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.

GroEL Ring Separation and Exchange in the Chaperonin Reaction.

The bacterial chaperonin GroEL and its cofactor, GroES, form a nano-cage for a single molecule of substrate protein (SP) to fold in isolation. GroEL and GroES undergo an ATP-regulated interaction cycle to close and open the folding cage. GroEL consists of two heptameric rings stacked back to back. Here, we show that GroEL undergoes transient ring separation, resulting in ring exchange between complexes. Ring separation occurs upon ATP-binding to the trans ring of the asymmetric GroEL:7ADP:GroES complex in the presence or absence of SP and is a consequence of inter-ring negative allostery. We find that a GroEL mutant unable to perform ring separation is folding active but populates symmetric GroEL:GroES2 complexes, where both GroEL rings function simultaneously rather than sequentially. As a consequence, SP binding and release from the folding chamber is inefficient, and E. coli growth is impaired. We suggest that transient ring separation is an integral part of the chaperonin mechanism.

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.

Allosteric differences dictate GroEL complementation of E. coli.

GroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies.

Chaperonin-assisted protein folding: a chronologue.

This chronologue seeks to document the discovery and development of an understanding of oligomeric ring protein assemblies known as chaperonins that assist protein folding in the cell. It provides detail regarding genetic, physiologic, biochemical, and biophysical studies of these ATP-utilizing machines from both in vivo and in vitro observations. The chronologue is organized into various topics of physiology and mechanism, for each of which a chronologic order is generally followed. The text is liberally illustrated to provide firsthand inspection of the key pieces of experimental data that propelled this field. Because of the length and depth of this piece, the use of the outline as a guide for selected reading is encouraged, but it should also be of help in pursuing the text in direct order.

Molecular chaperone GroEL-GroES enhances the soluble expression of biologically active ovine growth hormone in the prokaryotic system.

Growth hormone (GH) plays important roles in growth and development of mammalian animals and is valuable for many applications. This study aimed to express and purify biological active recombinant ovine growth hormone (roGH) through prokaryotic expression system. The roGH coding sequence was ligated into the prokaryotic expression vector and transformed into Escherichia coli (E. coli) for protein expression. Factors that influence the roGH expression were examined and the appropriate culture temperature (20 °C) and inducer (IPTG) concentration (25 µM) were determined. To enhance the soluble expression of the protein, co-expression with the molecular chaperone GroEL-GroES was utilized and eventually achieved a high yield of soluble roGH expressed in E. coli. Further, the fusion tag in expressed target protein could be efficiently removed through thrombin-specific cleavage. The expressed roGH was identified by Western blotting and the LC-MS spectrum confirmed its molecular weight of 22749.22 Da. Finally, the purified roGH had an expected biological activity when assayed in cell models in vitro and experimental mouse in vivo. In conclusion, the present study established an efficient and simple approach to produce recombinant GH, and facilitate relevant research and applications.

Synthesis of an Anti-CD7 Recombinant Immunotoxin Based on PE24 in CHO and E. coli Cell-Free Systems.

Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 StarTM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC50 value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody−toxin conjugate activity in a time-efficient workflow without cell lysis or purification required.

Exploiting the HSP60/10 chaperonin system as a chemotherapeutic target for colorectal cancer.

Over the past few decades, an increasing variety of molecular chaperones have been investigated for their role in tumorigenesis and as potential chemotherapeutic targets; however, the 60 kDa Heat Shock Protein (HSP60), along with its HSP10 co-chaperone, have received little attention in this regard. In the present study, we investigated two series of our previously developed inhibitors of the bacterial homolog of HSP60/10, called GroEL/ES, for their selective cytotoxicity to cancerous over non-cancerous colorectal cells. We further developed a third "hybrid" series of analogs to identify new candidates with superior properties than the two parent scaffolds. Using a series of well-established HSP60/10 biochemical screens and cell-viability assays, we identified 24 inhibitors (14%) that exhibited > 3-fold selectivity for targeting colorectal cancer over non-cancerous cells. Notably, cell viability EC50 results correlated with the relative expression of HSP60 in the mitochondria, suggesting a potential for this HSP60-targeting chemotherapeutic strategy as emerging evidence indicates that HSP60 is up-regulated in colorectal cancer tumors. Further examination of five lead candidates indicated their ability to inhibit the clonogenicity and migration of colorectal cancer cells. These promising results are the most thorough analysis and first reported instance of HSP60/10 inhibitors being able to selectively target colorectal cancer cells and highlight the potential of the HSP60/10 chaperonin system as a viable chemotherapeutic target.

