A molecular device for the redox quality control of GroEL/ES substrates.

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10 cofactor) to GroEL induces CnoX release. Cryoelectron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal α-helix. Furthermore, we identified complexes in which CnoX, bound to GroEL, forms mixed disulfides with GroEL substrates, indicating that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, suggesting that Hsp60 molecular plugins have been conserved through evolution.

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties.

The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.

Structural and Dynamic Disturbances Revealed by Molecular Dynamics Simulations Predict the Impact on Function of CCT5 Chaperonin Mutations Associated with Rare Severe Distal Neuropathies.

Mutations in genes encoding molecular chaperones, for instance the genes encoding the subunits of the chaperonin CCT (chaperonin containing TCP-1, also known as TRiC), are associated with rare neurodegenerative disorders. Using a classical molecular dynamics approach, we investigated the occurrence of conformational changes and differences in physicochemical properties of the CCT5 mutations His147Arg and Leu224Val associated with a sensory and a motor distal neuropathy, respectively. The apical domain of both variants was substantially but differently affected by the mutations, although these were in other domains. The distribution of hydrogen bonds and electrostatic potentials on the surface of the mutant subunits differed from the wild-type molecule. Structural and dynamic analyses, together with our previous experimental data, suggest that genetic mutations may cause different changes in the protein-binding capacity of CCT5 variants, presumably within both hetero- and/or homo-oligomeric complexes. Further investigations are necessary to elucidate the molecular pathogenic pathways of the two variants that produce the two distinct phenotypes. The data and clinical observations by us and others indicate that CCT chaperonopathies are more frequent than currently believed and should be investigated in patients with neuropathies.

A genome-wide CRISPR screen identifies the CCT chaperonin as a critical regulator of vesicle trafficking.

Vesicle trafficking is a fundamental cellular process that controls the transport of various proteins and cargos between cellular compartments in eukaryotes. Using a combination of genome-wide CRISPR screening in mammalian cells and RNAi screening in Caenorhabditis elegans, we identify chaperonin containing TCP-1 subunit 4 (CCT4) as a critical regulator of protein secretion and vesicle trafficking. In C. elegans, deficiency of cct-4 as well as other CCT subunits impairs the trafficking of endocytic markers in intestinal cells, and this defect resembles that of dyn-1 RNAi worms. Consistent with these findings, the silencing of CCT4 in human cells leads to defective endosomal trafficking, and this defect can be rescued by the dynamin activator Ryngo 1-23. These results suggest that the cytosolic chaperonin CCT may regulate vesicle trafficking by promoting the folding of dynamin in addition to its known substrate tubulin. Our findings establish an essential role for the CCT chaperonin in regulating vesicle trafficking, and provide new insights into the regulation of vesicle trafficking and the cellular function of the cytosolic chaperonin.

Mitochondrial respiration promotes Cdc37-dependent stability of the Cdk1 homolog Cdc28.

Cdc28, the homolog of mammalian Cdk1, is a conserved key regulatory kinase for all major cell cycle transitions in yeast. We have found that defects in mitochondrial respiration (including deletion of ATP2, an ATP synthase subunit) inhibit growth of cells carrying a degron allele of Cdc28 (cdc28td) or Cdc28 temperature-sensitive mutations (cdc28-1 and cdc28-1N) at semi-permissive temperatures. Loss of cell proliferation in the atp2Δcdc28td double mutant is associated with aggravated cell cycle arrest and mitochondrial dysfunction, including mitochondrial hyperpolarization and fragmentation. Unexpectedly, in mutants defective in mitochondrial respiration, steady-state protein levels of mutant cdc28 are strongly reduced, accounting for the aggravated growth defects. Stability of Cdc28 is promoted by the Hsp90-Cdc37 chaperone complex. Our results show that atp2Δcdc28td double-mutant cells, but not single mutants, are sensitive to chemical inhibition of the Hsp90-Cdc37 complex, and exhibit reduced levels of additional Hsp90-Cdc37 client kinases, suggesting an inhibition of this complex. In agreement, overexpression of CDC37 improved atp2Δcdc28td cell growth and Cdc28 levels. Overall, our study shows that simultaneous disturbance of mitochondrial respiration and Cdc28 activity reduces the capacity of Cdc37 to chaperone client kinases, leading to growth arrest.

Cdc37 as a Co-chaperone to Hsp90.

