Background For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. and immunoprecipitation (IP) experiment respectively. Interacting domains of GAPDH and its associated proteins were assessed by molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in SDF-5 cancer cells was evaluated by measuring enzyme activity Circular dichroism (CD) spectroscopy IP and mass spectrometry analyses. Result Here we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 1?mM MG association of GAPDH with PKM2 or GPI is not perturbed but the enzymatic activity of GAPDH is reduced to 26.8?±?5?% in 3MC induced tumor and 57.8?±?2.3?% in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only and changes the secondary structure of the protein complex. Conclusion PKM2 may regulate the Rebaudioside C enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 Rebaudioside C and GPI. Association of GAPDH with PKM2 Rebaudioside C and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2172-x) contains supplementary material which is available to authorized users. Fig.?2a). We quantified the expression of each of the enzyme and Fig.?2b shows that expression of GPI is higher by 2.2?±?0.45 fold whereas as that GAPDH is lower by 1.8?±?0.22 in 3MC induced tumor compared with normal tissue. On the other hand PKM2 was not detectable in normal tissue. Christofk et al. [25] have recently shown that PKM2 but not PKM1 (another alternative spliced isoform of PKM) is advantageous for tumor cell growth and critical for tumorigenesis. We checked the expression of PKM1 in 3MC induced tumor tissue. Additional file 1: Figure S2 shows that PKM1 is detectable only in normal tissue but not in the 3MC induced tumor tissue suggesting that tumor tissue expresses only PKM2. Fig. 2 Expression profile of three enzymes in mouse normal and 3MC induced tumor tissues. a Lysates were subjected to immunoblot analysis using Rebaudioside C anti-PKM2 (panel 1) ?GPI (panel 2) ?GAPDH (panel 3) and β-tubulin (panel 4) antibodies. … Association of GAPDH with PKM2 and GPI in tumor cell was validated by immunoprecipitation assay. We immunoprecipitated GAPDH in normal and 3MC induced tumor tissue lysates using antibody against GAPDH and the precipitate was further probed with antibodies against PKM2 GPI and GAPDH. In Fig.?3a (and Additional file 1: Figure S3A-B) panels 1 and 2 show that both PKM2 and GPI are detectable in the immunoprecipitate of GAPDH antibody but not of mouse IgG in 3MC induced tumor tissue (molecular docking analysis. 3D structure of human GAPDH (PDB code: 1U8F chain O) was docked onto PKM2 (PDB code: 1ZJH chain A) and GPI (PDB code: 1JLH chain A) independently without providing any prior information to the docking programs. Top docking solutions from each programs ClusPro [28 29 PatchDock [30] and SwarmDock [31] were screened and pooled together for interface analysis. Figure?4 and Additional file 1: Figure S4 plot the overall and average frequencies of N or C terminal domain/residue involvement of GAPDH PKM2 and GPI proteins within the GAPDH-PKM2 (Fig.?4 and Additional file 1: Figure S4A-C) and GAPDH-GPI (Fig.?4 and Additional file 1: Figure S4D-F) docking complexes respectively. Frequencies of C terminal domain of GAPDH are significantly higher in GAPDH-PKM2 (Fig.?4b) and GAPDH-GPI (Fig.?4e) docking complexes advocating the role of C terminal part of GAPDH in.
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Human immunodeficiency pathogen-1 (HIV-1) envelope proteins (Env) and influenza hemagglutinin (HA)
Human immunodeficiency pathogen-1 (HIV-1) envelope proteins (Env) and influenza hemagglutinin (HA) will be the surface area glycoproteins in charge of viral admittance into web host cells the first step in the trojan life cycle essential to start infection. concentrate their identification on the websites of equivalent function between your two infections: the receptor binding site and membrane fusion equipment. Nevertheless some sites of identification are unique towards the trojan neutralized like the thick shield of oligomannose sugars on HIV-1 Env. These observations are talked about in the framework of structure-based style strategies to assist in vaccine style or advancement of antivirals. family members which has two copies of single-stranded RNA. HIV-1 includes a huge spherical morphology of around 120 nm (3) and electron microscopy research have uncovered that just 9 to 14 irregularly distributed copies from the Env proteins are present around the viral membrane in addition to other proteins acquired from your host cell membrane during budding (4 5 Env therefore represents the only viral component on the surface of HIV-1 that is accessible for mounting a humoral immune response. Env is usually expressed as a gp160 precursor and cleaved in the Golgi; the mature Env glycoprotein consists of two non-covalently Rebaudioside C associated subunits gp120 and gp41 which assemble into Rebaudioside C a trimer of heterodimers (6). Env mediates the attachment and fusion of the computer virus to CD4+ T cells which is the first step in HIV-1 contamination. Obtaining an atomic structure of the full HIV-1 Env trimer has continued to be challenging and so much elusive but electron microscopy studies have allowed molecular-level characterization of the trimeric protein. Overall HIV-1 Env adopts a mushroom-shaped structure with the gp120 variable loops 1 and 2 (V1/V2) at its apex and the gp41 trimer inserting in the membrane (5 7 Structural knowledge of the HIV-1 Env has been further enhanced by the atomic structures of individual components. Crystal structures of unliganded gp120 monomer cores (i.e. devoid of the V1/V2 loops the V3 loop and truncations at both the N- and C-termini) were recently reported and found to adopt an overall architecture similar to that previously decided for the gp120 primary in the Compact disc4-bound condition (15 16 Lately structural information over the gp120 V1/V2 loops became obtainable from an antibody-scaffold Rebaudioside C crystal complicated structure and uncovered that this part of gp120 can adopt a four antiparallel disulfide-linked β-strand topology (17). Although many crystal buildings from the gp41 post-fusion six-helix pack have been driven along with crystal and alternative buildings from the gp41 MPER in a variety of conditions the conformation that gp41 adopts in the pre-fusion condition when connected with gp120 continues to be to be completely elucidated. A recently available cryo-EM framework at ~9 ? quality of the cleaved soluble SOSIP trimer in complicated with antibody 17b being a co-receptor imitate has provided a tantalizing glance of a suggested activated intermediate where in fact the gp41 N-terminal helices are noticeable in this open up conformation (18). Entirely the many molecular envelopes of HIV-1 Env along with several atomic-level buildings of its subunits give a workable style of the overall framework (Fig. 1A). Fig. 1 HIV-1 Env and influenza HA series variability and glycosylation Membrane fusion as mediated by Env is normally a complex procedure that is just partially understood and continues to be extensively reviewed somewhere else (19 20 Quickly the most recognized view from the HIV-cell membrane fusion procedure includes a two-step model which involves first the connections between the Compact disc4 receptor and gp120 which in turn induces conformational adjustments that permit connections from the Compact disc4-gp120 organic with another cell surface area co-receptor (generally CXCR4 or CCR5). The binding between gp120 and co-receptor after that triggers additional conformational adjustments in the gp41 transmembrane subunit resulting in formation from the expanded coil-coil pre-fusion intermediate. At this time destabilization from the cell membrane is set up by membrane insertion from the N-terminal peptide of gp41. Finally gp41 forms Rabbit Polyclonal to CYSLTR2. a six-helix bundle that draws the host and viral membranes jointly. It is this process that commercially available peptide access inhibitors such as Enfurvitide block by inserting an α-helical peptide into a postulated five-helix package intermediate and thus prevent six-helix package formation (6 21 For the cell and viral membranes to be pulled together efficiently mutational analysis offers revealed the Rebaudioside C importance of the gp41 membrane proximal external region (MPER) a flexible hinge region between the helix package and the transmembrane (TM) anchor (25). It is still unclear how many practical Env.