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: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:Human immunodeficiency virus 1 (HIV-1) infects blood monocytes that cross the blood-brain barrier to the central nervous system inducing neuronal damage. This damage is prompted by the secretion of viral and neurotoxic factors by HIV-infected macrophages and can result in HIV associated neurocognitive disorders (HAND). One of these neurotoxic factors secreted by HIV-infected macrophages is cathepsin B (CATB), a lysosomal cysteine protease that plays an important role in neurodegeneration. CATB interacts with Serum Amyloid P component (SAPC) contributing to HIV-induced neurotoxicity. However, the neuronal apoptosis pathways triggered by CATB and SAPC remain unknown. We aimed to elucidate these pathways in neurons exposed to HIV-infected macrophage conditioned media (MCM) before and after inhibition of CATB or SAPC using Tandem Mass Tag (TMT) proteomics labeling. Based on significant fold change (FC) ≥ ǀ2ǀ and p-value < 0.05 criteria, a total of 10, 48 and 13 proteins were deregulated after inhibiting CATB, SAPC antibodies and the cathepsin B inhibitor CA-074, respectively. We found that antibodies against CATB and SAPC, as well as the CATB inhibitor CA-074 downregulated apoptosis related proteins. CATB, SAPC or apoptotic related proteins could become potential targets against HIV-induced neuronal degeneration.
Project description:Cross-linking mass spectrometry has developed into an important method to study protein structures and interactions. The in-solution cross-linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross-links obtained from co-occurring protein oligomers, complexes, or conformers. Here we developed a cross-linking workflow combining blue native PAGE with in-gel cross-linking mass spectrometry (IGX-MS). This workflow circumvents steps, such as buffer exchange and cross-linker concentration optimization. Additionally, IGX-MS enables the parallel analysis of co-occurring protein complexes using only small amounts of sample. Another benefit of IGX-MS observed by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of artificial over-length cross-links when compared to in-solution cross-linking. We next used IGX-MS to investigate the complement components C5, C6, and their hetero-dimeric C5b6 complex. The obtained cross-links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX-MS can be used to provide new insights into the initial stages of the terminal complement pathway.
Project description:Suppressor of Cytokine Signaling 1 (SOCS1) functions as a tumor suppressor in hepatocellular carcinoma by regulating cytokine, growth factor and other signaling pathways. The tumor suppressor functions of SOCS1 are mediated partly via promoting ubiquitination and proteasomal degradation of several signaling proteins. In this study, we used an unbiased approach to characterize SOCS1-mediated changes in the protein profile of hepatocytes. The murine HCC cell line Hepa1-6 transduced with wildtype SOCS1, an SH2 domain mutant or the empty lentiviral vector were grown in Stable Isotopic Labelling of Amino acids in Cell culture (SILAC) media. The cells were stimulated with hepatocyte growth factor or left at steady state. Following cell lysis, proteins were separated on SDS-PAGE gels and peptides extracted by in-gel trypsinization were analyzed by mass spectrometry. Differentially modulated proteins identified and quantified were subjected to pathway enrichment analysis. In total, 3440 proteins were identified in Hepa cells in the presence of SOCS1 at steady state, of which 181 proteins were significantly modulated by SOCS1. The SH2 domain mutation and HGF stimulation increased the number of differentially modulated proteins, which showed only a limited overlap with SOCS1-modulated proteins. Protein interaction network analysis revealed enrichment of SOCS1-modulated proteins within multiprotein complexes such as ubiquitin conjugating enzymes, proteasome, mRNA spliceosome, mRNA exosome and mitochondrial ribosome. Further analysis indicated SOCS1-dependent regulation of the UBE2D ubiquitin conjugating enzymes, which are implicated in growth factor receptor signaling, indirectly via an unknown protein. Given the ubiquitous and highly regulated induction of SOCS1 by diverse cellular stimuli, our findings suggest a fundamental role of SOCS1 in regulating large macromolecular complexes that are important for cellular homeostasis.
Project description:It is generally believed that human mature erythrocytes do not possess functional ribosomes, and therefore cannot synthesize proteins. However, this dogma is not consistent with the long life of mature erythrocytes in the circulatory system. The mass-spectrometry analysis was done on highly pure preparation of human mature erythrocytes to identify the proteome. Results of the analysis show that there is no contamination from other cell types. We also demonstrate translation by polysome profiling, metabolic labelling and RiboPuromycylation. RNA-seq and quantitative RT-PCR assays revealed that HBA (alpha globin) and HBB (beta globin) transcripts are selectively translated. RNA-seq and translatome analyses revealed the presence of all necessary translation factors and aminoacyl tRNA synthetases.