The co-chaperone p50/Cdc37 is an important partner for Hsp90, assisting in molecular chaperone activities, particularly with regard to the regulation of protein kinases. Analysis of the structure of Hsp90-Cdc37-kinase complexes demonstrates the way in which Cdc37 interacts with and controls the folding of a large proportion of intracellular protein kinases. This co-chaperone thus stands at the hub of a multitude of intracellular signaling networks. Indeed, the influence of Cdc37 reaches beyond the housekeeping pathways of protein folding into the regulation of a wide range of cellular processes. This co-chaperone has attracted attention as a potential intermediate in carcinogenesis. Cdc37 is an attractive potential target in cancer due to (1) high expression in a number of tumor types and (2) control of multiple signaling pathways. These properties indicate (3) a potential for selectivity due to its elevated expression in malignant cells and (4) robustness, as the co-chaperone may control multiple growth signaling pathways and thus be less prone to evolution of resistance than less versatile oncoproteins. Cdc37 may also be involved in other aspects of pathophysiology and has been shown to be secreted in exosomes. Protein aggregation disorders have been linked to age-related declines in molecular chaperones and co-chaperones. Cdc37 also appears to be a potential agent in longevity due to its links to protein folding and autophagy, and it will be informative to study the role of Cdc37 maintenance/decline in aging organisms.

Chaperonin: Co-chaperonin Interactions.

Co-chaperonins function together with chaperonins to mediate ATP-dependent protein folding in a variety of cellular compartments. Chaperonins are evolutionarily conserved and form two distinct classes, namely, group I and group II chaperonins. GroEL and its co-chaperonin GroES form part of group I and are the archetypal members of this family of protein folding machines. The unique mechanism used by GroEL and GroES to drive protein folding is embedded in the complex architecture of double-ringed complexes, forming two central chambers that undergo conformational rearrangements that enable protein folding to occur. GroES forms a lid over the chamber and in doing so dislodges bound substrate into the chamber, thereby allowing non-native proteins to fold in isolation. GroES also modulates allosteric transitions of GroEL. Group II chaperonins are functionally similar to group I chaperonins but differ in structure and do not require a co-chaperonin. A significant number of bacteria and eukaryotes house multiple chaperonin and co-chaperonin proteins, many of which have acquired additional intracellular and extracellular biological functions. In some instances, co-chaperonins display contrasting functions to those of chaperonins. Human HSP60 (HSPD) continues to play a key role in the pathogenesis of many human diseases, in particular autoimmune diseases and cancer. A greater understanding of the fascinating roles of both intracellular and extracellular Hsp10 on cellular processes will accelerate the development of techniques to treat diseases associated with the chaperonin family.

HSP90-CDC37-PP5 forms a structural platform for kinase dephosphorylation.

Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, the kinase-specific co-chaperone CDC37, and the kinase client itself. Removal of regulatory phosphorylation from client kinases and their release from the HSP90-CDC37 system depends on the Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here, we present the cryoEM structure of the oncogenic protein kinase client BRAFV600E bound to HSP90-CDC37, showing how the V600E mutation favours BRAF association with HSP90-CDC37. Structures of HSP90-CDC37-BRAFV600E complexes with PP5 in autoinhibited and activated conformations, together with proteomic analysis of its phosphatase activity on BRAFV600E and CRAF, reveal how PP5 is activated by recruitment to HSP90 complexes. PP5 comprehensively dephosphorylates client proteins, removing interaction sites for regulatory partners such as 14-3-3 proteins and thus performing a 'factory reset' of the kinase prior to release.

Mechanistic insights into protein folding by the eukaryotic chaperonin complex CCT.

The cytosolic chaperonin CCT is indispensable to eukaryotic life, folding the cytoskeletal proteins actin and tubulin along with an estimated 10% of the remaining proteome. However, it also participates in human diseases such as cancer and viral infections, rendering it valuable as a potential therapeutic target. CCT consists of two stacked rings, each comprised of eight homologous but distinct subunits, that assists the folding of a remarkable substrate clientele that exhibits both broad diversity and specificity. Much of the work in recent years has been aimed at understanding the mechanisms of CCT substrate recognition and folding. These studies have revealed new binding sites and mechanisms by which CCT uses its distinctive subunit arrangement to fold structurally unrelated substrates. Here, we review recent structural insights into CCT-substrate interactions and place them into the broader context of CCT function and its implications for human health.

