Chaperonin System Research Articles

Expression of recombinant mouse cytosolic carboxypeptidase 6 in Escherichia coli

Cytosolic carboxypeptidase 6 (CCP6) is a member of cytosolic carboxypeptidase (CCP) family that catalyze the removal of polyglutamate side chains from protein substrates. Biochemical and biophysical characterization of CCPs requires large quantities of purified proteins. However, no method describing the expression of any mammalian CCP family member from bacteria has been published to our best knowledge. After considerable efforts to improve the solubility of mammalian CCPs expressed in bacteria, including the optimization of induction temperature and by using different receptive cells, we were able to get less expression of mouse CCP6 in soluble fraction of bacterial lysates. We report in this article, the bacterial expression of CCP6 using Arctic Express (DE3) competent cells that co-express the chaperonin system GroEL and GroES from Oleispira antarctica. However, to achieve a large number of soluble target proteins, the expression conditions still need to be further optimized.

Impact of bacterial chaperonin GroEL\u2013GroES on bacteriorhodopsin folding and membrane integration

Our understanding of molecular chaperone function in membrane protein biogenesis lags far behind that in soluble protein biogenesis. Through a combined approach including isothermal titration calorimetry, UV–Vis spectroscopy, and fluorescence spectroscopy, the behavior of ATP-dependent chaperonin GroEL–GroES, a paradigmatic chaperone of soluble protein folding, was investigated in the refolding of membrane protein bacteriorhodopsin (BR) and its membrane insertion. We found that BR bound asymmetrically to the double-ring GroEL, with a much higher affinity when it was partially denatured. GroEL alone showed a clear influence on BR refolding, but the presence of ATP was necessary to significantly enhance both the rate and yield of the GroEL-mediated folding, in contrast to the adverse effect of GroES on the folding yield. However, synergy between ATP and GroES was shown to be required not only for releasing high-affinity BR species from GroEL, but also for unfolding and rescuing the misfolded conformers complexed to GroEL. This is consistent with the observation that maximum rate enhancement of BR refolding or assembly with the prepared inverted membrane vesicles was achieved when the complete chaperonin system was used. Our results support the iterative unfolding mechanism of GroEL activity previously proposed for soluble proteins, whereby GroEL might perform repeated unfolding and release of BR, thus offering additional opportunities for timely folding or membrane integration. This work provides important information on the convergence of folding of membrane and soluble proteins in light of folding pathways and the role of molecular chaperones.

Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections.

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis - actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease - which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.

Hetero‐oligomeric CPN60 resembles highly symmetric group‐I chaperonin structure revealed by Cryo‐EM

The chloroplast chaperonin system is indispensable for the biogenesis of Rubisco, the key enzyme in photosynthesis. Using Chlamydomonas reinhardtii as a model system, we found that invivo the chloroplast chaperonin consists of CPN60α, CPN60β1 and CPN60β2 and the co-chaperonin of the three subunits CPN20, CPN11 and CPN23. In Escherichia coli, CPN20 homo-oligomers and all possible other chloroplast co-chaperonin hetero-oligomers are functional, but only that consisting of CPN11/20/23-CPN60αβ1β2 can fully replace GroES/GroEL under stringent stress conditions. Endogenous CPN60 was purified and its stoichiometry was determined to be 6:2:6 for CPN60α:CPN60β1:CPN60β2. The cryo-EM structures of endogenous CPN60αβ1β2/ADP and CPN60αβ1β2/co-chaperonin/ADP were solved at resolutions of 4.06 and 3.82Å, respectively. In both hetero-oligomeric complexes the chaperonin subunits within each ring are highly symmetric. Through hetero-oligomerization, the chloroplast co-chaperonin CPN11/20/23 forms seven GroES-like domains, which symmetrically interact with CPN60αβ1β2. Our structure also reveals an uneven distribution of roof-forming domains in the dome-shaped CPN11/20/23 co-chaperonin and potentially diversified surface properties in the folding cavity of the CPN60αβ1β2 chaperonin that might enable the chloroplast chaperonin system to assist in the folding of specific substrates.