The GroEL-GroES Chaperonin Machine: A Nano-Cage for Protein Folding.

The bacterial chaperonin GroEL and its cofactor GroES constitute the paradigmatic molecular machine of protein folding. GroEL is a large double-ring cylinder with ATPase activity that binds non-native substrate protein (SP) via hydrophobic residues exposed towards the ring center. Binding of the lid-shaped GroES to GroEL displaces the bound protein into an enlarged chamber, allowing folding to occur unimpaired by aggregation. GroES and SP undergo cycles of binding and release, regulated allosterically by the GroEL ATPase. Recent structural and functional studies are providing insights into how the physical environment of the chaperonin cage actively promotes protein folding, in addition to preventing aggregation. Here, we review different models of chaperonin action and discuss issues of current debate.

Analogs of nitrofuran antibiotics are potent GroEL/ES inhibitor pro-drugs.

In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.

Hydrogen deuterium exchange mass spectrometry applied to chaperones and chaperone-assisted protein folding.

Introduction: Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) is ideal for monitoring the protein folding and unfolding. The exchange of a deuterium in solution for an amide hydrogen in a protein can be very different depending on the degree of folding and protection of backbone amide positions. Molecular chaperones that assist with protein folding in vivo are necessary for folding of many substrate (client) proteins. HDX MS provides valuable insight into what chaperones are doing in protein folding and how they are doing it. Areas covered: Application of HDX MS to the protein folding problem was desirable from the outset of the technique, but technical issues prohibited many studies. In the last 20 years, conformational changes of chaperones themselves (e.g., GroEL/GroES, Hsp70, and Hsp90) have been studied. Studies of interactions between chaperones, co-chaperones, and substrate proteins have revealed binding interfaces, allosteric conformational changes, and remodeling of components during various chaperone cycles. Experiments elucidating how chaperones contribute to and enhance the folding pathway of substrate proteins have been demonstrated. Expert opinion: Technical issues that once prevented the analysis of chaperones have largely been resolved, permitting exciting comprehensive HDX MS studies of folding pathways during chaperone-assisted protein folding.

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.

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.

Rickettsia japonica Infections in Humans, Zhejiang Province, China, 2015.

We investigated 16 Japanese spotted fever cases that occurred in southeastern China during September-October 2015. Patients had fever, rash, eschar, and lymphadenopathy. We confirmed 9 diagnoses and obtained 2 isolates with high identity to Rickettsia japonica strain YH. R. japonica infection should be considered for febrile patients in China.

ATPase activity of GroEL is dependent on GroES and it is response for environmental stress in Riemerella anatipestifer.

Riemerella anatipestifer (Ra) is a serious gram-negative pathogen of birds and can cause considerable economic losses. The survival mechanisms of R. anatipestifer in the host and environment remain largely unknown. Previous results have demonstrated that GroEL is a molecular chaperone and an important component of the response to various stresses in most bacteria. This study focused on whether GroEL is implicated in this process in R. anatipestifer. The 1629 bp groEL is highly conserved among other gram-negative bacteria (levels of sequence similarity > 60%). A structural analysis and ATPase activity assay revealed that RaGroEL had weak ATPase activity and that the enzyme activity was temperature and ion dependent. GroES partially enhanced the GroEL ATPase activity in the same temperature range. In addition, we studied the mRNA expression of groEL under abiotic stresses caused by heat shock, pH, salt and hydrogen peroxide. These stresses increased the transcription of groEL to varying degrees. In R. anatipestifer, the ATPase activity of GroEL is dependent on GroES and temperature. The expression of groEL was strongly induced by heat, pH, hydrogen peroxide and salt stress. This study is the first to show that GroEL in R. anatipestifer might play a major role in response to environmental stress.