Proteomics analysis of cancer tissues identifies IGF2R as a potential therapeutic target in laryngeal carcinoma.

Background: Laryngeal cancer (LC) is a prevalent head and neck malignancy; however, the essential pathophysiological mechanism underlying its tumorigenesis and progression remains elusive. Due to the perduring scarcity of effective targeted drugs for laryngeal cancer, insights into the disease's pathophysiological mechanisms would substantially impact the treatment landscape of laryngeal cancer. Methods: To ensure quality consistency, 10 tumor and 9 non-tumor samples underwent proteomic analysis on a single mass spectrometer using a label-free technique. Subsequently, gene expression variations between laryngeal squamous cell carcinoma and normal tissues were analyzed using The Cancer Genome Atlas (TCGA) database. Immunohistochemical expressions of insulin-like growth factor 2 receptor (IGF2R), fibronectin (FN), vimentin, and α-smooth muscle actin (SMA) in LC tissues and normal tissues were determined. Results: In the tumor group, significant variations were detected for 433 upregulated and 61 downregulated proteins. Moreover, the heatmap revealed that the expressions of RNA translation-related proteins and proteins involved in RNA metabolism, such as IGF2R, tenascin C (TNC), periostin (POSTN), proteasome 26S subunit ATPase 4 (PSMC4), serpin family A member 3 (SERPINA3), heat shock protein family B (small) member 6 (HSPB6), osteoglycin (OGN), chaperonin containing TCP1 subunit 6A (CCT6A), and chaperonin containing TCP1 subunit 6B (CCT6B), were prominently elevated in the tumor group. Nonsense-mediated RNA decay (NMD), RNA translation, and protein stability were significantly altered in LC tumors. IGF2R was remarkably upregulated in LC tumors. In the TCGA database, the IGF2R mRNA level was significantly upregulated in LSCC tissues. Additionally, IGF2R mRNA expression was lowest in clinical grade 1 samples, with no significant difference between grades 2 and 3. In LSCC patients, a significant positive correlation between IGF2R expression and the stromal score was detected using the ESTIMATE algorithm to estimate the immune score, stromal score, and tumor purity in the tumor microenvironment. Lastly, immunohistochemical analysis revealed that IGF2R is overexpressed in LC. Conclusion: These results demonstrate the vital role of IGF2R in LC carcinogenesis and progression and may facilitate the identification of new therapeutic targets for the prevention and treatment of LC.

PSMG2-controlled proteasome-autophagy balance mediates the tolerance for MEK-targeted therapy in triple-negative breast cancer.

Although the MAPK pathway is aberrantly activated in triple-negative breast cancers (TNBCs), the clinical outcome of MEK-targeted therapy is still poor. Through a genome-wide CRISPR-Cas9 library screening, we find that inhibition of PSMG2 sensitizes TNBC cells BT549 and MB468 to the MEK inhibitor AZD6244. Mechanistically, PSMG2 knockdown impairs proteasome function, which in turn activates autophagy-mediated PDPK1 degradation. The PDPK1 degradation significantly enhances AZD6244-induced tumor cell growth inhibition by interrupting the negative feedback signals toward the AKT pathway. Consistently, co-targeting proteasomes and MEK with inhibitors synergistically suppresses tumor cell growth. The autophagy inhibitor chloroquine partially relieves the PDPK1 degradation and reverses the growth inhibition induced by combinatorial inhibition of MEK and proteasome. The combination regimen with the proteasome inhibitor MG132 plus AZD6244 synergistically inhibits tumor growth in a 4T1 xenograft mouse model. In summary, our study not only unravels the mechanism of MEK inhibitor resistance but also provides a combinatorial therapeutic strategy for TNBC in clinics.

CCT4 knockdown enhances the sensitivity of cisplatin by inhibiting glycolysis in human esophageal squamous cell carcinomas.

