Exposure of cells to various stresses often leads to the induction of a group of proteins called heat shock proteins (HSPs, molecular chaperones). Hsp70 is one of the most highly inducible molecular chaperones, but its expression must be maintained at low levels under physiological conditions to permit constitutive cellular activities to proceed. Heat shock transcription factor 1 (HSF1) is the transcriptional regulator of HSP gene expression, but it remains poorly understood how newly synthesized HSPs return to basal levels when HSF1 activity is attenuated. CHIP (carboxy terminus of Hsp70-binding protein), a dual-function co-chaperone/ubiquitin ligase, targets a broad range of chaperone substrates for proteasomal degradation. Here we show that CHIP not only enhances Hsp70 induction during acute stress but also mediates its turnover during the stress recovery process. Central to this dual-phase regulation is its substrate dependence: CHIP preferentially ubiquitinates chaperone-bound substrates, whereas degradation of Hsp70 by CHIP-dependent targeting to the ubiquitin-proteasome system occurs when misfolded substrates have been depleted. The sequential catalysis of the CHIP-associated chaperone adaptor and its bound substrate provides an elegant mechanism for maintaining homeostasis by tuning chaperone levels appropriately to reflect the status of protein folding within the cytoplasm.

The cytoplasm is protected against the perils of protein misfolding by two mechanisms: molecular chaperones (which facilitate proper folding) and the ubiquitin-proteasome system, which regulates degradation of misfolded proteins. CHIP (carboxyl terminus of Hsp70-interacting protein) is an Hsp70-associated ubiquitin ligase that participates in this process by ubiquitylating misfolded proteins associated with cytoplasmic chaperones. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. Using a proteomics approach, we have identified BAG2, a previously uncharacterized BAG domain-containing protein, as a common component of CHIP holocomplexes in vivo. Binding assays indicate that BAG2 associates with CHIP as part of a ternary complex with Hsc70, and BAG2 colocalizes with CHIP under both quiescent conditions and after heat shock. In vitro and in vivo ubiquitylation assays indicate that BAG2 is an efficient and specific inhibitor of CHIP-dependent ubiquitin ligase activity. This activity is due, in part, to inhibition of interactions between CHIP and its cognate ubiquitin-conjugating enzyme, UbcH5a, which may in turn be facilitated by ATP-dependent remodeling of the BAG2-Hsc70-CHIP heterocomplex. The association of BAG2 with CHIP provides a cochaperone-dependent regulatory mechanism for preventing unregulated ubiquitylation of misfolded proteins by CHIP.Cell viability is constantly threatened by protein misfolding within the cytoplasm due to imprecise de novo protein folding and the consequences of oxidative and thermal stresses and conformational chain reaction events that affect protein structure. Because of the cellular inefficiencies and toxicities associated with off-pathway protein conformations, tightly regulated systems have evolved to minimize protein misfolding. The molecular chaperones constitute one component of the cytoplasmic protein quality control process. These proteins (including Hsp70, Hsp90, TRiC, and other associated proteins) assist in cotranslational folding, maintain metastable protein conformations, and repair proteins that are structurally defective. The molecular regulation and coordination of cytoplasmic folding and refolding are becoming increasingly clear (1).In addition to promoting proper folding, a second requirement of protein quality control mechanisms is the efficient removal of proteins that are irreversibly damaged or extremely toxic. Degradation of misfolded proteins occurs predominantly through the ubiquitin-proteasome system. For proteins that are misfolded within the endoplasmic reticulum, the protein degradation pathways are well described (2). Less is known about degradation of misfolded proteins within the cytoplasm. Recently, the co-chaperone/ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) has been implicated in the degradation of a variety of chaperone-bound cytoplasmic proteins (3, 4). CHIP inhibits the ATPase activity of Hsp70 (5) and has U box-dependent ubiquitin ligaseactivitythattargetsarangeofchaperonesubstratesforp...

