Cell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins.

ClpB of Escherichia coli is an ATP-dependent ringforming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the Hsp100/Clp subfamily of AAA؉ proteins and is composed of an N-terminal domain and two AAA-domains that are separated by a "linker" region. Here we present a detailed structurefunction analysis of ClpB, dissecting the individual roles of ClpB domains and conserved motifs in oligomerization, ATP hydrolysis, and chaperone activity. Our results show that ClpB oligomerization is strictly dependent on the presence of the C-terminal domain of the second AAA-domain, while ATP binding to the first AAAdomains stabilized the ClpB oligomer. Analysis of mutants of conserved residues in Walker A and B and sensor 2 motifs revealed that both AAA-domains contribute to the basal ATPase activity of ClpB and communicate in a complex manner. Chaperone activity strictly depends on ClpB oligomerization and the presence of a residual ATPase activity. The N-domain is dispensable for oligomerization and for the disaggregating activity in vitro and in vivo. In contrast the presence of the linker region, although not involved in oligomerization, is essential for ClpB chaperone activity.

The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.

TorsinA is a membrane-associated AAA+ (ATPases associated with a variety of cellular activities) ATPase implicated in primary dystonia, an autosomal-dominant movement disorder. We reconstituted TorsinA and its cofactors in vitro and show that TorsinA does not display ATPase activity in isolation; ATP hydrolysis is induced upon association with LAP1 and LULL1, type II transmembrane proteins residing in the nuclear envelope and endoplasmic reticulum. This interaction requires TorsinA to be in the ATP-bound state, and can be attributed to the luminal domains of LAP1 and LULL1. This ATPase activator function controls the activities of other members of the Torsin family in distinct fashion, leading to an acceleration of the hydrolysis step by up to two orders of magnitude. The dystonia-causing mutant of TorsinA is defective in this activation mechanism, suggesting a loss-of-function mechanism for this congenital disorder.DYT1 dystonia | LINC complex | nuclear egress

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that bind denatured proteins in vitro, thereby facilitating their subsequent refolding by ATP-dependent chaperones. The mechanistic basis of this refolding process is poorly defined. We demonstrate that substrates complexed to sHsps from various sources are not released spontaneously. Dissociation and refolding of sHsp bound substrates relies on a disaggregation reaction mediated by the DnaK system, or, more efficiently, by ClpB/DnaK. While the DnaK system alone works for small, soluble sHsp/substrate complexes, ClpB/DnaK-mediated protein refolding is fastest for large, insoluble protein aggregates with incorporated sHsps. Such conditions reflect the situation in vivo, where sHsps are usually associated with insoluble proteins during heat stress. We therefore propose that sHsp function in cellular protein quality control is to promote rapid resolubilization of aggregated proteins, formed upon severe heat stress, by DnaK or ClpB/DnaK.

Summary YOD1 is a highly conserved deubiquitinating enzyme of the ovarian tumor (otubain) family, whose function has yet to be assigned in mammalian cells. YOD1 is a constituent of a multiprotein complex with p97 as its nucleus, suggesting a functional link to a pathway responsible for the dislocation of misfolded proteins from the endoplasmic reticulum. Expression of a YOD1 variant deprived of its deubiquitinating activity imposes a halt on the dislocation reaction, as judged by the stabilization of various dislocation substrates. Accordingly, we observe an increase in polyubiquitinated dislocation intermediates in association with p97 in the cytosol. This dominant negative effect is dependent on the UBX and Zinc finger domains, appended to the N- and C-terminus of the catalytic otubain core domain, respectively. The assignment of a p97-associated ubiquitin processing function to YOD1 adds to our understanding of p97’s role in the dislocation process.

The addition of ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers to proteins serves to modulate function and is a key step in protein degradation, epigenetic modification and intracellular localization. Deubiquitinating enzymes and Ubl-specific proteases, the proteins responsible for the removal of Ub and Ubls, act as an additional level of control over the ubiquitin-proteasome system. Their conservation and widespread occurrence in eukaryotes, prokaryotes and viruses shows that these proteases constitute an essential class of enzymes. Here, we discuss how chemical tools, including activity-based probes and suicide inhibitors, have enabled (i) discovery of deubiquitinating enzymes, (ii) their functional profiling, crystallographic characterization and mechanistic classification and (iii) development of molecules for therapeutic purposes.

The largest tegument protein of herpes simplex virus 1 (HSV-1), UL36, contains a novel deubiquitinating activity embedded in it. All members of the Herpesviridae contain a homologue of HSV-1 UL36, the N-terminal segments of which show perfect conservation of those residues implicated in catalysis. For murine cytomegalovirus and Epstein-Barr virus, chosen as representatives of the beta-and gammaherpesvirus subfamilies, respectively, we here show that the homologous modules indeed display deubiquitinating activity in vitro. The conservation of this activity throughout all subfamilies is indicative of an important, if not essential, function.Modification of proteins by ubiquitin (Ub) plays a pivotal role in a multitude of cellular processes, including proteolysis, cell cycle control, receptor internalization, and sorting within the endo/lysosomal system (7,14,16). Ubiquitination is achieved by an enzymatic cascade comprising a Ub-activating enzyme (E1), several Ub-carrier proteins (E2s), and hundreds of Ub ligases (E3s). Ubiquitination can be reversed by several families of enzymes collectively designated deubiquitinating enzymes (DUBs) (1,15).A number of viruses have evolved strategies to manipulate the ubiquitination status of host cell proteins, both through conjugation and deconjugation (2,4,6,10,13). Recently, we reported the identification of a novel viral ubiquitin-specific protease (USP), UL36 USP , encoded by the herpes simplex virus 1 (HSV-1) genome (9). UL36USP is a polypeptide of approximately 420 amino acids (aa) carried within the N-terminal portion of UL36, the largest tegument protein (3,164 aa) of HSV-1. This activity was detected through the use of mechanism-based, activesite-directed probes and confirmed by expression in Escherichia coli of a corresponding fragment that cleaves ubiquitin-based substrates. UL36USP activity peaks at late stages of viral replication and appears to require proteolytic processing from fulllength UL36 (9). The N-terminal UL36 fragment is well conserved in alphaherpesviruses, and a low homology to corresponding genes of the betaherpesvirus and gammaherpesvirus subfamilies was apparent in sequence alignments, but with strict conservation of the proposed catalytic residues. DUB activity may therefore be well conserved across the herpesvirus family and, if this is proven to be correct, would suggest an important function for this type of activity.We therefore set out to investigate the possible DUB activity of two phylogenetically distant homologues of HSV-1 UL36 USP , each representing a different subfamily of the Herpesviridae. We chose UL36 homologues encoded by mouse cytomegalovirus (MCMV, M48) and Epstein-Barr virus (EBV, BPLF1) as representatives of the beta-and gammaherpesvirus subfamilies, respectively. In order to assess the degree of homology between UL36 from HSV-1 and its MCMV and EBV counterparts, a sequence alignment was generated, covering the first 336 aa (the numbering refers to HSV-1) of UL36 (Fig. 1). Overall, the homology to HSV-1 is rather low, with on...