HSP60/10 chaperonin systems are inhibited by a variety of approved drugs, natural products, and known bioactive molecules

All living organisms contain a unique class of molecular chaperones called 60 kDa heat shock proteins (HSP60 – also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus – MRSA). Intriguingly, during our studies we found that three known antibiotics – suramin, closantel, and rafoxanide – were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.

Contact Order Is a Determinant for the Dependence of GFP Folding on the Chaperonin GroEL

The GroE chaperonin system facilitates protein folding in an ATP-dependent manner. It has remained unclear why some proteins are obligate clients of the GroE system, whereas other closely related proteins are able to fold efficiently in its absence. Factors that cause folding to be slower affect kinetic partitioning between spontaneous folding and chaperone binding in favor of the latter. One such potential factor is contact order (CO), which is the average separation in sequence between residues that are in contact in the native structure. Here, we generated variants of enhanced green fluorescent protein with different COs using circular permutations. We found that GroE dependence in vitro and in vivo increases with increasing CO. Thus, our results show that CO is relevant not only for folding in vitro of relatively simple model systems but also for chaperonin dependence and folding in vivo.

The chaperonin TRiC/CCT is essential for the action of bacterial glycosylating protein toxins like Clostridium difficile toxins A and B

Various bacterial protein toxins, including Clostridium difficile toxins A (TcdA) and B (TcdB), attack intracellular target proteins of host cells by glucosylation. After receptor binding and endocytosis, the toxins are translocated into the cytosol, where they modify target proteins (e.g., Rho proteins). Here we report that the activity of translocated glucosylating toxins depends on the chaperonin TRiC/CCT. The chaperonin subunits CCT4/5 directly interact with the toxins and enhance the refolding and restoration of the glucosyltransferase activities of toxins after heat treatment. Knockdown of CCT5 by siRNA and HSF1A, an inhibitor of TRiC/CCT, blocks the cytotoxic effects of TcdA and TcdB. In contrast, HSP90, which is involved in the translocation and uptake of ADP ribosylating toxins, is not involved in uptake of the glucosylating toxins. We show that the actions of numerous glycosylating toxins from various toxin types and different species depend on TRiC/CCT. Our data indicate that the TRiC/CCT chaperonin system is specifically involved in toxin uptake and essential for the action of various glucosylating protein toxins acting intracellularly on target proteins.

Expression, Functional Characterization, and Preliminary Crystallization of the Cochaperone Prefoldin from the Thermophilic Fungus Chaetomium thermophilum.

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits, and has the appearance of a jellyfish: Its body consists of a double β-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. Based on structural information from archaeal prefoldins, mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the structure and mechanisms of eukaryotic prefoldins remain unknown. In this study, we succeeded in obtaining recombinant prefoldin from a thermophilic fungus, Chaetomium thermophilum (CtPFD). The recombinant CtPFD could not protect citrate synthase from thermal aggregation. However, CtPFD formed a complex with actin from chicken muscle and tubulin from porcine brain, suggesting substrate specificity. We succeeded in observing the complex formation of CtPFD and the group II chaperonin of C. thermophilum (CtCCT) by atomic force microscopy and electron microscopy. These interaction kinetics were analyzed by surface plasmon resonance using Biacore. Finally, we have shown the transfer of actin from CtPFD to CtCCT. The study of the folding pathway formed by CtPFD and CtCCT should provide important information on mechanisms of the eukaryotic prefoldin–chaperonin system.

Pathway of Actin Folding Directed by the Eukaryotic Chaperonin TRiC

The hetero-oligomeric chaperonin of eukarya, TRiC, is required to fold the cytoskeletal protein actin. The simpler bacterial chaperonin system, GroEL/GroES, is unable to mediate actin folding. Here, we use spectroscopic and structural techniques to determine how TRiC promotes the conformational progression of actin to the native state. We find that actin fails to fold spontaneously even in the absence of aggregation but populates a kinetically trapped, conformationally dynamic state. Binding of this frustrated intermediate to TRiC specifies an extended topology of actin with native-like secondary structure. In contrast, GroEL stabilizes bound actin in an unfolded state. ATP binding to TRiC effects an asymmetric conformational change in the chaperonin ring. This step induces the partial release of actin, priming it for folding upon complete release into the chaperonin cavity, mediated by ATP hydrolysis. Our results reveal how the unique features of TRiC direct the folding pathway of an obligate eukaryotic substrate.

