(Molecular cellular proteomics[TA]) articles in PubMed
- The Primary Effect on the Proteome of ARID1A-Mutated Ovarian Clear Cell Carcinoma is Downregulation of the Mevalonate Pathway at the Post-Transcriptional Level. [Journal Article]
- Mol Cell Proteomics 2016 Sep 21MC
- Inactivating mutations in ARID1A, which encodes a subunit of the SWI/SNF chromatin-remodeling complex, are found in over half of ovarian clear cell carcinoma cases and more broadly across most types ...
Inactivating mutations in ARID1A, which encodes a subunit of the SWI/SNF chromatin-remodeling complex, are found in over half of ovarian clear cell carcinoma cases and more broadly across most types of cancers. To identify ARID1A-dependent changes in intracellular signaling pathways, we performed proteome analyses of isogenic ovarian clear cell carcinoma cell lines with or without ARID1A expression. Knockout of ARID1A in an ovarian clear cell carcinoma cell line with wild-type ARID1A, OVCA429, primarily resulted in downregulation of the mevalonate pathway, an important metabolic pathway involved in isoprenoid synthesis, cholesterol synthesis, and other downstream pathways. In a complementary experiment, expression of wild-type ARID1A in an ovarian clear cell carcinoma cell line containing mutated ARID1A, OVISE, affected the mevalonate pathway in a reciprocal manner. A striking aspect of these analyses was that, although only 5% of the detected proteome showed significant abundance changes, most proteins in the mevalonate pathway were coordinately affected by ARID1A status. There were generally corresponding changes when comparing the proteomics data to our previously published microarray data for ectopic expression of ARID1A in the OVISE cell line. However, ARID1A-dependent changes were not detected for genes within the mevalonate pathway. This discrepancy suggests that the mevalonate pathway is not regulated directly by ARID1A-mediated transcription and may be regulated post-transcriptionally. We conclude that ARID1A status indirectly influences the mevalonate pathway and probably influences other processes including glycogen metabolism and 14-3-3-mediated signaling. Further, our findings demonstrate that changes in mRNA levels are sometimes poor indicators of signaling pathways affected by gene manipulations in cancer cells.
- Comparative ploidy proteomics of Candida albicans biofilms unraveled the role of AHP1 in the biofilm persistence against amphotericin B. [Journal Article]
- Mol Cell Proteomics 2016 Sep 19MC
- Candida albicans is a major fungal pathogen causing lethal infections in immunocompromised patients. C. albicans forms antifungal tolerant biofilms contributing significantly to therapeutic failure. ...
Candida albicans is a major fungal pathogen causing lethal infections in immunocompromised patients. C. albicans forms antifungal tolerant biofilms contributing significantly to therapeutic failure. The recently established haploid C. albicans biofilm model provides a new toolbox to uncover the mechanism governing the higher antifungal tolerance of biofilms. Here, we comprehensively examined the proteomics and antifungal susceptibility of standard diploid (SC5314 and BWP17) and stable haploid (GZY792 and GZY803) strains of C. albicans biofilms. Subsequent downstream analyses identified AHP1 as a critical determinant of C. albicans biofilm's tolerance of amphotericin B. At 32 µg/ml of amphotericin B, GZY803 haploid biofilms showed 0.1% of persister population as compared to 1% of the diploid biofilms. AHP1 expression was found to be lower in GZY803 biofilms, and AHP1 overexpression in GZY803 restored the percentage of persister population. Consistently, deleting AHP1 in the diploid strain BWP17 caused a similar increase in amphotericin B susceptibility. AHP1 expression was also positively correlated with the antioxidant potential. Furthermore, C. albicans ira2Δ/Δ biofilms were susceptible to amphotericin B and had a diminished antioxidant capacity. Interestingly, AHP1 overexpression in the ira2Δ/Δ strain restored the antioxidant potential and enhanced the persister population against amphotericin B, and shutting down the AHP1 expression in ira2Δ/Δ biofilms reversed the effect. In conclusion, we provide evidence that the AHP1 gene critically determines the amphotericin B tolerance of C. albicans biofilms possibly by maintaining the persisters' antioxidant capacity. This finding will open up new avenues for developing therapies targeting the persister population of C. albicans biofilms. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD004274.
- Identification of 2-oxohistidine interacting proteins using E. coli proteome chips. [Journal Article]
- Mol Cell Proteomics 2016 Sep 19MC
- Cellular proteins are constantly damaged by reactive oxygen species generated by cellular respiration. Due to its metal-chelating property, the histidine residue is easily oxidized in the presence of...
