Project description:O-linked β-N-acetylglucosamine (O-GlcNAc) functions as a nutrition rheostat to mediate cellular signaling pathways. It fluctuates in response to various nutritional factors, for instance, glucose. Previous investigations have shown that both glucose surplus and glucose deprivation upregulate O-GlcNAcylation levels. Meanwhile, starvation also activates autophagy, including chaperone-mediated autophagy (CMA). In the CMA pathway, heat shock cognate 70 kDa protein (HSC70) recognizes client proteins that bear a KFERQ pentapeptide motif, and delivers them to lysosome associated membrane protein type 2A (Lamp2a) on the lysosome, allowing lysosomal degradation of client proteins. Herein we show that Hsc70 interacts with O-GlcNAc transferase (OGT) and is O-GlcNAcylation, both of which are elevated when glucose is depleted. We further validated that Hsc70 O-GlcNAcylation occurs at T430, according to a previous chemoproteomic screen. T430 sits at the hydrophobic pocket of Hsc70. Moreover, we used label-free quantitative mass spectrometry to analyze HSC70-WT and HSC70-T430A interactome, identified a new CMA substrate, Ataxin-10, and mapped a potential KFERQ motif on Ataxin-10. In sum, our work suggests that CMA and O-GlcNAcylation intersect at HSC70, the CMA chaperone. HSC70 O-GlcNAcylation is essential to alter its client profile in response to nutrient availability.
Project description:In this study, we performed a ChIP-chip experiment to determine the respective regulons of RpoHI and RpoHII in Rhodobacter sphaeroides. We grew R. sphaeroides in aerobic conditions and induced either proteins ectopically and immuno-precipitated the regions of the genomic DNA interacting with the sigma factors. DNA immunoprecipitated with antibodies against RpoHI or RpoHII was labelled with Cy5 and hybridized on two-color tilling arrays (triplicates for each) with genomic DNA as an input control labelled with Cy3.
Project description:To identify the late endosome LE/MVB proteome related to the endosomal microautophagy eMI activity we purified subcellular organelles including cytosol, late endosomes (LE/MVBs) and lysosomes (CMA+) from the rat liver and performed co-Ip experiment with antibodies directed against the major cellular chaperones mediating CMA and eMI. As such, we analyzed Hsc70-interacting proteins in LE/MVB, CMA+ lysosomes and cytosol by performing pull-down experiments (co-IP) using anti-Hsc70 antibodies. A comparative analysis of the Hsc-70 interactomes revealed that proteins bound to Hsc70 in cytosol and LE/MVBs or in cytosol and CMA+ lysosomes were considered eMI or CMA substrates, respectively.
Project description:Using a three-pronged MS-based proteomic approach, we assessed the mode-of-action of four clinically-relevant tyrosine kinase inhibitors in an epithelial cancer cell line. Here we defined the: (I) semi-quantitative tyrosine kinome; (II) drug interactome; and (III) drug-induced effects on the tyrosine signalome. We first examined the entire proteome and kinome profile expressed in the A431 cell line, and determined the relative abundance of all possible protein targets of the tyrosine kinase inhibitors. We therefore utilized a ‘shot-gun’ approach that involved a multidimensional separation-based proteomic strategy. To ensure maximal protein identification, we reduced sample complexity by strong cation exchange (SCX) chromatography, and analyzed all fractions by LC-MS/MS. To determine the direct targets of the four selected TKIs, we subjected the proteins extracted from the A431 cancer cell line to drug-coupled affinity matrices. By comparing the pull-down results from a lysate with a second identical lysate to which 20 uM of the inhibitor had been added to block binding to the beads, we were able to identify selective binding interactors using LC-MS/MS. This differential affinity pull-downs approach allowed us to also quantitatively evaluate the binding affinity of the tyrosine kinases to each of the drugs. To evaluate the global tyrosine phosphorylation dynamics that occurs in A431 cells upon treatment with each of the four inhibitors, phosphotyrosine peptides were enriched and identified by quantitative mass spectrometry. For this purpose, we treated the cells with each inhibitor for 2 h at appropriate concentrations adopted from reported studies. Cells were lysed, and the proteins digested with the proteases Lys-C and trypsin. To allow quantitation of the phosphorylated peptides originating from the three different peptide pools (control/imatinib/dasatinib or control/bosutinib/nilotinib), we used stable isotope dimethyl labeling. Subsequently, tyrosine-phosphorylated peptides were enriched by immunoprecipitation and analyzed by LC-MS/MS.
Project description:HSC70 is the cytosolic isoform of plant HSP70. We have found that HSC70 family proteins bind to the heat shock transcription factor A1s (HsfA1s), which are the master regulators of the heat shock response in plants, and supress their activity. To investigate the role of HSC70s in the regulation of HsfA1s and heat shock responses, we evaluated the effect of triple knock out of HSC70s on the transcriptome under the normal growth condition.
Project description:HSC70 is the cytosolic isoform of plant HSP70. We have found that HSC70 family proteins bind to the heat shock transcription factor A1s (HsfA1s), which are the master regulators of the heat shock response in plants, and suppress their activity. We additionally found that the triple knock out of HSC70s alters responses of Arabidopsis plants to salt stress. To investigate the role of the HSC70s in salt stress responses, we evaluated the effects of the triple knock out on the transcriptome under salt stress.
