Hereditary inclusion body myopathy associated with early-onset Paget disease of bone and frontotemporal dementia (hIBMPFTD) is a degenerative disorder caused by single substitutions in highly conserved residues of p97/VCP. All mutations identified thus far cluster within the NH 2 domain or the D1 ring, which are both required for communicating conformational changes to adaptor protein complexes. In this study, biochemical approaches were used to identify the consequences of the mutations R155P and A232E on p97/VCP structure. Assessment of p97/VCP oligomerization revealed that p97 R155P and p97 A232E formed hexameric ring-shaped structures of ϳ600 kDa. p97 R155P and p97 A232E exhibited an ϳ3-fold increase in ATPase activity compared to wild-type p97 (p97 WT ) and displayed increased sensitivity to heat-induced upregulation of ATPase activity. Protein fluorescence analysis provided evidence for conformational differences in the D2 rings of both hIBMPFTD mutants. Furthermore, both mutations increased the proteolytic susceptibility of the D2 ring. The solution structures of all p97/VCP proteins revealed a didispersed distribution of a predominant hexameric population and a minor population of large-diameter complexes. ATP binding significantly increased the abundance of large-diameter complexes for p97 R155P and p97 A232E , but not p97 WT or the ATP-binding mutant p97 K524A . Therefore, we propose that hIBMPFTD p97/VCP mutants p97 R155P and p97 A232E possess structural defects that may compromise the mechanism of p97/VCP activity within large multiprotein complexes.The ubiquitous valosin-containing protein (p97/VCP) is a prominent member of the highly conserved AAA ϩ (ATPases associated with diverse cellular activities) proteins, which are known for their oligomeric structure and chaperone-like activities. p97/VCP is an essential biochemical component of a wide range of ubiquitin-linked cell biological reactions, including ubiquitin-proteasome system-mediated protein degradation (8), Golgi and endoplasmic reticulum (ER) membrane fusion (1, 18), transcription factor activation (19), and DNA repair (10, 16). In these processes, p97 acts as a molecular segregase that utilizes ATP-powered conformational changes in the assembly and disassembly of macromolecular machineries (8, 11).p97 ATPase activity is contingent on the assembly of an inherently stable hexamer (24) comprised of two highly homologous D1 and D2 nucleotide-binding rings and regulatory NH 2 -and COOH-terminal domains (4). ATP hydrolysis in the D2 ring mediates p97 major ATPase activity, while the D1 ring is involved in the regulation of p97 hexamerization (24). Recently, p97 has been linked to a severe degenerative disorder identified as hereditary inclusion body myopathy associated with early-onset Paget disease of bone and frontotemporal dementia (hIBMPFTD). The pathogenesis of hIBMPFTD is attributed to autosomal-dominant single-amino-acid substitutions in highly conserved residues within the p97 NH 2 domain and D1 ring (28). Patients present with hallma...

The multifunctional AAA-ATPase p97/VCP is one of the most extensively studied members of this protein family, yet it presents the field with many perplexing questions surrounding its mechanism of substrate engagement and processing. Recent discoveries have unmasked a new purgatorial identity for this molecule in the ubiquitin-proteasome pathway, specifically its role in linking ubiquitylated substrates with competing ubiquitin conjugation and deconjugation machineries. Furthermore, biochemical studies surprisingly identify the C-terminal D2 ring as essential for substrate interaction, thus bringing p97 one step closer to its prokaryotic AAA protease relatives.