Combined thermodynamic and kinetic analysis of GroEL interacting with CXCR4 transmembrane peptides.

GroEL along with ATP and its co-chaperonin GroES has been demonstrated to significantly enhance the folding of newly translated G-protein-coupled receptors (GPCRs). This work extends the previous studies to explore the guest capture and release processes in GroEL-assisted folding of GPCRs, by the reduced approach of employing CXCR4 transmembrane peptides as model substrates. Each of the CXCR4-derived peptides exhibited high affinity for GroEL with a binding stoichiometry near seven. It is found that the peptides interact with the paired α helices in the apical domain of the chaperonin which are similar with the binding sites of SBP (strongly binding peptide: SWMTTPWGFLHP). Complementary binding study with a single-ring version of GroEL indicates that each of the two chaperonin rings is competent for accommodating all the seven CXCR4 peptides bound to GroEL under saturation condition. Meanwhile, the binding kinetics of CXCR4 peptides with GroEL was also examined; ATP alone, or in combination of GroES evidently promoted the release of the peptide substrates from the chaperonin. The results obtained would be beneficial to understand the thermodynamic and kinetic nature of GroEL-GPCRs interaction which is the central molecular event in the assisted folding process.

GroEL and the GroEL-GroES Complex.

Chaperonin is categorized as a molecular chaperone and mediates the formation of the native conformation of proteins by first preventing folding during synthesis or membrane translocation and subsequently by mediating the step-wise ATP-dependent release that result in proper folding. In the GroEL-GroES complex, a single heptameric GroEL ring binds one GroES ring in the presence of ATP/ADP, in this vein, the double ring GroEL tetradecamer is present in two distinct types of GroEL-GroES complexes: asymmetric 1:1 "bullet"-shaped GroEL:GroES and symmetric 1:2 "football" (American football)-shaped GroEL:GroES2. There have been debates as to which complex is critical to the productive protein folding mediated by the GroEL-GroES complex, and how GroES coordinates with GroEL in the chaperonin reaction cycle in association with regulation by adenine nucleotides and through the interplay of substrate proteins. A lot of knowledge on chaperonins has been accumulating as if expanding as ripples spread around the GroEL-GroES from Escherichia coli. In this article, an overview is presented on GroEL and the GroEL-GroES complex, with emphasis on their morphological variations, and some potential applications to the fabrication of nanocomposites using GroEL as a nano-block. In parallel, a guideline is presented that supports the recognition that the E. coli and its GroEL-GroES complex do not always receive in standard literature because the biochemical features of chaperonins derived from others special, such as mammals, are not always the same as those confirmed using GroEL-GroES derived from E. coli.

[Limited Trypsinolysis of GroES: The Effect on the Interaction with GroEL and Assembly In Vitro].

GroES is a heptameric partner of tetradecameric molecular chaperone GroEL, which ensures the correct folding and assembly of numerous cellular proteins both in vitro and in vivo. This work demonstrates the results of a study of structural aspects of GroES that affect its interaction with GroEL and reassembly. The effect of limited trypsinolysis of GroES on these processes has been studied. It has been shown that limited trypsinolysis of GroES is only strongly pronounced outside the complex with GroEL and results in the cleavage of the peptide bond between Lys20 and Ser21. The N-terminal fragment (~2 kDa) is retained in the GroES particle, which maintains its heptaoligomeric structure but loses the ability to interact with GroEL and dissociates upon a change in the pH from 7 to 8. Trypsin-nicked GroES cannot reassemble after urea-induced unfolding, while the urea-induced unfolding of intact GroES is fully reversible. The reported results indicate the important role of the N-terminal part of GroES subunit in the assembly of its heptameric structure and the interaction with GroEL.

Biochemical and Genetic Analysis of the Chlamydia GroEL Chaperonins.