Prefoldin, a jellyfish-like molecular chaperone: functional cooperation with a group II chaperonin and beyond.

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits and has the appearance of a jellyfish: its body consists of a double beta-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. The prefoldin-group II chaperonin system is thought to be important for the folding of newly synthesized proteins and for their maintenance, or proteostasis, in the cytosol. Based on structural information of archaeal prefoldins, the mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the role and mechanism of eukaryotic PFDs remain unknown. Recent studies have shown that prefoldin plays an important role in proteostasis and is involved in various diseases. In this paper, we review a series of studies on the molecular mechanisms of archaeal prefoldins and introduce recent findings about eukaryotic prefoldin.

Chloroplast Chaperonin: An Intricate Protein Folding Machine for Photosynthesis.

Group I chaperonins are large cylindrical-shaped nano-machines that function as a central hub in the protein quality control system in the bacterial cytosol, mitochondria and chloroplasts. In chloroplasts, proteins newly synthesized by chloroplast ribosomes, unfolded by diverse stresses, or translocated from the cytosol run the risk of aberrant folding and aggregation. The chloroplast chaperonin system assists these proteins in folding into their native states. A widely known protein folded by chloroplast chaperonin is the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), an enzyme responsible for the fixation of inorganic CO2 into organic carbohydrates during photosynthesis. Chloroplast chaperonin was initially identified as a Rubisco-binding protein. All photosynthetic eucaryotes genomes encode multiple chaperonin genes which can be divided into α and β subtypes. Unlike the homo-oligomeric chaperonins from bacteria and mitochondria, chloroplast chaperonins are more complex and exists as intricate hetero-oligomers containing both subtypes. The Group I chaperonin requires proper interaction with a detachable lid-like co-chaperonin in the presence of ATP and Mg2+ for substrate encapsulation and conformational transition. Besides the typical Cpn10-like co-chaperonin, a unique co-chaperonin consisting of two tandem Cpn10-like domains joined head-to-tail exists in chloroplasts. Since chloroplasts were proposed as sensors to various environmental stresses, this diversified chloroplast chaperonin system has the potential to adapt to complex conditions by accommodating specific substrates or through regulation at both the transcriptional and post-translational levels. In this review, we discuss recent progress on the unique structure and function of the chloroplast chaperonin system based on model organisms Chlamydomonas reinhardtii and Arabidopsis thaliana. Knowledge of the chloroplast chaperonin system may ultimately lead to successful reconstitution of eukaryotic Rubisco in vitro.

Functionally Significant Amino Acid Motifs of Heat Shock Proteins: Structural and Bioinformatics Analyses of Hsp60/Hsp10 in Five Classes of Chordata

The Hsp60/Hsp10 chaperonin system is one of the most studied systems of cell emergency responses to stresses associated with changes in environmental conditions. In this regard, we have performed a bioinformatics analysis of 164 amino acid sequences of Hsp60 and 125 amino acid sequences of Hsp10 in five classes of chordata. This enabled uncovering the relationship between the identity of the amino acid composition of Hsp60/Hsp10 and the evolutionary distance between classes of chordata. In the course of the study of the chaperonin crystal structure, potentially significant amino acid motifs responsible for the oligomerization of Hsp60 and Hsp10 monomers and the association/dissociation of the Hsp60 and Hsp10 heterodimer have been identified. In addition, we have established that Hsp60 and Hsp10 can form amyloid fibrils due to structural features through the alternative using of the oligomerization sites of monomers as well as association/dissociation sites.

