Project description:Na+/H+ exchanger 1 (NHE1; SLC9A1) is a transport protein responsible for pH regulation in various types of cells. In cardiac myocytes, NHE1 generates the largest transmembrane acid-extrusion flux and is therefore the most significant controller of the intracellular acid/base milieu. The protein itself is regulated by various enzymes, including kinases. A number of critically important sites have been identified, coupling NHE1 to signalling cascades such as cAMP. NHE1 activity is very sensitive to oxygen tension, thus linking metabolism with pH. However, the sites implicated in this effect are not known. This study identified novel phosphorylation sites on NHE1. The state of these residues regulates NHE1 activity, hence pH and a myriad of pH sensitive downstream processes. Dysregulated NHE1 activity has been shown in diseases of the heart. Identification of novel regulatory sites can help demarcate the mechanisms of disease and suggest possible interventions to correct NHE1 activity.
Project description:Shellfish allergy is a significant public health concern, yet the molecular basis of allergenicity in molluscs remains poorly defined. In this study, we performed an in-depth proteomic and immunoinformatic analysis of two widely consumed blood clam species, Anadara broughtonii and Tegillarca granosa, to identify and characterize novel allergen candidates. Using high-resolution tandem mass spectrometry, we generated comprehensive protein profiles and applied three complementary allergen prediction algorithms (SEP-AlgPro™, AllerCatPro,and AlgPred 2.0) to screen for potential IgE-binding proteins. Immunoblotting with sera from shrimp-sensitized individuals confirmed IgE reactivity for nine proteins, including catalase and several hemoglobin isoforms—none of which are currently recognized as molluscan allergens. Homology modeling and sequence alignment revealed conserved structural motifsimplicated in allergenicity and suggested cross-reactivity with known crustacean allergens. These findings expand the repertoire of putative molluscan allergens and demonstrate theutility of integrated proteomic and immunoinformatic strategies for allergen discovery.
Project description:Purpose: Delonix regia or Gulmohor commonly grows here and there in Indian villages as well as it is used for megacity beautification and environmental management due to its evergreen nature and vibrant flower colour. However, an increasing incidence of seasonal pollinosis was observed among the inhabitants living in close vicinity to this tree suggesting a possible link between the airborne pollen load and the concomitant respiratory hazards. This prompted us to investigate the allergens in the pollen of this dominant avenue tree. Methodology: Allergenicity of D. regia pollen grains was first checked by Skin Prick Test (SPT) and further confirmed by in vitro tests, such as, ELISA, Dot Blot and histamine release assay. The total proteome profiling was done by 2D PAGE and it was confronted with the pooled sera of 10 patients. The IgE reactive proteins were identified by MALDI TOF/TOF in Autoflex speed (Bruker,Germany). The raw spectra were searched against NCBInr database using MASCOT search engine. Result: Delonix regia, pollen grains have been found in considerable amount in the air during its flowering season (May to July). Approximately 31% of atopic individuals were found allergic to D. regia, pollen with elevated level of specific IgE and histamine in the serum. Total 8 IgE reactive proteins have been identified by homology driven proteomics. These proteins are ATP synthase beta subunit (spot no 5), Actin (spot no 6), Hypothetical actin like protein (spot no 7), Recombinant S-adenosylmethionine synthase 2 (spot no 9), UDP-arabinopyranose mutase (spot no 13), Luminal-binding 5 (spot no 2), ELF3-like protein (spot no 8) and hypothetical protein OsJ_04810 (spot no 11). Among these eight identified allergenic proteins, five were previously reported as allergen from different sources whereas the rest three have been reported for the first time as novel allergens. Conclusion: Novelty of this study was to identify 8 allergens from D.regia for the first time using immune-biochemical and proteomic techniques. Further studies will open up new avenues in component resolved diagnosis of pollen allergy.
Project description:Shotgun proteomics focusing on the membrane proteomes, of four Synechococcus spp. strains namely CC9311 (clade I), CC9605 (Clade II), WH8102 (clade III) and CC9902 (clade IV) were conducted to gain insight into the amount of resources these unicellular organisms invest into adaptation strategies.