Chaperonins are essential for cellular growth under normal and stressful conditions and consequently represent one of the most conserved and ancient protein classes. The paradigm Escherichia coli chaperonin, EcGroEL, and its cochaperonin, EcGroES, assist in the folding of proteins via an ATP-dependent mechanism. In addition to the presence of groEL and groES homologs, groEL paralogs are found in many bacteria, including pathogens, and have evolved poorly understood species-specific functions. Chlamydia spp., which are obligate intracellular bacteria, have reduced genomes that nonetheless contain three groEL genes, Chlamydia groEL (ChgroEL), ChgroEL2, and ChgroEL3 We hypothesized that ChGroEL is the bona fide chaperonin and that the paralogs perform novel Chlamydia-specific functions. To test our hypothesis, we investigated the biochemical properties of ChGroEL and its cochaperonin, ChGroES, and queried the in vivo essentiality of the three ChgroEL genes through targeted mutagenesis in Chlamydia trachomatis ChGroEL hydrolyzed ATP at a rate 25% of that of EcGroEL and bound with high affinity to ChGroES, and the ChGroEL-ChGroES complex could refold malate dehydrogenase (MDH). The chlamydial ChGroEL was selective for its cognate cochaperonin, ChGroES, while EcGroEL could function with both EcGroES and ChGroES. A P35T ChGroES mutant (ChGroESP35T) reduced ChGroEL-ChGroES interactions and MDH folding activities but was tolerated by EcGroEL. Both ChGroEL-ChGroES and EcGroEL-ChGroESP35T could complement an EcGroEL-EcGroES mutant. Finally, we successfully inactivated both paralogs but not ChgroEL, leading to minor growth defects in cell culture that were not exacerbated by heat stress. Collectively, our results support novel functions for the paralogs and solidify ChGroEL as a bona fide chaperonin that is biochemically distinct from EcGroEL.IMPORTANCEChlamydia is an important cause of human diseases, including pneumonia, sexually transmitted infections, and trachoma. The chlamydial chaperonin ChGroEL and chaperonin paralog ChGroEL2 have been associated with survival under stress conditions, and ChGroEL is linked with immunopathology elicited by chlamydial infections. However, their exact roles in bacterial survival and disease remain unclear. Our results further substantiate the hypotheses that ChGroEL is the primary chlamydial chaperonin and that the paralogs play specialized roles during infection. Furthermore, ChGroEL and the mitochondrial GroEL only functioned with their cochaperonin, in contrast to the promiscuous nature of GroEL from E. coli and Helicobacter pylori, which might indicate a divergent evolution of GroEL during the transition from a free-living organism to an obligate intracellular lifestyle.

Conversion of an inactive xylose isomerase into a functional enzyme by co-expression of GroEL-GroES chaperonins in Saccharomyces cerevisiae.

BACKGROUND: Second-generation ethanol production is a clean bioenergy source with potential to mitigate fossil fuel emissions. The engineering of Saccharomyces cerevisiae for xylose utilization is an essential step towards the production of this biofuel. Though xylose isomerase (XI) is the key enzyme for xylose conversion, almost half of the XI genes are not functional when expressed in S. cerevisiae. To date, protein misfolding is the most plausible hypothesis to explain this phenomenon. RESULTS: This study demonstrated that XI from the bacterium Propionibacterium acidipropionici becomes functional in S. cerevisiae when co-expressed with GroEL-GroES chaperonin complex from Escherichia coli. The developed strain BTY34, harboring the chaperonin complex, is able to efficiently convert xylose to ethanol with a yield of 0.44 g ethanol/g xylose. Furthermore, the BTY34 strain presents a xylose consumption rate similar to those observed for strains carrying the widely used XI from the fungus Orpinomyces sp. In addition, the tetrameric XI structure from P. acidipropionici showed an elevated number of hydrophobic amino acid residues on the surface of protein when compared to XI commonly expressed in S. cerevisiae. CONCLUSIONS: Based on our results, we elaborate an extensive discussion concerning the uncertainties that surround heterologous expression of xylose isomerases in S. cerevisiae. Probably, a correct folding promoted by GroEL-GroES could solve some issues regarding a limited or absent XI activity in S. cerevisiae. The strains developed in this work have promising industrial characteristics, and the designed strategy could be an interesting approach to overcome the non-functionality of bacterial protein expression in yeasts.