An In Vivo Metabolic Approach for Deciphering the Product Specificity of Glycerate Kinase Proves that Both E. coli’s Glycerate Kinases Generate 2-Phosphoglycerate

Apart from addressing humanity’s growing demand for fuels, pharmaceuticals, plastics and other value added chemicals, metabolic engineering of microbes can serve as a powerful tool to address questions concerning the characteristics of cellular metabolism. Along these lines, we developed an in vivo metabolic strategy that conclusively identifies the product specificity of glycerate kinase. By deleting E. coli’s phosphoglycerate mutases, we divide its central metabolism into an ‘upper’ and ’lower’ metabolism, each requiring its own carbon source for the bacterium to grow. Glycerate can serve to replace the upper or lower carbon source depending on the product of glycerate kinase. Using this strategy we show that while glycerate kinase from Arabidopsis thaliana produces 3-phosphoglycerate, both E. coli’s enzymes generate 2-phosphoglycerate. This strategy represents a general approach to decipher enzyme specificity under physiological conditions

HIV-1 gp41 and TCRα Trans-Membrane Domains Share a Motif Exploited by the HIV Virus to Modulate T-Cell Proliferation

Viruses have evolved several strategies to modify cellular processes and evade the immune response in order to successfully infect, replicate, and persist in the host. By utilizing in-silico testing of a transmembrane sequence library derived from virus protein sequences, we have pin-pointed a nine amino-acid motif shared by a group of different viruses; this motif resembles the transmembrane domain of the α-subunit of the T-cell receptor (TCRα). The most striking similarity was found within the immunodeficiency virus (SIV and HIV) glycoprotein 41 TMD (gp41 TMD). Previous studies have shown that stable interactions between TCRα and CD3 are localized to this nine amino acid motif within TCRα, and a peptide derived from it (TCRα TMD, GLRILLLKV) interfered and intervened in the TCR function when added exogenously. We now report that the gp41 TMD peptide co-localizes with CD3 within the TCR complex and inhibits T cell proliferation in vitro. However, the inhibitory mechanism of gp41 TMD differs from that of the TCRα TMD and also from the other two known immunosuppressive regions within gp41

Design principles of autocatalytic cycles constrain enzyme kinetics and force low substrate saturation at flux branch points

A set of chemical reactions that require a metabolite to synthesize more of that metabolite is an autocatalytic cycle. Here, we show that most of the reactions in the core of central carbon metabolism are part of compact autocatalytic cycles. Such metabolic designs must meet specific conditions to support stable fluxes, hence avoiding depletion of intermediate metabolites. As such, they are subjected to constraints that may seem counter-intuitive: the enzymes of branch reactions out of the cycle must be overexpressed and the affinity of these enzymes to their substrates must be relatively weak. We use recent quantitative proteomics and fluxomics measurements to show that the above conditions hold for functioning cycles in central carbon metabolism of E. coli. This work demonstrates that the topology of a metabolic network can shape kinetic parameters of enzymes and lead to seemingly wasteful enzyme usage.ISSN:2050-084

Gp41 TMD inhibits T-cell proliferation <i>in vitro</i>.

<p>T-cells were activated with the MOG 35–55 peptide and APC in the presence of gp41 TMD (black) or FP_5–13 AAAA as control peptide (grey) in three different concentrations (50, 25 and 5 µg/ml) and the proliferative responses were assayed. The data are presented as mean inhibitions+SD (<i>n</i> = 4 or more, ** <i>p</i><0.01). The uninhibited T-cell proliferative responses were 9382±891 cpm. The background proliferation in the absence of antigen was 232±36 cpm.</p

HIV-1 gp41 extra-cellular domain.

<p>A scheme showing the functional regions within the extra-cellular portion of gp41 (aa 512–684).</p

Gp41 TMD does not inhibit T-cell activation induced by PMA/ionomycin.

<p>T cells were stimulated with PMA/ionomycin or in the presence of gp41 TMD or TCRα CP, and T cell proliferation was studied. The uninhibited T-cell proliferative responses were 12220±871 cpm. The background proliferation in the absence of antigen was 246±38 cpm. The data are presented as normalized mean proliferation percentage (control, no peptide = 100%)+SD (<i>n</i> = 5 or more). There was no significant statistical difference between all groups.</p

Gp41 TMD co-localizes with CD3 within the TCR complex.

<p>Gp41 TMD-NBD and AMP-NBD were used to study peptide binding to the membranes of resting and activated T cells in combination with Sy5-labeled antibodies to CD3. The T cells had been activated by incubation with APC and the MOG 35–55 peptide. (<b>A</b>) Distribution of gp41 TMD-NBD, AMP-NBD and CD3 molecules in resting and activated T cells. (<b>B</b>) Co-localization of gp41 TMD-NBD with the CD3 molecules. (<b>C</b>) Co-localization of AMP-NBD with the CD3 molecules.</p