Project description:There remains a need for analysis of CD4 helper T cells differentiation in vivo. To this end ovalbumin (OVA)-specific CD4 (OTII) T cells transferred into congenic mice were studied. Live attenuated OVA-expressing Salmonella (SalOVA) induce T-bet and IFN-g in OTII cells, while alum-precipitated OVA (alumOVA) induces GATA-3 and IL-4. Although 70% of alumOVA-responding OTII cells express GATA-3, only 7% produce IL-4. Thus Th2-polarization defined solely by IL-4 production does not recognize the diversity of GATA-3-expressing effectors. Low-density arrays were designed to assess the expression of 384 genes by real-time RT-PCR. Extensive early diversification occurred in both responses. SalOVA selectively induced many chemokines and pro-inflammatory cytokines, while alumOVA induced few Th2-associated cytokines. Several cytokines and molecules associated with Th17 cells and follicular helper cells were also induced by both antigens. The transcription factor Helios was exclusively induced in alumOVA-responding OTII cells, and critically not in standard in vitro Th2-polarization systems. Early synchronous up-regulation of Helios and GATA-3 mRNA is paralleled at protein level with largely coincident localization in specific nuclear foci of OTII cells responding to alumOVA. This appears to be consistent with a key role for both transcription regulators in the direction of Th2 responses in vivo. Keywords: In vivo T cell polarization Ovalbumin (OVA)-specific CD4 (OTII) T cells were transferred into C57BL/6 mice that were immunized either with live attenuated OVA-expressing Salmonella (Sal) or with alum-precipitated OVA (alum), or not (Naïve). Gene expression assay was performed on FACS sorted OTII cells (Naïve, Sal, Alum). OTII cells were purified from three independent groups of ten naïve, or SalOVA-immunized or alumOVA-immunized mice.
Project description:Anti-tick vaccines represent a promising alternative to chemical acaricides for the management of ticks on wildlife; however little progress has been made to produce an effective vaccine in hosts relevant to tick reproduction. Further, most of the antigens described are expressed during feeding in domesticated hosts and may not activate the immune system in wild hosts. We recently tested Amblyomma americanum salivary (SG) and midgut (MG) extracellular vesicles (EVs) as vaccine candidates in white-tailed deer (Odocoileus virginianus; WTD), which resulted in on-host female tick mortality. Herein, we describe our efforts to identify the antigenic proteins found within these vesicles. Within both SG- and MG-EVs, an enriched “core- cargo” was identified within both SG- and MG-EVs, including chaperones, small GTPases, and other proteins previously reported in small EVs was discovered. Label-free quantitative proteomics showed significant differences in protein cargo between MG and SG-EVs (333 proteins out of 516). Serum from three vaccinated WTD (pre-vaccinated and day 57 post vaccination) and one control WTD was used to immunoprecipitated antigenic proteins from SG- and MG-EV preparations. Proteomic analysis of immunoprecipitated proteins identified seven antigenic proteins in SG-EVs and one in MG-EVs that were statistically significantly different from the pre-vaccinated serum. Moreover, these proteins were unique to vaccinated animals (not precipitated by control serums). Additionally, two MG-EVs and 24 SG-EV proteins show antigenic potential. These proteins represent promising candidates for anti-tick vaccine design in WTD and other wildlife.
Project description:The heat shock response is a universal transcriptional response to proteotoxic stress orchestrated by heat shock transcription factor Hsf1 in all eukaryotic cells. Despite over 40 years of intense research, the mechanism of HSF1 activity regulation remains poorly understood at a molecular level. In metazoa Hsf1 trimerizes upon heat shock through a leucin-zipper domain and binds to DNA. How Hsf1 is dislodged from DNA and monomerized remained enigmatic. Here, using purified proteins we demonstrate that unmodified trimeric Hsf1 is dissociated from DNA in vitro by Hsc70 and DnaJB1. Hsc70 binds to multiple sites in Hsf1 with different affinities. Hsf1 trimers are monomerized by successive cycles of entropic pulling, unzipping the triple leucinezipper. Starting this unzipping at several protomers of the Hsf1 trimer results in faster monomerization. This process directly monitors the concentration of Hsc70 and DnaJB1. During heat shock adaptation Hsc70 first binds to a high affinity site in the transactivation domain leading to partial attenuation of the response and subsequently, at higher concentrations, Hsc70 removes Hsf1 from DNA to restore the resting state.
Project description:Human biology is tightly linked to proteins, yet most measurements do not precisely determine their full sequence and post-translational modifications. Here, we present the primary structures of 30,000 unique proteoforms expressed from 1,690 human genes across 21 cell types and plasma from human blood and bone marrow compiled in the Blood Proteoform Atlas (BPA). Our results indicate that while a given protein can be expressed across multiple cell types, the proteoform functions as a more specific indicator of differentiation. These results provide a better biochemical description of protein-level biology expressed through gene transcription and translation. We demonstrate the utility of the BPA by focusing on cell- and proteoform-specific signatures within 58 liver transplant recipients having healthy graft function or undergoing acute organ rejection or dysfunction.