Molecular chaperones: Inside and outside the Anfinsen cage

The GroEL/GroES chaperonin system acts as a passive anti-aggregation cage for refolding rubisco and rhodanese, and not as an active unfolding device. Refolding aconitase is too large to enter the cage but reversible binding to GroEL reduces its aggregration. Unexpectedly, confinement in the cage increases the rate of refolding of rubisco, but not rhodanese.     

Chaperoning osteogenesis: new protein-folding disease paradigms

Recent discoveries of severe bone disorders in patients with deficiencies in several endoplasmic reticulum chaperones are reshaping the discussion of type I collagen folding and related diseases. Type I collagen is the most abundant protein in all vertebrates and a crucial structural molecule for bone and other connective tissues. Its misfolding causes bone fragility, skeletal deformity and other tissue failures. Studies of newly discovered bone disorders indicate that collagen folding, chaperones involved in the folding process, cellular responses to misfolding and related bone pathologies might not follow conventional protein folding paradigms. In this review, we examine the features that distinguish collagen folding from that of other proteins and describe the findings that are beginning to reveal how cells manage collagen folding and misfolding. We discuss implications of these studies for general protein folding paradigms, unfolded protein response in cells and protein folding diseases.     

Chaperoning ribosome assembly

Reconstitution of bacterial ribosomes in vitro from RNA and protein constituents requires a heating step to rearrange conformation of an intermediate. In this issue of Molecular Cell, Maki et al. demonstrate that the DnaK chaperone system circumvents the requirement for heating.     

Chaperoning through the mitochondrial intermembrane space

The first high-resolution structure of a mitochondrial translocase complex, the Tim9-Tim10 chaperone, is reported by Webb et al. (2006) in a recent issue of Molecular Cell, providing important insight in the transport of hydrophobic proteins through the aqueous intermembrane space and the mechanisms of protein assembly.     

Chaperoning transmembrane helices in the lipid bilayer

Elimination of membrane proteins often requires recognition of their transmembrane domains (TMDs) in the lipid bilayer. In this issue, Arines et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.202001116) show that in Saccharomyces cerevisiae, the vacuole-associated Rsp5 ubiquitin ligase uses a TMD in substrate adaptor Ssh4 to recognize membrane helices in Ypq1, which targets this lysine transporter for lysosomal degradation during lysine starvation.