Local energetic frustration affects the dependence of green fluorescent protein folding on the chaperonin GroEL

The GroE chaperonin system in Escherichia coli comprises GroEL and GroES and facilitates ATP-dependent protein folding in vivo and in vitro Proteins with very similar sequences and structures can differ in their dependence on GroEL for efficient folding. One potential but unverified source for GroEL dependence is frustration, wherein not all interactions in the native state are optimized energetically, thereby potentiating slow folding and misfolding. Here, we chose enhanced green fluorescent protein as a model system and subjected it to random mutagenesis, followed by screening for variants whose in vivo folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with in vitro folding assays. Strikingly, mutations at positions predicted to be highly frustrated were found to correlate with decreased GroEL dependence. Conversely, mutations at positions with low frustration were found to correlate with increased GroEL dependence. Further support for this finding was obtained by showing that folding of an enhanced green fluorescent protein variant designed computationally to have reduced frustration is indeed less GroEL-dependent. Our results indicate that changes in local frustration also affect partitioning in vivo between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels.

The versatile mutational "repertoire" of Escherichia coli GroEL, a multidomain chaperonin nanomachine.

The bacterial chaperonins are highly sophisticated molecular nanomachines, controlled by the hydrolysis of ATP to dynamically trap and remove from the environment unstable protein molecules that are susceptible to denaturation and aggregation. Chaperonins also act to assist in the refolding of these unstable proteins, providing a means by which these proteins may return in active form to the complex environment of the cell. The Escherichia coli GroE chaperonin system is one of the largest protein supramolecular complexes known, whose quaternary structure is required for segregating aggregation-prone proteins. Over the course of more than two decades of research on GroE, it has become accepted that GroE, more specifically the GroEL subunit, is a "high-tolerance" molecular system, capable of accommodating numerous mutations, while retaining its molecular integrity. In some cases, a given site of mutation was revealed to be absolutely required for GroEL function, providing hints regarding the network of signals and triggers that propel this unique system. In other instances, however, a mutation has produced a more delicate response, altering only part of, or in some cases, only a single facet of, the molecular mechanism, and these mutants have often provided invaluable hints on the extent of the complexity underlying chaperonin-assisted protein folding. In this review, we highlight some examples of the latter type of GroEL mutants which compose the unique "mutational repertoire" of GroEL and touch upon the important clues that each mutant provided to the overall effort to elucidate the details of GroE action.

From chaperonins to Rubisco assembly and metabolic repair.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO2 in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Despite its pivotal role, Rubisco is an inefficient enzyme and thus has been a key target for bioengineering. However, efforts to increase crop yields by Rubisco engineering remain unsuccessful, due in part to the complex machinery of molecular chaperones required for Rubisco biogenesis and metabolic repair. While the large subunit of Rubisco generally requires the chaperonin system for folding, the evolution of the hexadecameric Rubisco from its dimeric precursor resulted in the dependence on an array of additional factors required for assembly. Moreover, Rubisco function can be inhibited by a range of sugar-phosphate ligands. Metabolic repair of Rubisco depends on remodeling by the ATP-dependent Rubisco activase and hydrolysis of inhibitors by specific phosphatases. This review highlights our work toward understanding the structure and mechanism of these auxiliary machineries.

Creating the Functional Single-Ring GroEL-GroES Chaperonin Systems via Modulating GroEL-GroES Interaction

Chaperonin and cochaperonin, represented by E. coli GroEL and GroES, are essential molecular chaperones for protein folding. The double-ring assembly of GroEL is required to function with GroES, and a single-ring GroEL variant GroELSR forms a stable complex with GroES, arresting the chaperoning reaction cycle. GroES I25 interacts with GroEL; however, mutations of I25 abolish GroES-GroEL interaction due to the seven-fold mutational amplification in heptameric GroES. To weaken GroELSR-GroES interaction in a controlled manner, we used groES7, a gene linking seven copies of groES, to incorporate I25 mutations in selected GroES modules in GroES7. We generated GroES7 variants with different numbers of GroESI25A or GroESI25D modules and different arrangements of the mutated modules, and biochemically characterized their interactions with GroELSR. GroES7 variants with two mutated modules participated in GroELSR–mediated protein folding in vitro. GroES7 variants with two or three mutated modules collaborated with GroELSR to perform chaperone function in vivo: three GroES7 variants functioned with GroELSR under both normal and heat-shock conditions. Our studies on functional single-ring bacterial chaperonin systems are informative to the single-ring human mitochondrial chaperonin mtHsp60-mtHsp10, and will provide insights into how the double-ring bacterial system has evolved to the single-ring mtHsp60-mtHsp10.

