Unfolding or aggregation, that is the question.

Cellular processes accompanying protein aggregation are diverse and entangled, making it difficult to investigate the underlying molecular processes in a time-resolved way. Gottlieb, Thompson, and colleagues address this shortcoming using a chemical biology approach to monitor ubiquitination within the first 10 min after the initiation of protein aggregation. Intriguingly, unfolding rather than aggregation seems to trigger the observed events. This work might provide a method to answer open questions regarding the regulation of the proteostasis network upon protein misfolding.

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis.

Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed.

Protein feature analysis of heat shock induced ubiquitination sites reveals preferential modification site localization.

Protein aggregation is indicative of failing protein quality control systems. These systems are responsible for the refolding or degradation of aberrant and misfolded proteins. Heat stress can cause proteins to misfold, triggering cellular responses including a marked increase in the ubiquitination of proteins. This response has been characterized in yeast, however more studies are needed within mammalian cells. Herein, we examine proteins that become ubiquitinated during heat shock in human tissue culture cells using diGly enrichment coupled with mass spectrometry. A majority of these proteins are localized in the nucleus or cytosol. Proteins which are conjugated under stress display longer sequence lengths, more interaction partners, and more hydrophobic patches than controls but do not show lower melting temperatures. Furthermore, heat-induced conjugation sites occur less frequently in disordered regions and are closer to hydrophobic patches than other ubiquitination sites; perhaps providing novel insight into the molecular mechanism mediating this response. Nuclear and cytosolic pools of modified proteins appear to have different protein features. Using a pulse-SILAC approach, we found that both long-lived and newly-synthesized proteins are conjugated under stress. Modified long-lived proteins are predominately nuclear and were distinct from newly-synthesized proteins, indicating that different pathways may mediate the heat-induced increase of polyubiquitination. SIGNIFICANCE: The maintenance of protein homeostasis requires a balance of protein synthesis, folding, and degradation. Under stress conditions, the cell must rapidly adapt by increasing its folding capacity to eliminate aberrant proteins. A major pathway for proteolysis is mediated by the ubiquitin proteasome system. While increased ubiquitination after heat stress was observed over 30 years ago, it remains unclear which proteins are conjugated during heat shock in mammalian cells and by what means this conjugation occurs. In this study, we combined SILAC-based mass spectrometry with computational analyses to reveal features associated to proteins ubiquitinated while under heat shock. Interestingly, we found that conjugation sites induced by the stress are less often located within disordered regions and more often located near hydrophobic patches. Our study showcases how proteomics can reveal distinct feature associated to a cohort of proteins that are modified post translationally and how the ubiquitin conjugation sites are preferably selected in these conditions. Our work opens a new path for delineating the molecular mechanisms leading to the heat stress response and the regulation of protein homeostasis.

Molecular determinants of microbial resistance to thiopeptide antibiotics.

Ribosomally produced thiopeptide antibiotics are highly promising lead compounds targeting the GTPase-associated region (GAR) of the bacterial ribosome. A representative panel of GAR mutants suspected to confer resistance against thiopeptide antibiotics was reconstituted in vitro and quantitatively studied with fluorescent probes. It was found that single-site mutations of the ribosomal 23S rRNA binding site region directly affect thiopeptide affinity. Quantitative equilibrium binding data clearly identified A1067 as the base contributing most strongly to the binding environment. The P25 residue on the ribosomal protein L11 was essential for binding of the monocyclic thiopeptides micrococcin and promothiocin B, confirming that the mutation of this residue in the producer organism confers self-resistance. For the bicyclic thiopeptides thiostrepton and nosiheptide, all studied single-site resistance mutations on the L11 protein were still fully capable of ligand binding in the upper pM range, both in the RNA-protein complex and in isolated 70S ribosomes. These single-site mutants were then specifically reconstituted in Bacillus subtilis, confirming their efficacy as resistance-conferring. It is thus reasoned that, in contrast to modifications of the 23S rRNA in the GAR, mutations of the L11 protein do not counteract binding of bicyclic thiopeptides, but allow the ribosome to bypass the protein biosynthesis blockade enforced by these antibiotics in the wild type.

Structural Analysis of the Bacterial Proteasome Activator Bpa in Complex with the 20S Proteasome.

Mycobacterium tuberculosis harbors proteasomes that recruit substrates for degradation through an ubiquitin-like modification pathway. Recently, a non-ATPase activator termed Bpa (bacterial proteasome activator) was shown to support an alternate proteasomal degradation pathway. Here, we present the cryo-electron microscopy (cryo-EM) structure of Bpa in complex with the 20S core particle (CP). For docking into the cryo-EM density, we solved the X-ray structure of Bpa, showing that it forms tight four-helix bundles arranged into a 12-membered ring with a 40 Å wide central pore and the C-terminal helix of each protomer protruding from the ring. The Bpa model was fitted into the cryo-EM map of the Bpa-CP complex, revealing its architecture and striking symmetry mismatch. The Bpa-CP interface was resolved to 3.5 Å, showing the interactions between the C-terminal GQYL motif of Bpa and the proteasome α-rings. This docking mode is related to the one observed for eukaryotic activators with features specific to the bacterial complex.

Bacterial proteasome activator bpa (rv3780) is a novel ring-shaped interactor of the mycobacterial proteasome.

The occurrence of the proteasome in bacteria is limited to the phylum of actinobacteria, where it is maintained in parallel to the usual bacterial compartmentalizing proteases. The role it plays in these organisms is still not fully understood, but in the human pathogen Mycobacterium tuberculosis (Mtb) the proteasome supports persistence in the host. In complex with the ring-shaped ATPase Mpa (called ARC in other actinobacteria), the proteasome can degrade proteins that have been post-translationally modified with the prokaryotic ubiquitin-like protein Pup. Unlike for the eukaryotic proteasome core particle, no other bacterial proteasome interactors have been identified to date. Here we describe and characterize a novel bacterial proteasome activator of Mycobacterium tuberculosis we termed Bpa (Rv3780), using a combination of biochemical and biophysical methods. Bpa features a canonical C-terminal proteasome interaction motif referred to as the HbYX motif, and its orthologs are only found in those actinobacteria encoding the proteasomal subunits. Bpa can inhibit degradation of Pup-tagged substrates in vitro by competing with Mpa for association with the proteasome. Using negative-stain electron microscopy, we show that Bpa forms a ring-shaped homooligomer that can bind coaxially to the face of the proteasome cylinder. Interestingly, Bpa can stimulate the proteasomal degradation of the model substrate ß-casein, which suggests it could play a role in the removal of non-native or damaged proteins.