Cellular proteins are constantly damaged by reactive oxygen species generated by cellular respiration. Due to its metal-chelating property, the histidine residue is easily oxidized in the presence of Cu/Fe ions and H2O2 via metal-catalyzed oxidation, usually converted to 2-oxohistidine. We hypothesized that cells may have evolved antioxidant defenses against the generation of 2-oxohistidine residues on proteins, and therefore there would be cellular proteins which specifically interact with this oxidized side chain. Using two chemically synthesized peptide probes containing 2-oxohistidine, high-throughput interactome screening was conducted using the E. coli K12 proteome microarray containing >4200 proteins. Ten interacting proteins were successfully validated using a third peptide probe, fluorescence polarization assays, as well as binding constant measurements. We discovered that 9 out of 10 identified proteins seemed to be involved in redox-related cellular functions. We also built the functional interaction network to reveal their interacting proteins. The network showed that our interacting proteins were enriched in oxido-reduction processes, ion binding, and carbon metabolism. A consensus motif was identified among these 10 bacterial interacting proteins based on bioinformatic analysis, which also appeared to be present on human S100A1 protein. Besides, we found that the consensus binding motif among our identified proteins, including bacteria and human, were located within α-helices and faced the outside of proteins. The combination of chemically engineered peptide probes with proteome microarrays proves to be an efficient discovery platform for protein interactomes of unusual post-translational modifications, and sensitive enough to detect even the insertion of a single oxygen atom in this case.
- Dynamic protein interactions of the Polycomb Repressive Complex 2 during differentiation of pluripotent cells. [Journal Article]
- Mol Cell Proteomics 2016 Sep 15MC
- Polycomb proteins assemble to form complexes with important roles in epigenetic regulation. The Polycomb Repressive Complex 2 (PRC2) modulates the di- and tri-methylation of lysine 27 on histone H3, ...
Polycomb proteins assemble to form complexes with important roles in epigenetic regulation. The Polycomb Repressive Complex 2 (PRC2) modulates the di- and tri-methylation of lysine 27 on histone H3, each of which are associated with gene repression. Although three subunits, EZH1/2, SUZ12 and EED, form the catalytic core of PRC2, a wider group of proteins associate with low stoichiometry. This raises the question of whether dynamic variation of the PRC2 interactome results in alternative forms of the complex during differentiation. Here we compared the physical interactions of PRC2 in undifferentiated and differentiated states of NTERA2 pluripotent embryonic carcinoma cells. Label-free quantitative proteomics was used to assess endogenous immunoprecipitation of the EZH2 and SUZ12 subunits of PRC2. A high stringency dataset reflecting the endogenous state of PRC2 was produced that included all previously reported core and associated PRC2 components, and several novel interacting proteins. Comparison of the interactomes obtained in undifferentiated and differentiated cells revealed candidate proteins that were enriched in complexes isolated from one of the two states. For example, SALL4 and ZNF281 associate with PRC2 in pluripotent cells, while PCL1 and SMAD3 preferentially associate with PRC2 in differentiating cells. Analysis of the mRNA and protein levels of these factors revealed that their association with PRC2 correlated with their cell state-specific expression. Taken together, we propose that dynamic changes to the PRC2 interactome during differentiation may contribute to directing its activity during cell fate transitions.
- GAPP: a proteogenomic software for genome annotation and global profiling of posttranslational modifications in prokaryotes. [Journal Article]
- Mol Cell Proteomics 2016 Sep 14MC
- While the number of sequenced prokaryotic genomes is growing rapidly, experimentally verified annotation of prokaryotic genome remains patchy and challenging. To facilitate genome annotation efforts ...
While the number of sequenced prokaryotic genomes is growing rapidly, experimentally verified annotation of prokaryotic genome remains patchy and challenging. To facilitate genome annotation efforts for prokaryotes, we developed an open source software called GAPP for genome annotation and global profiling of posttranslational modifications (PTMs) in prokaryotes. With a single command, it provides a standard workflow to validate and refine predicted genetic models and discover diverse PTM events. We demonstrated the utility of GAPP using proteomic data from Helicobacter pylori, one of the major human pathogens that is responsible for many gastric diseases. Our results confirmed 84.9% of the existing predicted H. pylori proteins, identified 20 novel protein coding genes, and corrected four existing gene models with regard to translation initiation sites. In particular, GAPP revealed a large repertoire of PTMs using the same proteomic data and provided a rich resource that can be used to examine the functions of reversible modifications in this human pathogen. This software is a powerful tool for PTM genome annotation and global discovery and is applicable to any sequenced prokaryotic organism; we expect that it will become an integral part of ongoing genome annotation efforts for prokaryotes. GAPP is freely available at https://sourceforge.net/projects/gappproteogenomic/.