In eukaryotic cells, protein quality control (PQC) mediates the degradation of not only aberrant but also unwanted polypeptides, safeguarding both the quality and quantity of the cellular proteome (1). A central goal in PQC research is to delineate the mechanism of substrate selection, which, if inappropriately executed, could lead to undesired destruction of functional proteins and thus the collapse of the proteostasis network. For soluble proteins that succumb to PQC, it is usually the surface exposure of hydrophobic elements that alerts cellular chaperones to potential folding catastrophe (2). Chaperones often serve a dual triaging role: while giving their clients additional time to fold, they can also interface with degradation machineries such as the ubiquitin proteasome system or lysosomes, causing the elimination of terminally misfolded or unwanted polypeptides.Unlike PQC of soluble proteins, substrate recognition for membrane proteins bearing abnormal transmembrane domain (TMD) is largely unknown, even for the best characterized PQC process, ER-associated degradation (ERAD; 3). Early studies on PQC of unassembled T cell receptor α chain (TCRα) showed that the single TMD of TCRα contains two charged residues, which are thermodynamically unfavored in the lipid environment and thus must be shielded when TCRα assembles with CD3σ. Accordingly, unassembled TCRα is eliminated by ERAD via a mechanism dependent on these charged residues (4), but TMD-specific chaperones responsible for recognizing charged residues in the lipid bilayer have not been identified. Likewise, recent investigations into the function of the Hrd1 ubiquitin ligase suggested a role for the TMDs of Hrd1 in recognition of specific aberrant membrane proteins in ERAD (5). Cryo-EM studies further showed two juxtaposed central cavities with a lateral gate poised to receive TMDs in the yeast Hrd1 complex (6), but how aberrant TMDs in ERAD substrates are recognized and retrotranslocated by Hrd1 remains an open question.The issue of substrate recognition becomes even more complex for feedback-regulated degradation of unwanted membrane proteins. In this case, substrates are initially stable and functionally essential, but a change in environmental cues renders them dispensable and results in a short-lived fate. One such example is the sterol-regulated degradation of a sterol-synthesizing enzyme called HMG-CoA reductase (HMGR). HMGR is a stable ER protein when the sterol level is low, but an increase in membrane sterol abundance alters the conformation of a sterol-sensing domain in HMGR, exposing an element functionally equivalent to a degron in short-lived proteasomal substrates (7). Despite extensive studies, the molecular signature of the degron in HMGR is still undefined, let alone the molecular basis of its recognition. In this issue, Arines and colleagues investigate how Ypq1, a multi-spanning lysine transporter of the yeast vacuole, is regulated by lysine availability, a regulated membrane protein turnover event analogous to HMGR degradation. Their study identifies critical residues in Ypq1 TMDs for its turnover and establishes the Rsp5 ubiquitin ligase adaptor Ssh4 as a TMD-specific chaperone that recognizes these elements (8).Ypq1 is a seven-transmembrane, PQ loop–containing lysine transporter localized to the yeast vacuole membrane. Under lysine-replete conditions, Ypq1 is stable as it uses a PQ loop–dependent conformational cycle to import excess lysine into the vacuole. When lysine is depleted, Ypq1 is sorted into the multivesicular body (MVB) for degradation (Fig. 1). This process is initiated once Ypq1 is ubiquitinated by the ubiquitin ligase complex Rsp5–Ssh4, but how Ypq1 is targeted by Rsp5–Ssh4 has been unclear (9).Open in a separate windowFigure 1.Regulated recognition of Ypq1 by Ssh4. When lysine in the cytosol is abundant, Ypq1 undergoes a rapid conformational cycle to transport lysine from the cytosol into the vacuole lumen. Under lysine-depleted conditions, the transporter is trapped in a conformation recognizable by Ssh4, which recruits Rsp5 to catalyze Ypq1 ubiquitination and internalization into the MVB. Ub, ubiquitin.To understand the mechanism of Ypq1 recognition, Arines et al. first engineered a Ypq1 mutant that uncouples ligase-mediated degradation from lysine availability. This Ypq1 mutant is constitutively degraded in an Ssh4-dependent manner even under lysine-replete conditions. With this tool in hand, they performed a random mutagenesis-based suppressor screen, which identified many suppressor mutants. Mapping these mutations revealed several elements in Ypq1 that are critical for ligase recognition, which include two TMDs (TM5 and TM7) and a cytosolic loop. Importantly, when these mutations were introduced back into wild-type Ypq1, they also block Ssh4-dependent, lysine-regulated Ypq1 degradation. As expected, coimmunoprecipitation showed that Ypq1 suppressor mutants have reduced affinity to Ssh4. Since the cytosolic loop contains a previously known Rsp5 recognition motif, they further characterized the role of Ypq1 TMDs in ligase recruitment.Structural modeling suggests that TM5 and TM7 are juxtaposed to each other. Systematic mutagenesis targeting each residue of these two TMDs further consolidated the residues essential for Ssh4-mediated degradation. A similar mutagenesis study on Ssh4 revealed an important role for the Ssh4 TMD in Ypq1 degradation. Interestingly, for both Ssh4 and Ypq1, many identified residues are clustered on one side of the membrane helices. Arines et al. propose that Ssh4 uses its TMD to recognize TM5 and TM7 in Ypq1 based on a charge complementation experiment: a charged residue introduced into TM5 of Ypq1 abolished Ssh4-mediated degradation, but introducing an opposite charge into the TMD of Ssh4 restored lysine-regulated Ypq1 degradation.The recognition of Ypq1 by Ssh4 appears to occur when Ypq1 adopts a specific conformation during lysine transport because charge complementarity–based degradation of Ypq1 depends on the PQ loop, which is required for lysine transport. Additionally, structural modeling of Ypq1 suggested that in the inward-open and occluded conformations, TM5 is packed against TM7, but the two TMDs become distant from each other in the outward-open conformation, which exposes residues critical for Ssh4 recognition. These findings suggest that the rapid conformational cycling during lysine transport may prevent Ssh4 recognition, but lysine depletion stalls Ypq1 in a conformation recognizable by Ssh4 (Fig. 1).The study, together with the recent discovery of the ER membrane protein complex (EMC) in the biogenesis of multi-spanning membrane proteins at the ER, suggests a new class of chaperones that recognize specific features in TMDs. While emerging evidence suggests that the EMC recognizes exposed charged or polar residues in TMDs (10), the molecular basis of Ssh4 substrate interaction remains unclear. Like cytosolic chaperones, the EMC at the ER appears to play a dual role: while initially shielding charged/polar residues to facilitate TMD assembly, it may eventually target misassembled membrane proteins for degradation. By contrast, TMD-specific chaperones in other organelles like Ssh4 may have a more dedicated function in PQC. Clearly, more TMD-specific chaperones await to be discovered. Additionally, future studies will surely reveal not only the range of substrates and functions for each TMD-specific chaperone but also the structural basis of TMD recognition.     