Esophageal squamous cell carcinoma (ESCC) is a common human malignancy characterized by late-stage diagnosis, metastasis, and poor prognosis. Cisplatin (DDP)-based chemotherapy has been the most predominant treatment for patients with ESCC. However, the high rate of DDP resistance and toxicity seriously hinder its clinical application. Then, the optimized strategy and mechanisms for ESCC to enhance DDP sensitivity are in great demand. Accumulating evidence have shown that chaperone proteins are closely related to the tumorigenesis and drug resistance of cancers. Chaperonin containing TCP1 complex 4 (CCT4) is a recent identified member of the family. However, its expression and function in ESCC have not been well illustrated. In this study, we found that CCT4 was highly expressed in human ESCC tissues and cell lines, and closely related to the poor prognosis. Moreover, CCT4 silence raised oxidative stress and inhibited glycolysis of ESCC cells, which significantly inhibited cell proliferation and migration, promoted apoptosis and caused cell cycle arrest in ESCC cells. Interestingly, CCT4 knockdown enhanced the sensitivity of KYSE150 cells to DDP by regulating AMPK/AKT/Nrf2 signaling pathway and inhibiting glycolysis ability. Taken together, our results indicate that targeting CCT4 may be a therapeutic target in ESCC patients, which provides a theoretical basis to enhance the sensitivity of DDP in ESCC.

Progressive retinal degeneration of rods and cones in a Bardet-Biedl syndrome type 10 mouse model.

Bardet-Biedl syndrome (BBS) is a multi-organ autosomal-recessive disorder caused by mutations in at least 22 different genes. A constant feature is early-onset retinal degeneration leading to blindness. Among the most common forms is BBS type 10 (BBS10), which is caused by mutations in a gene encoding a chaperonin-like protein. To aid in developing treatments, we phenotyped a Bbs10 knockout (Bbs10-/-) mouse model. Analysis by optical coherence tomography (OCT), electroretinography (ERG) and a visually guided swim assay (VGSA) revealed a progressive degeneration (from P19 to 8 months of age) of the outer nuclear layer that is visible by OCT and histology. Cone ERG was absent from at least P30, at which time rod ERG was reduced to 74.4% of control levels; at 8 months, rod ERG was 2.3% of that of controls. VGSA demonstrated loss of functional vision at 9 months. These phenotypes progressed more rapidly than retinal degeneration in the Bbs1M390R/M390R knock-in mouse. This study defines endpoints for preclinical trials that can be utilized to detect a treatment effect in the Bbs10-/- mouse and extrapolated to human clinical trials.

Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation.

RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer, while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding RAF1 follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities in attempting the pharmacological degradation of RAF1 for therapeutic purposes.

Multi-Omics Studies Unveil Extraciliary Functions of BBS10 and Show Metabolic Aberrations Underlying Renal Disease in Bardet-Biedl Syndrome.

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive ciliopathy resulting in multiple organ dysfunctions, including chronic kidney disease (CKD). Despite the recent progress in the 'ciliopathy' field, there is still little information on the mechanisms underlying renal disease. To elucidate these pathomechanisms, we conducted a translational study, including (i) the characterization of the urine metabolomic pattern of BBS patients and controls in a pilot and confirmation study and (ii) the proteomic analysis of the BBS10 interactome, one of the major mutated BBS genes in patients, in a renal-epithelial-derived cell culture model. The urine metabolomic fingerprinting of BBS patients differed from controls in both pilot and confirmation studies, demonstrating an increased urinary excretion of several monocarboxylates, including lactic acid (LA), at both early and late CKD stages. Increased urine LA was detected in the absence of both increased plasmatic LA levels and generalized proximal tubular dysfunction, suggesting a possible renal-specific defective handling. The inner medulla renal epithelial (IMCD3) cell line, where Bbs10 was stably invalidated, displayed an increased proliferative rate, increased ATP production, and an up-regulation of aerobic glycolysis. A mass spectrometry-based analysis detected several putative BBS10 interactors in vitro, indicating a potential role of BBS10 in several biological processes, including renal metabolism, RNA processing, and cell proliferation. The present study suggests that the urine metabolomic pattern of BBS patients may reflect intra-renal metabolic aberrations. The analysis of BBS10 interactors unveils possible novel functions, including cell metabolism.

