Studies on interactions of Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) with anti apoptotic protein Bcl-2.

Bcl-2 regulates apoptosis by controlling luminal Ca2+ concentration of endoplasmic reticulum (ER). Dremina et al. reported that Bcl-2 interacts with SERCA, the Ca2+ pump in SR/ER membrane, causing inactivation and translocation. This work reports the characteristics of the SERCA/Bcl-2 interactions using wild type and three mutants, G145E, S24C/C158S and S205C/C158S, of the truncated protein, Bcl-2Δ21. Protein cross-linking, Ca2+-ATPase activity assay, Sucrose Density Gradient fractionation, immunoprecipitation and the fusion protein binding assay are the approaches used. Results reveal that the two proteins can interact with both 1:1 and 2:1 (Bcl-2Δ21: SERCA) molar ratios. The hydrophobic groove of Bcl-2Δ21 is involved in the interactions. The BH1 domain of Bcl-2Δ21 interacts with the ATP binding domain of SERCA. The G145E mutant is a loss-of-function whereas the two Cys-mutants are gain-of-function on SERCA inactivation and translocation. Therefore the conserved residue G145 is a critical hot spot for the Bcl-2Δ21-mediated inactivation and translocation of SERCA

Methods development for Analysis of Partially Deglycosylated Proteins and Application to an HIV Envelope Protein Vaccine Candidate

The work presented herein describes the first comprehensive analysis of a partially deglycosylated HIV vaccine candidate envelope protein (Env). The Env, JRFL gp140 ΔCF, with 27 potential glycosylation sites, was partially deglycosylated with PNGase F as part of a strategy to generate a more immunogenic HIV vaccine, and the resulting protein’s glycosylation was characterized in a unique workflow using two different glycosidases, Endo H and Endo F3. This unique analysis protocol provided for coverage on 26 of the 27 glycosylation sites, and the data showed that the biochemical treatment with PNGase F resulted in a highly heterogeneous glycoprotein product that had been partially deglycosylated at most of the glycosylation sites. The protocols described in this work could be useful for characterizing the glycosylation site occupancy of other native or biochemically deglycosylated proteins

Characterization of Glycosylation Profiles of HIV-1 Transmitted/Founder Envelopes by Mass Spectrometry

This is the published version, also available here: http://dx.doi.org/10.1128/JVI.05053-11.The analysis of HIV-1 envelope carbohydrates is critical to understanding their roles in HIV-1 transmission as well as in binding of envelope to HIV-1 antibodies. However, direct analysis of protein glycosylation by glycopeptide-based mass mapping approaches involves structural simplification of proteins with the use of a protease followed by an isolation and/or enrichment step before mass analysis. The successful completion of glycosylation analysis is still a major analytical challenge due to the complexity of samples, wide dynamic range of glycopeptide concentrations, and glycosylation heterogeneity. Here, we use a novel experimental workflow that includes an up-front complete or partial enzymatic deglycosylation step before trypsin digestion to characterize the glycosylation patterns and maximize the glycosylation coverage of two recombinant HIV-1 transmitted/founder envelope oligomers derived from clade B and C viruses isolated from acute infection and expressed in 293T cells. Our results show that both transmitted/founder Envs had similar degrees of glycosylation site occupancy as well as similar glycan profiles. Compared to 293T-derived recombinant Envs from viruses isolated from chronic HIV-1, transmitted/founder Envs displayed marked differences in their glycosylation site occupancies and in their amounts of complex glycans. Our analysis reveals that the glycosylation patterns of transmitted/founder Envs from two different clades (B and C) are more similar to each other than they are to the glycosylation patterns of chronic HIV-1 Envs derived from their own clades