An interaction map of endoplasmic reticulum chaperones and foldases

Chaperones and foldases in the endoplasmic reticulum (ER) ensure correct protein folding. Extensive protein-protein interaction maps have defined the organization and function of many cellular complexes, but ER complexes are under-represented. Consequently, chaperone and foldase networks in the ER are largely uncharacterized. Using complementary ER-specific methods, we have mapped interactions between ER-lumenal chaperones and foldases and describe their organization in multiprotein complexes. We identify new functional chaperone modules, including interactions between protein-disulfide isomerases and peptidyl-prolyl cis-trans-isomerases. We have examined in detail a novel ERp72-cyclophilin B complex that enhances the rate of folding of immunoglobulin G. Deletion analysis and NMR reveal a conserved surface of cyclophilin B that interacts with polyacidic stretches of ERp72 and GRp94. Mutagenesis within this highly charged surface region abrogates interactions with its chaperone partners and reveals a new mechanism of ER protein-protein interaction. This ability of cyclophilin B to interact with different partners using the same molecular surface suggests that ER-chaperone/foldase partnerships may switch depending on the needs of different substrates, illustrating the flexibility of multichaperone complexes of the ER folding machinery.     

Chaperoning ribonucleoprotein biogenesis in health and disease

The survival motor neuron (SMN) protein is part of a macromolecular complex that functions in the biogenesis of small nuclear ribonucleoproteins (snRNPs)--the essential components of the pre-messenger RNA splicing machinery--as well as probably other RNPs. Reduced levels of SMN expression cause the inherited motor neuron disease spinal muscular atrophy (SMA). Knowledge of the composition, interactions and functions of the SMN complex has advanced greatly in recent years. The emerging picture is that the SMN complex acts as a macromolecular chaperone of RNPs to increase the efficiency and fidelity of RNA-protein interactions in vivo, and to provide an opportunity for these interactions to be regulated. In addition, it seems that RNA metabolism deficiencies underlie SMA. Here, a dual dysfunction hypothesis is presented in which two mechanistically and temporally distinct defects--that are dependent on the extent of SMN reduction in SMA--affect the homeostasis of specific messenger RNAs encoding proteins essential for motor neuron development and function.     

Chaperones and foldases in endoplasmic reticulum stress signaling in plants

Molecular chaperones and foldases are a diverse group of proteins that in vivo bind to misfolded or unfolded proteins (non-native or unstable proteins) and play important role in their proper folding. Stress conditions compel altered and heightened chaperone and foldase expression activity in the endoplasmic reticulum (ER), which highlights the role of these proteins, due to which several of the proteins under these classes were identified as heat shock proteins. Different chaperones and foldases are active in different cellular compartment performing specific tasks. The review will discuss the role of ER chaperones and foldases under stress conditions, to maintain proper protein folding dynamics in the plant cells and recent advances in the field. The ER chaperones and foldases, which are described in article, are binding protein (BiP), glucose regulated protein (GRP94), protein-disulfide isomerase (PDI), peptidyl-prolyl isomerases (PPI) or immunophilins, calnexin and calreticulin.Key words: Abiotic stress, chaperones, endoplasmic reticulum, foldases, immunophilins, protein folding, signal transduction     

Chaperoning the synapse—NMNAT protects Bruchpilot from crashing

Maintaining active zone structure is crucial for synaptic function. In this issue of EMBO reports, NMNAT is shown to act as a chaperone that protects the active zone structural protein Bruchpilot from degradation.EMBO reports (2013) 14 1, 87–94 doi:10.1038/embor.2012.181Synapses perform several tasks independently from the cell body of the neuron, including synaptic vesicle recycling through endocytosis or local protein maturation and degradation. Failure to regulate protein function locally is detrimental to the nervous system as evidenced by neuronal dysfunctions that arise as a consequence of synaptic ageing. This relative synaptic autonomy comes with a need for mechanisms that ensure correct protein (re)folding, and there is accumulating evidence that key chap-erones have a central role in the regulation and maintenance of synaptic structural integrity and function [1]. Work by Grace Zhai''s group, published in this issue of EMBO reports, demonstrates a key role of the Drosophila nicotinamide mononucleotide adenylyltransferase (NMNAT) chaperone in the protection of active zone components against activity-induced degeneration (Fig 1; [2]).Open in a separate windowFigure 1Results reported by Zang and colleagues [2] reveal a specific role of nicotinamide mononucleotide adenylyltransferase (NMNAT) in preserving active zone structure against use-dependent decline. This protection is exerted by direct interaction with BRP and protection of this key structural protein against ubiquitination and subsequent degradation. BRP, Bruchpilot; Ub, ubiquitin.Active zones, the specialized sites for neurotransmitter release at presynaptic terminals, are characterized by a dense protein network called the cytomatrix at the active zone (CAZ). The protein machinery of the CAZ is responsible for efficient synaptic vesicle tethering, docking and fusion with the presynaptic membrane and, thus, for reliable signal transmission from the neuron to the postsynaptic cell. Clearly, proteins in the CAZ are tightly regulated, especially in response to external cues such as synaptic activity [3,4]. Yet, this particularly crowded protein environment might be favourable for the formation of non-functional—and sometimes toxic—protein aggregates. Chaperones that act at the synapse reduce the probability of crucial protein aggregation by preventing and reverting these inappropriate interactions, which happen as a result of environmental stress.One of these chaperones, the Drosophila neuroprotective NMNAT, was identified in a genetic screen for factors involved in synapse function [5]. Its chaperone activity was later confirmed by using in vitro and in vivo protein folding assays [6]. NMNAT null mutants show severe and early onset neurodegeneration, whereas neurodevelopment does not seem to be strongly affected. Interestingly, degeneration of photoreceptors lacking NMNAT can be significantly attenuated by limiting synaptic activity, either by rearing flies in the dark or by introducing the no receptor potential A (norpA) mutation that blocks phototransduction [5]. These results indicate that NMNAT protects adult neurons from activity-induced degeneration.In this issue of EMBO reports, Zang and colleagues report a role for NMNAT at the synapse. They observed that loss or reduced levels of NMNAT leads to a concomitant loss of several synaptic markers including cysteine-string protein (CSP), synaptotagmin and the active zone structural protein Bruchpilot (BRP). Remarkably, BRP was the only one of these proteins found to co-immunoprecipitate with NMNAT from brain lysates. Both proteins show approximately 50% co-localization at the neuromuscular junction when imaged by 3D-SIM^™ super-resolution microscopy, suggesting that NMNAT might act directly as a chaperone for maintaining a functional BRP conformation.Consistent with a protective role of NMNAT against BRP degradation, RNA interference-mediated NMNAT knockdown leads to BRP ubiquitination, whereas this modification was not detected in control brain lysates. Given the involvement of the ubiquitin proteasome pathway in regulating synaptic development and function [1], the authors tested the effect of the proteasome inhibitor MG-132 on BRP ubiquitination. They observed an increased level of BRP ubiquitination in wild-type flies fed with this drug, suggesting a role for the proteasome in the clearance of ubiquitinated BRP. By contrast, overexpression of NMNAT reduces the level of BRP ubiquitination both in the absence and the presence of MG-132, providing further evidence for the protective role of this chaperone against ubiquitination of BRP (Fig 1).a key role of the […] nicotinamide mononucleotide adenylyltransferase (NMNAT) chaperone in the protection of active zone components against activity-induced degenerationBRP is a cytoskeletal-like protein that is an integral component of T-bars—electron-dense structures that project from the presynaptic membrane and around which synaptic vesicles cluster. In agreement with a protective role of NMNAT against BRP ubiquitination, reduced levels of this chaperone give rise to a marked decrease in T-bar size in an age-dependent manner (Fig 1). Active zones are known to show dynamic changes in response to synaptic activity, and NMNAT was previously reported to protect photoreceptors against activity-induced degeneration [5]. The authors thus tested the effect of minimizing photoreceptor activity on active zone structure by keeping flies in the dark or inhibiting phototransduction by means of the norpA mutation. Both manipulations largely reversed the effect of NMNAT knockdown on T-bar size. Absence of light exposure also significantly reduced the amount of BRP that co-immunoprecipitates with NMNAT, indicating that neuronal activity regulates NMNAT–BRP interaction. Further experiments are needed to examine whether there is a positive correlation between synaptic activity and BRP ubiquitination levels, and whether NMNAT can indeed keep T-bar structure intact by protecting BRP against this modification under conditions of high synaptic activity.Finally, the study shows that reduced NMNAT levels not only caused a loss of BRP from the synapse but also a specific mislocalization of this protein to the cell body, where it accumulates in clusters together with the remaining NMNAT protein. Under these conditions BRP co-immunoprecipitated with the stress-induced Hsp70, a chaperone classically used as a marker for protein aggregation. It is still unclear whether these BRP clusters form as a result of defective anterograde trafficking and/or of enhanced retrograde transport of BRP. In the absence of light stimulation T-bars are properly assembled in nmnat null photoreceptors, but at this stage a role of NMNAT in regulating the axonal transport of BRP under conditions of normal synaptic activity cannot be excluded. Noticeably, two independent recent reports show involvement of NMNAT in mitochondrial mobility [7,8].As BRP and NMNAT co-localize and interact with one another, the simplest model that accounts for all the observations by Zang et al is that NMNAT directly prevents activity-induced ubiquitination of BRP and subsequent degradation. Yet, as its name indicates, this chaperone is an essential enzyme in NAD synthesis. It was previously shown by the Bellen lab that mutant versions of NMNAT, impaired for NAD production, rescue photoreceptor degeneration caused by loss of NMNAT [5]. This strongly suggests that NAD production is not required for stabilization of BRP but this might need further scrutiny [9].…reduced levels of this chaperone [NMNAT] give rise to a marked decrease in T-bar sizeWhile providing further insights into the role of NMNAT at the active zone in Drosophila, the paper by Zang et al might also have important implications for neurodegeneration in mammals. When ectopically expressed in mice, Nmnat has a protective role against Wallerian degeneration, that is, synapse and axon degeneration that rapidly occurs distal from an axonal wound in wild-type animals. This process is significantly delayed in mice overexpressing a chimaeric protein consisting of the amino-terminal 70 residues of the ubiquitination factor E4B (Ube4b) fused through a linker to Nmnat1, known as the Wallerian degeneration slow (Wld^s) protein. Conversely, mutations in the human NMNAT1 gene were characterized in several families with Leber congenital amaurosis—a severe, early-onset neurodegenerative disease of the retina [10,11,12,13]. As Wld^s or Nmnat1 overexpression protects axons from degeneration in various disease models [9], Nmnat1 emerges as a promising candidate for developing protective strategies against axonal degeneration in peripheral neuropathies such as amyotrophic lateral sclerosis but also in glaucoma, AIDS and other diseases [9].     

Chaperoning the Cln3 cyclin prevents promiscuous activation of start

In a recent issue of Molecular Cell, Vergés et al. (2007) described a new mechanism of cell-cycle control. Nuclear translocation of the G1 cyclin Cln3 is prevented by its retention at the endoplasmic reticulum (ER), and its release requires growth-associated increases in chaperone activity.