Metabolic pathways contributing to adiposity and aging are activated by the mammalian target of rapamycin complex 1 (mTORC1) and p70 ribosomal protein S6 kinase 1 (S6K1) axis1–3. However, known mTORC1-S6K1 targets do not account for observed loss-of-function phenotypes, suggesting additional downstream effectors4–6. Here we identify glutamyl-prolyl tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes importantly to adiposity and aging. EPRS phosphorylation at Ser999 by mTORC1-S6K1 induces its release from the aminoacyl tRNA multisynthetase complex (MSC), required for execution of noncanonical functions beyond protein synthesis7,8. To investigate physiological function of EPRS phosphorylation, we generated EPRS knock-in mice bearing phospho-deficient Ser999-to-Ala (S999A) and phospho-mimetic (S999D) mutations. Homozygous S999A mice exhibited low body weight, reduced adipose tissue mass, and increased lifespan, thereby displaying notable similarities with S6K1-deficient mice9–11 and mice with adipocyte-specific deficiency of raptor, an mTORC1 constituent12. Substitution of the EPRS S999D allele in S6K1-deficient mice normalized body mass and adiposity, indicating EPRS phosphorylation mediates S6K1-dependent metabolic responses. In adipocytes, insulin stimulated S6K1-dependent EPRS phosphorylation and release from the MSC. Interaction screening revealed phospho-EPRS binds Slc27a1 (i.e., fatty acid transport protein 1, FATP1)13–15, inducing its translocation to the plasma membrane and long-chain fatty acid uptake. Thus, EPRS and FATP1 are terminal mTORC1-S6K1 axis effectors critical for metabolic phenotypes.

Background: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with β-TrCP, a component of the SCF β-TrCP E3 Ub ligase complex.

When endoplasmic reticulum (ER) homeostasis is perturbed, an adaptive mechanism is triggered and named the unfolded protein response (UPR). Thus far, three known UPR signaling branches (IRE-1, PERK, and ATF-6) mediate the reestablishment of ER functions but can also lead to apoptosis if ER stress is not alleviated. However, the understanding of the molecular mechanisms integrating the UPR to other ER functions, such as membrane traffic or endomembrane signaling, remains incomplete. We consequently sought to identify new regulators of UPR-dependent transcriptional mechanisms and focused on a family of proteins known to mediate, among other, ER-related functions: the small GTP-binding proteins of the RAS superfamily. To this end, we used transgenic UPR reporter Caenorhabditis elegans strains as a model to specifically silence small-GTPase expression. We show that the Rho subfamily member CRP-1 is an essential component of UPR-induced transcriptional events through its physical and genetic interactions with the AAA ؉ ATPase CDC-48. In addition, we describe a novel signaling module involving CRP-1 and CDC-48 which may directly link the UPR to DNA remodeling and transcription control.The endoplasmic reticulum (ER) is an organelle found in eukaryotic cells, which is mainly involved in calcium sequestration, lipid biosynthesis and translation, folding, and transport of secretory proteins (9). These functions require specialized and integrated molecular machines (7). Most of the proteins distributed in organelles of the secretory pathway, expressed at the cell surface or secreted, transit through the ER before reaching their final destination. Indeed, polypeptide chains, translated on ER membrane-bound ribosomes, are first translocated into the ER lumen via the translocon and then processed through the ER folding machineries, which include a chaperone component (e.g., BiP or GRP94), a posttranslational modification machinery (e.g., N glycosylation with the oligosaccharyl transferase complex, S-S bound formation with the protein disulfide isomerases), and a quality control component (e.g., calnexin, UDP-glucose:glycoprotein glucosyltransferase). Proteins which do not acquire a correct conformation are retained in the ER by ER-specific quality control mechanisms and are consequently granted further folding attempts. If this fails again, terminally misfolded proteins are degraded via the ER-associated degradation (ERAD) machinery (9).Under basal conditions, these integrated mechanisms maintain the ER protein load in equilibrium with ER's folding and export capacities. However, if one of those components is dysfunctional, the entire chain reaction is perturbed and ER homeostasis is disrupted. This leads to an increased amount of improperly folded proteins, which accumulate within the ER. As a mechanism for adaption to this phenomenon, cells have evolved the unfolded protein response (UPR), which aims at restoring ER homeostasis (38, 41) by (i) attenuating protein translation, (ii) increasing ER folding capacity, (iii) incre...