Biogenesis and Metabolic Maintenance of Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO2 in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Rubisco is a remarkably inefficient enzyme, fixing only 2-10 CO2 molecules per second. Efforts to increase crop yields by bioengineering Rubisco remain unsuccessful, owing in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. The large subunit of Rubisco requires the chaperonin system for folding, and recent studies have shown that assembly of hexadecameric Rubisco is mediated by specific assembly chaperones. Moreover, Rubisco function can be inhibited by a range of sugar-phosphate ligands, including RuBP. Metabolic repair depends on remodeling of Rubisco by the ATP-dependent Rubisco activase and hydrolysis of inhibitory sugar phosphates by specific phosphatases. Here, we review our present understanding of the structure and function of these auxiliary factors and their utilization in efforts to engineer more catalytically efficient Rubisco enzymes.

Targeting the HSP60/10 chaperonin systems of Trypanosoma brucei as a strategy for treating African sleeping sickness

Trypanosoma brucei are protozoan parasites that cause African sleeping sickness in humans (also known as Human African Trypanosomiasis—HAT). Without treatment, T. brucei infections are fatal. There is an urgent need for new therapeutic strategies as current drugs are toxic, have complex treatment regimens, and are becoming less effective owing to rising antibiotic resistance in parasites. We hypothesize that targeting the HSP60/10 chaperonin systems in T. brucei is a viable anti-trypanosomal strategy as parasites rely on these stress response elements for their development and survival. We recently discovered several hundred inhibitors of the prototypical HSP60/10 chaperonin system from Escherichia coli, termed GroEL/ES. One of the most potent GroEL/ES inhibitors we discovered was compound 1. While examining the PubChem database, we found that a related analog, 2e-p, exhibited cytotoxicity to Leishmania major promastigotes, which are trypanosomatids highly related to Trypanosoma brucei. Through initial counter-screening, we found that compounds 1 and 2e-p were also cytotoxic to Trypanosoma brucei parasites (EC50=7.9 and 3.1μM, respectively). These encouraging initial results prompted us to develop a library of inhibitor analogs and examine their anti-parasitic potential in vitro. Of the 49 new chaperonin inhibitors developed, 39% exhibit greater cytotoxicity to T. brucei parasites than parent compound 1. While many analogs exhibit moderate cytotoxicity to human liver and kidney cells, we identified molecular substructures to pursue for further medicinal chemistry optimization to increase the therapeutic windows of this novel class of chaperonin-targeting anti-parasitic candidates. An intriguing finding from this study is that suramin, the first-line drug for treating early stage T. brucei infections, is also a potent inhibitor of GroEL/ES and HSP60/10 chaperonin systems.

In vivo aspects of protein folding and quality control.

Most proteins must fold into unique three-dimensional structures to perform their biological functions. In the crowded cellular environment, newly synthesized proteins are at risk of misfolding and forming toxic aggregate species. To ensure efficient folding, different classes of molecular chaperones receive the nascent protein chain emerging from the ribosome and guide it along a productive folding pathway. Because proteins are structurally dynamic, constant surveillance of the proteome by an integrated network of chaperones and protein degradation machineries is required to maintain protein homeostasis (proteostasis). The capacity of this proteostasis network declines during aging, facilitating neurodegeneration and other chronic diseases associated with protein aggregation. Understanding the proteostasis network holds the promise of identifying targets for pharmacological intervention in these pathologies.

Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components.

Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of β-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes.