The rejuvenating power of the Buena Vista Social Club

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes

The uncoupled ATPase activity of the ABC transporter BtuC2D2 leads to a hysteretic conformational change, conformational memory, and improved activity

ABC transporters comprise a large and ubiquitous family of proteins. From bacteria to man they translocate solutes at the expense of ATP hydrolysis. Unlike other enzymes that use ATP as an energy source, ABC transporters are notorious for having high levels of basal ATPase activity: they hydrolyze ATP also in the absence of their substrate. It is unknown what are the effects of such prolonged and constant activity on the stability and function of ABC transporters or any other enzyme. Here we report that prolonged ATP hydrolysis is beneficial to the ABC transporter BtuC2D2. Using ATPase assays, surface plasmon resonance interaction experiments, and transport assays we observe that the constantly active transporter remains stable and functional for much longer than the idle one. Remarkably, during extended activity the transporter undergoes a slow conformational change (hysteresis) and gradually attains a hyperactive state in which it is more active than it was to begin with. This phenomenon is different from stabilization of enzymes by ligand binding: the hyperactive state is only reached through ATP hydrolysis, and not ATP binding. BtuC2D2 displays a strong conformational memory for this excited state, and takes hours to return to its basal state after catalysis terminates

Mixed-linkage ubiquitin chains send mixed messages

SummaryResearch on ubiquitin (Ub) signaling has focused primarily on homogeneously linked polyUb. Although polyUb containing different linkages within the same chain exist, their structures and signaling properties are unknown. These mixed-linkage chains could be unbranched (i.e., no more than one lysine or methionine linkage per Ub) or branched. Here, we examined the structure, dynamics, receptor selectivity, and disassembly of branched and unbranched tri-Ub containing both K48 and K63 linkages. Each linkage was virtually indistinguishable from its counterpart in homogeneously linked polyUb. Linkage-selective receptors from hHR23A and Rap80 preferentially bound to the K48 or K63 linkages in the branched trimer. Linkage-selective deubiquitinases specifically cleaved their cognate Ub-Ub linkages in mixed-linkage chains, and the 26S proteasome recognized and processed branched tri-Ub. We conclude that mixed-linkage chains retain the distinctive signaling properties of their K48 and K63 components and that these multiple signals can be recognized by multiple linkage-specific receptors. Finally, we propose a new, comprehensive notation for Ub and Ub-like polymers

L-glutamine Induces Expression of Listeria monocytogenes Virulence Genes.

The high environmental adaptability of bacteria is contingent upon their ability to sense changes in their surroundings. Bacterial pathogen entry into host poses an abrupt and dramatic environmental change, during which successful pathogens gauge multiple parameters that signal host localization. The facultative human pathogen Listeria monocytogenes flourishes in soil, water and food, and in ~50 different animals, and serves as a model for intracellular infection. L. monocytogenes identifies host entry by sensing both physical (e.g., temperature) and chemical (e.g., metabolite concentrations) factors. We report here that L-glutamine, an abundant nitrogen source in host serum and cells, serves as an environmental indicator and inducer of virulence gene expression. In contrast, ammonia, which is the most abundant nitrogen source in soil and water, fully supports growth, but fails to activate virulence gene transcription. We demonstrate that induction of virulence genes only occurs when the Listerial intracellular concentration of L-glutamine crosses a certain threshold, acting as an on/off switch: off when L-glutamine concentrations are below the threshold, and fully on when the threshold is crossed. To turn on the switch, L-glutamine must be present, and the L-glutamine high affinity ABC transporter, GlnPQ, must be active. Inactivation of GlnPQ led to complete arrest of L-glutamine uptake, reduced type I interferon response in infected macrophages, dramatic reduction in expression of virulence genes, and attenuated virulence in a mouse infection model. These results may explain observations made with other pathogens correlating nitrogen metabolism and virulence, and suggest that gauging of L-glutamine as a means of ascertaining host localization may be a general mechanism

Pathogenesis of Human Mitochondrial Diseases Is Modulated by Reduced Activity of the Ubiquitin/Proteasome System