A temporal gradient of cytonuclear coordination of chaperonins and chaperones during RuBisCo biogenesis in allopolyploid plants.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) has long been studied from many perspectives. As a multisubunit (large subunits [LSUs] and small subunits[SSUs]) protein encoded by genes residing in the chloroplast (rbcL) and nuclear (rbcS) genomes, RuBisCo also is a model for cytonuclear coevolution following allopolyploid speciation in plants. Here, we studied the genomic and transcriptional cytonuclear coordination of auxiliary chaperonin and chaperones that facilitate RuBisCo biogenesis across multiple natural and artificially synthesized plant allopolyploids. We found similar genomic and transcriptional cytonuclear responses, including respective paternal-to-maternal conversions and maternal homeologous biased expression, in chaperonin/chaperon-assisted folding and assembly of RuBisCo in different allopolyploids. One observation is about the temporally attenuated genomic and transcriptional cytonuclear evolutionary responses during early folding and later assembly process of RuBisCo biogenesis, which were established by long-term evolution and immediate onset of allopolyploidy, respectively. Our study not only points to the potential widespread and hitherto unrecognized features of cytonuclear evolution but also bears implications for the structural interaction interface between LSU and Cpn60 chaperonin and the functioning stage of the Raf2 chaperone.

Chaperone requirements for de novo folding of Saccharomyces cerevisiae septins.

Polymers of septin protein complexes play cytoskeletal roles in eukaryotic cells. The specific subunit composition within complexes controls functions and higher-order structural properties. All septins have globular GTPase domains. The other eukaryotic cytoskeletal NTPases strictly require assistance from molecular chaperones of the cytosol, particularly the cage-like chaperonins, to fold into oligomerization-competent conformations. We previously identified cytosolic chaperones that bind septins and influence the oligomerization ability of septins carrying mutations linked to human disease, but it was unknown to what extent wild-type septins require chaperone assistance for their native folding. Here we use a combination of in vivo and in vitro approaches to demonstrate chaperone requirements for de novo folding and complex assembly by budding yeast septins. Individually purified septins adopted nonnative conformations and formed nonnative homodimers. In chaperonin- or Hsp70-deficient cells, septins folded slower and were unable to assemble posttranslationally into native complexes. One septin, Cdc12, was so dependent on cotranslational chaperonin assistance that translation failed without it. Our findings point to distinct translation elongation rates for different septins as a possible mechanism to direct a stepwise, cotranslational assembly pathway in which general cytosolic chaperones act as key intermediaries.

Recognition of BRAF by CDC37 and Re-Evaluation of the Activation Mechanism for the Class 2 BRAF-L597R Mutant.

The kinome specific co-chaperone, CDC37 (cell division cycle 37), is responsible for delivering BRAF (B-Rapidly Accelerated Fibrosarcoma) to the Hsp90 (heat shock protein 90) complex, where it is then translocated to the RAS (protooncogene product p21) complex at the plasma membrane for RAS mediated dimerization and subsequent activation. We identify a bipartite interaction between CDC37 and BRAF and delimitate the essential structural elements of CDC37 involved in BRAF recognition. We find an extended and conserved CDC37 motif, 20HPNID---SL--W31, responsible for recognizing the C-lobe of BRAF kinase domain, while the c-terminal domain of CDC37 is responsible for the second of the bipartite interaction with BRAF. We show that dimerization of BRAF, independent of nucleotide binding, can act as a potent signal that prevents CDC37 recognition and discuss the implications of mutations in BRAF and the consequences on signaling in a clinical setting, particularly for class 2 BRAF mutations.

Effect of bacteriophage-encoded chaperonins on amyloid transformation of α-synuclein.

Controversial information about the role of chaperonins in the amyloid transformation of proteins and, in particular, α-synuclein, requires a more detailed study of the observed effects due to the structure and functional state of various chaperonins. In this work, two types of phage chaperonins, the double-ring EL and the single-ring OBP, were shown to stimulate α-synuclein fibrillation in an ATP-dependent manner. Chaperonin morphology does not affect the stimulation of α-synuclein amyloid transformation. However, the ATP-dependent effect of single- and double-ring chaperonins on this process differs, which can lead to different morphology of resulting fibrils. Fibril formation seems to proceed without substrate encapsulation in the internal cavity of chaperonin, because of the structural features of phage chaperonins and their ability to function without co-chaperonins. In the absence of ATP, both chaperonins, on the contrary, completely prevent α-synuclein amyloid transformation, which provides the possibility of their use as anti-amyloid agents, in the form of incomplete molecules or mutants with suppressed ATPase activity.

Chaperonins, collaborations, and challenging dogmas: An interview with Dr. Arthur L. Horwich.

For our 25th anniversary of Molecular Cell, we talk to one of our original board members, Dr. Arthur L. Horwich, about the discovery of chaperones, the importance of challenging "dogmas," and the benefits of scientific collaboration. He also shares anecdotes from the early days of Molecular Cell, some of his favorite papers, and his advice for junior scientists.