- Molecular profiling of phagocytic immune cells in Anopheles gambiae reveals integral roles for hemocytes in mosquito innate immunity. [Journal Article]
- Mol Cell Proteomics 2016 Sep 13MC
- The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are ...
The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are important components of the mosquito innate immune response, yet critical aspects of their biology have remained uncharacterized. Using a novel method of enrichment, we isolated phagocytic granulocytes and quantified their proteomes by mass spectrometry. The data demonstrate that phagocytosis, blood-feeding, and Plasmodium falciparum infection promote dramatic shifts in the proteomic profiles of An. gambiae granulocyte populations. Of interest, large numbers of immune proteins were induced in response to blood feeding alone, suggesting that granulocytes have an integral role in priming the mosquito immune system for pathogen challenge. In addition, we identify several granulocyte proteins with putative roles as membrane receptors, cell signaling, or immune components that when silenced, have either positive or negative effects on malaria parasite survival. Integrating existing hemocyte transcriptional profiles, we also compare differences in hemocyte transcript and protein expression to provide new insight into hemocyte gene regulation and discuss the potential that post-transcriptional regulation may be an important component of hemocyte gene expression. These data represent a significant advancement in mosquito hemocyte biology, providing the first comprehensive proteomic profiling of mosquito phagocytic granulocytes during homeostasis blood-feeding, and pathogen challenge. Together, these findings extend current knowledge to further illustrate the importance of hemocytes in shaping mosquito innate immunity and their principal role in defining malaria parasite survival in the mosquito host.
- Physiological response of Escherichia coli O157:H7 Sakai to dynamic changes in temperature and water activity as experienced during carcass chilling. [Journal Article]
- Mol Cell Proteomics 2016 Sep 11MC
- Enterohaemorrhagic Escherichia coli is a leading cause of foodborne illness, with the majority of cases linked to foods of bovine origin. Currently, no completely effective method for controlling thi...
Enterohaemorrhagic Escherichia coli is a leading cause of foodborne illness, with the majority of cases linked to foods of bovine origin. Currently, no completely effective method for controlling this pathogen during carcass processing exists. Understanding how this pathogen behaves under those stress conditions experienced on the carcass during chilling in cold air could offer opportunities for development or improvement of effective decontamination processes. Therefore, we studied the growth kinetics and physiological response of exponential phase E. coli O157:H7 Sakai cultures upon an abrupt downshift in temperature and water activity (from 35°C aw 0.993 to 14°C aw 0.967). A parallel Biolog study was conducted to follow the phenotypic responses to 190 carbon sources. Exposure of E. coli to combined cold and water activity stresses resulted in a complex pattern of population changes. This pattern could be divided into two main phases, including adaptation and re-growth phases, based on growth kinetics and clustering analyses. The transcriptomic and proteomic studies revealed that E. coli exhibited a window of cell susceptibility (i.e., weaknesses) during adaptation phase. This included apparent DNA damage, the down-regulation of molecular chaperones and proteins associated with responses to oxidative damage. However, E. coli also displayed a transient induction in the RpoE-controlled envelope stress response and activation of the master stress regulator RpoS and the Rcs phosphorelay system involved in colanic acid biosynthesis. Increased expression was observed for several genes and/or proteins involved in DNA repair, protein and peptide degradation, amino acid biosynthesis and carbohydrate catabolism and energy generation. Furthermore, the Biolog study revealed reduced carbon source utilisation during adaptation phase, indicating the disruption of energy-generating processes. This study provides insight into the physiological response of E. coli during exposure to combined cold and water activity stress, which could be exploited to enhance the microbiological safety of carcasses and related foods.
- Novel aquaporin regulatory mechanisms revealed by interactomics. [Journal Article]
- Mol Cell Proteomics 2016 Sep 8MC
- PIP1;2 and PIP2;1 are aquaporins which are highly expressed in roots and bring a major contribution to root water transport and its regulation by hormonal and abiotic factors. Interactions between ce...
PIP1;2 and PIP2;1 are aquaporins which are highly expressed in roots and bring a major contribution to root water transport and its regulation by hormonal and abiotic factors. Interactions between cellular proteins or with other macromolecules contribute to forming molecular machines. Proteins that molecularly interact with PIP1;2 and PIP2;1 were searched to get new insights into regulatory mechanisms of root water transport. For that, a immunopurification strategy coupled to protein identification and quantification by mass spectrometry (IP-MS) of PIPs was combined with data from the literature, to build thorough PIP1;2 and PIP2;1 interactomes, sharing about 400 interacting proteins. Such interactome revealed PIPs to behave as a platform for recruitment of a wide range of transport activities and provided novel insights into regulation of PIP cellular trafficking by osmotic and oxidative treatments. This work also pointed a role of lipid signaling in PIP function and enhanced our knowledge of protein kinases involved in PIP regulation. In particular we show that 2 members of the receptor-like kinase (RLK) family [RKL1 (At1g48480) and Feronia (At3g51550)] differentially modulate PIP activity through distinct molecular mechanisms. The overall work opens novel perspectives in understanding PIP regulatory mechanisms and their role in adjustment of plant water status.
- TNIP2 is a hub protein in the NF-&(kappa)B network with both protein and RNA mediated interactions. [Journal Article]
- Mol Cell Proteomics 2016 Sep 8MC
- The NF-κB family of transcription factors is pivotal in controlling cellular responses to environmental stresses; abnormal NF-κB signaling features in many autoimmune diseases and cancers. Several co...
The NF-κB family of transcription factors is pivotal in controlling cellular responses to environmental stresses; abnormal NF-κB signaling features in many autoimmune diseases and cancers. Several components of the NF-κB signaling pathway have been reported to interact with the protein TNIP2 (also known as ABIN2), and TNIP2 can both positively and negatively regulate NF-κB- dependent transcription of target genes. However, the function of TNIP2 remains elusive and the cellular machinery associating with TNIP2 has not been systematically defined. Here we first used a broad MudPIT/Halo AP-MS approach to map the network of proteins associated with the NF-κB transcription factors, and establish TNIP2 as an NF-κB network hub protein. We then combined AP-MS with biochemical approaches in a more focused study of truncated and mutated forms of TNIP2 to map protein associations with distinct regions of TNIP2. NF-κB interacted with the N-terminal region of TNIP2. A central region of TNIP2 interacted with the endosomal sorting complex ESCRT-I via its TSG101 subunit, a protein essential for HIV-1 budding, and a single point mutant in TNIP2 disrupted this interaction. The major gene ontology category for TNIP2 associated proteins was mRNA metabolism, and several of these associations, like KHDRBS1, were lost upon depletion of RNA. Given the major association of TNIP2 with mRNA metabolism proteins, we analyzed the RNA content of affinity purified TNIP2 using RNA-Seq. Surprisingly, a specific limited number of mRNAs was associated with TNIP2. These RNAs were enriched for transcription factor binding, transcription factor co-factor activity, and transcription regulator activity. They included mRNAs of genes in the Sin3A complex, the Mediator complex, JUN, HOXC6, and GATA2. Taken together, our findings suggest an expanded role for TNIP2, establishing a link between TNIP2, cellular transport machinery, and RNA transcript processing.
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- Multi-species identification of polymorphic peptide variants via propagation in spectral networks. [Journal Article]
- Mol Cell Proteomics 2016 Sep 8MC
- Peptide and protein identification remains challenging in organisms with poorly annotated or rapidly evolving genomes, as are commonly encountered in environmental or biofuels research. Such limitati...
Peptide and protein identification remains challenging in organisms with poorly annotated or rapidly evolving genomes, as are commonly encountered in environmental or biofuels research. Such limitations render tandem mass spectrometry (MS/MS) database search algorithms ineffective as they lack corresponding sequences required for peptide-spectrum matching. We address this challenge with the spectral networks approach to a) match spectra of orthologous peptides across multiple related species and then b) propagate peptide annotations from identified to unidentified spectra. We here present algorithms to assess the statistical significance of spectral alignments (Align-GF), reduce the impurity in spectral networks, and accurately estimate the error rate in propagated identifications. Analyzing three related Cyanothece species, a model organism for biohydrogen production, spectral networks identified peptides from highly divergent sequences from networks with dozens of variant peptides, including thousands of peptides in species lacking a sequenced genome. Our analysis further detected the presence of many novel putative peptides even in genomically characterized species, thus suggesting the possibility of gaps in our understanding of their proteomic and genomic expression. A web-based pipeline for spectral networks analysis is available at http://proteomics.ucsd.edu/software.