Background: Association of the proteasome core with activators regulates proteasome activity. Results: Blm10 association increases proteasome activity toward peptides and the unstructured proteasome substrate tau-441. This process is mediated by the C terminus of Blm10. Conclusion: C-terminal docking-mediated proteasome activation by Blm10 facilitates the turnover of peptide and protein substrates. Significance: Blm10 contributes to the regulation of proteasome activity.

The polycomb repressive complex (PRC) 1 protein Ring1B is an ubiquitin ligase that modifies nucleosomal histone H2A, a modification which plays a critical role in regulation of gene expression. We have shown that self-ubiquitination of Ring1B generates multiply branched, "noncanonical" polyubiquitin chains that do not target the ligase for degradation, but rather stimulate its activity toward histone H2A. This finding implies that Ring1B is targeted by a heterologous E3. In this study, we identified E6-AP (E6-associated protein) as a ligase that targets Ring1B for "canonical" ubiquitination and subsequent degradation. We further demonstrated that both the self-ubiquitination of Ring1B and its modification by E6-AP target the same lysines, suggesting that the fate of Ring1B is tightly regulated (e.g., activation vs. degradation) by the type of chains and the ligase that catalyzes their formation. As expected, inactivation of E6-AP affects downstream effectors: Ring1B and ubiquitinated H2A levels are increased accompanied by repressed expression of HoxB9, a PRC1 target gene. Consistent with these findings, E6-AP knockout mice display an elevated level of Ring1B and ubiquitinated histone H2A in various tissues, including cerebellar Purkinje neurons, which may have implications to the pathogenesis of Angelman syndrome, a neurodevelopmental disorder caused by deficiency of E6-AP in the brain.polycomb complexes | ubiquitin-proteasome system

Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests that Mtb relies mainly on fatty acid catabolism in the host. However, Mtb also maintains a functional glycolytic pathway and its role in the cellular metabolism of Mtb has yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and the Mtb genome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show that pykA encodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion of pykA prevents Mtb growth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the ␣-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism in Mtb.Mycobacterium tuberculosis (Mtb) 3 pathogenesis has been studied for decades, however, our knowledge concerning the metabolism and physiology of the bacterium during host infection is still limited (1-4). Specifically, we lack understanding of nutrient availability in the host microenvironments during the different phases of infection and consequently, which nutrients (e.g. carbon and nitrogen sources) are used by the bacterium for growth and maintenance of replicative and non-replicative states (5). In vitro evidence suggests that Mtb does not utilize carbon catabolite repression, a regulatory mechanism that allows bacteria and single-cell eukaryotes to gain growth advantage through prioritized metabolism of one carbon source over the other (6), but rather co-catabolizes multiple carbon sources at once (4). Several lines of evidence suggest that Mtb relies mainly on fatty acid metabolism in the non-replicative state within the host (1, 7-12). During growth on fatty acids Mtb bypasses the carbon dioxide releasing steps of the TCA cycle by running the glyoxylate shunt to conserve carbon (4). Nevertheless, Mtb maintains a functional and intact glycolytic pathway, suggesting that glycolysis plays a role under certain in vivo conditions. Recent studies with the adenosine triphosphate (ATP) synthase inhibitor, Bedaquiline, showed a delayed cidal effect when Mtb was grown on fermentable carbon sources (13), suggesting that Mtb can produce ATP through substrate level phosphorylation when oxidative-phosph...

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Acetyl-CoA synthetase (ACS) catalyzes the formation of AcCoA from acetate, ATP and Coenzyme A, allowing the organism to grow on acetate as the sole carbon source. ACS was the first enzyme in Mycobacterium tuberculosis shown to be regulated by posttranslational acetylation by the cAMP-dependent protein acetyltransferase. This modification results in the inactivation of the enzyme and can be reversed in the presence of NAD+ and a mycobacterial sirtuin-like deacetylase. In this study we characterize the kinetic mechanism of MtACS, where the overall reaction can be divided into two half-reactions: the acetyl-adenylate forming reaction and the thiol-ligation reaction. We also provide evidence for the existence of the acetyl-adenylate intermediate via 31P-NMR spectroscopy. Furthermore, we dissect the regulatory role of K617 acetylation and show that acetylation inhibits only the first, adenylation half-reaction while leaving the second half reaction unchanged. Finally, we demonstrate that the chemical mechanism of the enzyme relies on a conformational change which is controlled by the protonation state of aspartate 525. Together with our earlier results, this suggests a degree of regulation of enzyme activity that is appropriate for the role of the enzyme in central carbon metabolism.