Project description:To characterize the nature of the cytochrome c1 (CYC1) processivity defect in native CIII2 assemblies we identified CYC1 peptides using mass spectrometry analysis of blue native (BN)-PAGE. Gel slices ranging from ~600-900kDa and containing CIII2 assemblies were excised from U2OS control cells, U2OS OCIAD1 knockdown cells, and U2OS OCIAD1 knockdown cells rescued with wildtype OCIAD1. Gel slices were then digested and analyzed by mass spectrometry.
Project description:Glaucoma is an optic neuropathy, the second leading cause of irreversible blindness worldwide. In our earlier studies, we demonstrated the isolation and functional efficacy (wound healing and anti-oxidant potential) of small extracellular vesicles (sEV) from the adult tissue-resident stem cells of the trabecular meshwork (TMSC). This study aimed to elucidate the protein profile of sEV derived from TMSCs to correlate these functional differences for a better understanding of their potential as a cell-free therapeutic agents for primary open angle glaucoma (POAG). TM and TMSC sEV were isolated by ultracentrifugation and characterized. The protein cargo of these sEV were analyzed by Mass spectrometry. Mass spectrometry analysis identified 2802 proteins in TMSC sEV and 2848 in TM sEV. Further Maxquant analysis revealed that 511 proteins were reproducibly quantified across the samples. and 18% of the proteins were significantly altered between TMSC and TM sEV. Differential expression analysis identified distinct protein profiles between TMSC and TM sEV. Notably, sEV from TMSCs were enriched with proteins associated with wound healing, cell proliferation, migration, anti-oxidant and anti-apoptotic activities, consistent with the findings in other mesenchymal stem cells. Pathway analysis highlighted the enrichment of proteins associated with PI3K-AKT and MAPK signaling pathways. Further validation by western blotting confirmed that TMSC sEV effectively modulated these pathways in TM cells, which are essential for cell proliferation and survival under oxidative stress. To our knowledge, this is the first comprehensive protein profiling of TMSC-derived sEV, demonstrating the presence of functional proteins and their capacity to regulate the MAPK and PI3K-AKT signaling pathways in the recipient TM cells. This highlights the potential of TMSC sEV in advancing cell-free therapeutic strategies for POAG in the future.
Project description:We here provide a comprehensive paired proteome and transcriptome data set including major stages (egg, larva, pupa and adult) across the whole developmental life cycle of the silkworm Bombyx mori. Our data constitutes a rich resource enabling comparative analysis of developmental regulatory dynamics. Analyzing this paired proteome and transcriptome data we revealed similarities and differences between both levels of gene expression in B. mori. We were also able to examine protein-transcript correlations and characterize stage-specific dynamics. Integrating similar publicly available data for D. melanogaster were also compared the two holometabolous insects at the level of the developmental proteome and transcriptome.
Project description:The intracellular O-linked N-acetylglucosamine (O-GlcNAc) modification is known to be enriched in the nucleus and in particular on chromatin, but many of its chromatin targets remain to be identified. Herein we demonstrate the O-GlcNAcylation of YEATS Domain Containing 2 (YEATS2), a subunit of the chromatin Ada-two-A-containing (ATAC) complex and a reader of histone H3K27ac. We show that YEATS2 interacts with the O-GlcNAc transferase (OGT) and further pinpoint its major O-GlcNAcylation site to be Thr604 using electron transfer dissociation mass spectrometry. O-GlcNAcylation promotes the chromatin association of YEATS2, and the affinity between YEATS2 and other ATAC components on chromatin, such as ZZZ3, GCN5 and PCAF. Downstream, YEATS2-T604A attenuated the ATAC-dependent histone H3K9ac levels and inactivated the expression of essential ribosomal genes as shown in chromatin immunoprecipitation assays. Further, xenograft experiments show that YEATS2 O-GlcNAcylation promotes lung cancer tumorigenesis. Our work reveals the critical role of YEATS2 O-GlcNAcylation in stabilizing the ATAC complex on chromatin and expands the chromatin substrates of OGT.