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Balanced protein synthesis and degradation are crucial for proper cellular function. Protein synthesis is tightly coupled to energy status and nutrient levels by the mammalian target of rapamycin complex 1 (mTORC1). Quality of newly synthesized polypeptides is maintained by the molecular chaperone and ubiquitin-proteasome systems. Little is known about how cells integrate information about the quantity and quality of translational products simultaneously. We demonstrate that cells distinguish moderate reductions in protein quality from severe protein misfolding using molecular chaperones to differentially regulate mTORC1 signaling. Moderate reduction of chaperone availability enhances mTORC1 signaling, whereas stress-induced complete depletion of chaperoning capacity suppresses mTORC1 signaling. Molecular chaperones regulate mTORC1 assembly in coordination with nutrient availability. This mechanism enables mTORC1 to rapidly detect and respond to environmental cues while also sensing intracellular protein misfolding. The tight linkage between protein quality and quantity control provides a plausible mechanism coupling protein misfolding with metabolic dyshomeostasis.The eukaryotic translational initiation machinery is a tightly controlled system that regulates protein synthesis based on many factors including the availability of growth factors, nutrients, and glucose (1). Accordingly, when there are shortages of these factors, protein synthesis stalls. The molecular pathway responsible for linking the environmental cues and translational control is the mammalian target of rapamycin (mTOR) 3 signaling pathway (2, 3). mTOR is an evolutionarily conserved serine/threonine kinase that links environmental status with a host of other cellular processes such as cellular growth, proliferation, metabolism, autophagy, and translational control (4). mTOR is present in two functionally and structurally distinct multiprotein complexes termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (5). mTORC1 consists of mTOR, Raptor, PRAS40, and mLST8 and is sensitive to rapamycin (6, 7). mTORC2 contains mTOR, Rictor, mSIN1, and mLST8 and is not directly inhibited by rapamycin (8). The pivotal role of mTOR in cellular and organismal homeostasis is reflected in the fact that dysregulation of this pathway results in unrestrained signaling activity in mammals and is associated with the occurrence of disease states including inflammation, cancer, and diabetes (9).A common theme underlying a variety of stress conditions is the accumulation of misfolded proteins in cells. In response to stress, or heat shock, cells increase the expression of molecular chaperones. Molecular chaperones are essential for protecting nascent polypeptide chains from misfolding, facilitating co-and posttranslational folding, assisting in assembly and disassembly of macromolecular complexes, and regulating translocation (10, 11). The concentration of molecular chaperones is titrated closely to the folding requirements within a specific cell type (12), and it ha...

Protein ubiquitination regulates numerous cellular functions in eukaryotes. The prevailing view about the role of RING or U-box ubiquitin ligases (E3) is to provide precise positioning between the attached substrate and the ubiquitin-conjugating enzyme (E2). However, the mechanism of ubiquitin transfer remains obscure. Using the carboxyl terminus of Hsc70-interacting protein as a model E3, we show herein that although U-box binding is required, it is not sufficient to trigger the transfer of ubiquitin onto target substrates. Furthermore, additional regions of the E3 protein that have no direct contact with E2 play critical roles in mediating ubiquitin transfer from E2 to attached substrates. By combining computational structure modeling and protein engineering approaches, we uncovered a conformational flexibility of E3 that is required for substrate ubiquitination. Using an engineered version of the carboxyl terminus of Hsc70-interacting protein ubiquitin ligase as a research tool, we demonstrate a striking flexibility of ubiquitin conjugation that does not affect substrate specificity. Our results not only reveal conformational changes of E3 during ubiquitin transfer but also provide a promising approach to custom-made E3 for targeted proteolysis.Protein modification by ubiquitin and ubiquitin-like proteins is a common mechanism through which numerous cellular pathways are regulated (1). The canonical cascade of ubiquitination involves the action of three enzymes, termed E1, E2, and E3, which activate and then conjugate ubiquitin to its substrates (2, 3). The E3 ligase catalyzes the final step in ubiquitin transfer in a substrate-specific manner. Despite advances in understanding the enzymatic cascade of ubiquitination, the mechanism of ubiquitin transfer to the substrate remains an outstanding issue (4). In particular, the role of E3 ubiquitin ligases and how they adapt to progressively modified substrates to maintain specific ubiquitin chain topology is still a mystery.The known E3s belong to three protein families: HECT, RING, and U-box. HECT domain enzymes form a covalent intermediate with ubiquitin before the final transfer of ubiquitin to substrates. In contrast, RING and U-box E3s have been suggested to function as adaptors that position the substrate in close proximity to the E2-ubiquitin thioester (E2-Ub) (5). It has become common "wisdom" that the substrate has to be precisely positioned to get ubiquitinated (6). The positioning hypothesis originally predicted that E3 substrates would have a specific ubiquitination site. However, the absence of "consensus" ubiquitination sites has become apparent in an increasing list of E3 substrates (7-9). In addition, the crystal structures of several ubiquitination machinery components have revealed a puzzling gap (ϳ50 Å) between the substrate binding sites and the E2 active sites (10, 11). This raises a fundamental question in ubiquitin transfer. How does the ubiquitin molecule shuttle from the E2 to substrates? Though several interesting models for ubiquitin tra...