Mitochondria maintain cellular homeostasis by coordinating ATP synthesis with metabolic activity, redox signaling, and apoptosis. Excessive levels of mitochondria-derived reactive oxygen species (ROS) promote mitochondrial dysfunction, triggering numerous metabolic disorders. However, the molecular basis for the harmful effects of excessive ROS formation is largely unknown. Here, we identify a link between mitochondrial stress and ubiquitin-dependent proteolysis, which supports cellular surveillance both in Caenorhabditis elegans and humans. Worms defective in respiration with elevated ROS levels are limited in turnover of a GFP-based substrate protein, demonstrating that mitochondrial stress affects the ubiquitin/proteasome system (UPS). Intriguingly, we observed similar proteolytic defects for disease-causing IVD and COX1 mutations associated with mitochondrial failure in humans. Together, these results identify a conserved link between mitochondrial metabolism and ubiquitin-dependent proteostasis. Reduced UPS activity during pathological conditions might potentiate disease progression and thus provides a valuable target for therapeutic intervention

Base-CP proteasome can serve as a platform for stepwise lid formation

Synopsis 26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP . A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11-m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11-Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes

The <i>LMRG_02270–1</i> operon is required for activation of the Type I interferon response during macrophage cell infection.

<p>(A) Schematic representation of the <i>LMRG_02270–1</i> operon, which contains fused transmembrane and substrate binding protein (SBP) domains and an ATPase that together form the complete transporter. The transposon insertion site and the E164A and R105A mutations are indicated. RT-qPCR analysis of transcription of IFN-β (B) or IL-6 (C) in BMDMs 6 h post infection with WT <i>L</i>. <i>monocytogenes</i> (WT) or Δ<i>LMRG_02270–1</i> mutant, are indicated. Transcription levels are represented as relative quantity (RQ), relative to uninfected cells. Data represents at least 3 biological repeats. Error bars represent 95% confidence interval. (D) Intracellular growth curves, described in Colony Forming Units (CFU), of WT <i>L</i>. <i>monocytogenes</i> (circles) or Δ<i>LMRG_02270–1</i> mutant (squares) in BMDM cells. Representative growth curves of 3 biological repeats are shown. Error bars represent standard deviation of triplicates.</p

The SBP domain of LMRG_02270 specifically binds L-glutamine.

<p>Isothermal titration calorimetry was used to determine the binding of (A) L-glutamine or (B) D-glutamine to the SBP domain, or (C) of L-glutamine to the R105A mutant of the SBP domain. Shown are the consecutive injections of 2 μL aliquots from a 500 μM solution of the indicated amino acid, into 200 μL of 50 μM SBP. The upper panels show the calorimetric titration and the lower panels display the integrated injection heat derived from the titrations, for which the best-fit curve was used to calculate the <i>K</i>_<i>D</i>. The experiments were conducted five times, and the <i>K</i>_<i>D</i> value is mean ±SD of 5 independent experiments.</p

The transcription and activity of virulence genes depends on L-glutamine.

<p>(A) RT-qPCR transcription analysis of <i>hly</i>, <i>plcA</i>, <i>plcB</i>, and <i>actA</i> in WT bacteria grown in MDM with either 4 mM L-glutamine (gray bars) or 4 mM NH₄Cl (black bars) as a sole nitrogen source. Transcription levels are presented as relative quantity (RQ), relative to levels in WT. Results are mean ±SD of 3 independent experiments performed in triplicates. (B) Hemolysis activity of LLO in culture supernatants of WT and <i>ΔglnPQ</i> bacteria grown on minimal defined media with 1 mM L-glutamine (grey) or 4 mM NH₄Cl (black) as the sole nitrogen source. (C) Analysis of PI-PLC activity of PlcA in culture supernatants of WT (circles) and <i>ΔglnPQ</i> (squares) bacteria grown on minimal defined media with 1 mM L-glutamine (full symbols) or 4 mM NH₄Cl (empty symbols) as the sole nitrogen source. Representative graphs of 3 biological repeats are shown. Error bars represent standard deviation of the triplicate.</p