-confidence set of host proteins associated with SMM. This comprehensive analysis allowed us to reconstruct the interactions of Salmonella with host membranes. For example, we found that Salmonella redirect ER membrane trafficking to its intracellular niche, which has not been described for Salmonella. Our system-wide approach has therefore the potential to rapidly close gaps in our knowledge of the infection process of intracellular pathogens and demonstrates a hitherto unrecognized complexity in the formation of Salmonella host niches.
- The metacaspase Mca1p has a dual role in farnesol-induced apoptosis in Candida albicans. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 27.
Manipulating the apoptotic response of Candida albicans may help in the control of this opportunistic pathogen. The metacaspase Mca1p has been described as a key protease for apoptosis in C. albicans but little is known about its cleavage specificity and substrates. We therefore initiated a series of studies to describe its function. We used a strain disrupted for the MCA1 gene and compared its proteome to that of a wild-type isogenic strain, in the presence and absence of a known inducer of apoptosis, the quorum-sensing molecule farnesol. Label-free and TMT labeling quantitative proteomic analyses showed that both mca1 disruption and farnesol treatment significantly affected the proteome of the cells. The combination of both conditions led to an unexpected biological response: the strong overexpression of proteins implicated in the general stress. We studied sites cleaved by Mca1p using native peptidomic techniques, and a bottom-up approach involving GluC endoprotease: there appeared to be a K/R substrate specificity in P1 and a D/E specificity in P2. We also found 80 potential substrates of Mca1p, implicated in protein folding, protein aggregate resolubilization, glycolysis and a number of mitochondrial functions. These various results indicate that Mca1p is involved in a limited and specific proteolysis program triggered by apoptosis. One of the main functions of Mca1p appears to be the degradation of several major Heat Shock Proteins, thereby contributing to weakening cellular defenses and amplifying the cell death process. Finally, Mca1p appears to contribute significantly to the control of mitochondria biogenesis and degradation. Consequently, Mca1p may be a link between the extrinsic and the intrinsic programmed cell death pathways in C. albicans.
- Glycosylation of Skp1 promotes formation of Skp1/Cullin-1/F-box protein complexes in Dictyostelium. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Oct 23.
O2-sensing in diverse protozoa depends on the prolyl 4-hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of 5 sugars. In yeast, plants and animals, Skp1 is associated with F-box proteins. The Skp1/F-box protein heterodimer can, for many F-box proteins, dock onto Cullin-1 en route to assembly of the Skp1/Cullin-1/F-box protein/Rbx1 subcomplex of E3SCFUb-ligases. E3SCFUb-ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S-proteasome. In the social amoeba Dictyostelium, we show that O2-availability is rate limiting for hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knock-out mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells that can fully glycosylate Skp1 have greater abundance of an SCF complex containing the Cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, relative to the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA/Skp1CulA complex was also more abundant in glycosylation competent cells. The CulE interactome also includes higher levels of proteasomal regulatory particles when Skp1 is glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding consistent with an impact on the abundance of potential substrate proteins. We propose that O2-dependent posttranslational glycosylation of Skp1 promotes association with F-box proteins and their engagement in functional E3SCFUb-ligases that regulate O2-dependent developmental progression.
- 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.