Structural basis of substrate progression through the bacterial chaperonin cycle.

The bacterial chaperonin GroEL-GroES promotes protein folding through ATP-regulated cycles of substrate protein binding, encapsulation, and release. Here, we have used cryoEM to determine structures of GroEL, GroEL-ADP·BeF3, and GroEL-ADP·AlF3-GroES all complexed with the model substrate Rubisco. Our structures provide a series of snapshots that show how the conformation and interactions of non-native Rubisco change as it proceeds through the GroEL-GroES reaction cycle. We observe specific charged and hydrophobic GroEL residues forming strong initial contacts with non-native Rubisco. Binding of ATP or ADP·BeF3 to GroEL-Rubisco results in the formation of an intermediate GroEL complex displaying striking asymmetry in the ATP/ADP·BeF3-bound ring. In this ring, four GroEL subunits bind Rubisco and the other three are in the GroES-accepting conformation, suggesting how GroEL can recruit GroES without releasing bound substrate. Our cryoEM structures of stalled GroEL-ADP·AlF3-Rubisco-GroES complexes show Rubisco folding intermediates interacting with GroEL-GroES via different sets of residues.

Dominant-negative chaperonin mutation ptCPN60α1S57F uncovers redundancy in chloroplast rRNA processing.

The biogenesis of functional forms of chloroplast ribosomal RNAs (rRNAs) is crucial for the translation of chloroplast mRNAs into polypeptides. However, the molecular mechanisms underlying the proper processing and maturation of chloroplast rRNA species are poorly understood. Through a genetic approach, we isolated and characterized an Arabidopsis mutant, α1-4, harboring a missense mutation in the plastid chaperonin-60α1 gene. Using allelism tests and transgenic manipulation, we determined functional redundancy among ptCPN60 subunits. The ptCPN60α1S57F mutation caused specific defects in the formation of chloroplast rRNA species, including 23S, 5S, and 4.5S rRNAs, but not 16S rRNAs. Allelism tests suggested that the dysfunctional ptCPN60α1S57F competes with other members of the ptCPN60 family. Indeed, overexpression of the ptCPN60α1S57F protein in wild-type plants mimicked the phenotypes observed in the α1-4 mutant, while increasing the endogenous transcriptional levels of ptCPN60α2, ß1, ß2, and ß3 in the α1-4 mutant partially mitigated the abnormal fragmentation processing of chloroplast 23S, 5S, and 4.5S rRNAs. Furthermore, we demonstrated functional redundancy between ptCPN60ß1 and ptCPN60ß2 in chloroplast rRNA processing through double-mutant analysis. Collectively, our data reveal a novel physiological role of ptCPN60 subunits in generating the functional rRNA species of the large 50S ribosomal subunit in Arabidopsis chloroplasts.

A diminished hydrophobic effect inside the GroEL/ES cavity contributes to protein substrate destabilization.

Confining compartments are ubiquitous in biology, but there have been few experimental studies on the thermodynamics of protein folding in such environments. Recently, we reported that the stability of a model protein substrate in the GroEL/ES chaperonin cage is reduced dramatically by more than 5 kcal mol-1 compared to that in bulk solution, but the origin of this effect remained unclear. Here, we show that this destabilization is caused, at least in part, by a diminished hydrophobic effect in the GroEL/ES cavity. This reduced hydrophobic effect is probably caused by water ordering due to the small number of hydration shells between the cavity and protein substrate surfaces. Hence, encapsulated protein substrates can undergo a process similar to cold denaturation in which unfolding is promoted by ordered water molecules. Our findings are likely to be relevant to encapsulated substrates in chaperonin systems, in general, and are consistent with the iterative annealing mechanism of action proposed for GroEL/ES.

Chaperone Machineries of Rubisco - The Most Abundant Enzyme.

A major challenge faced by human civilization is to ensure that agricultural productivity keeps pace with population growth and a changing climate. All food supply is generated, directly or indirectly, through the process of photosynthesis, with the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzing the assimilation of atmospheric CO2. Despite its pivotal role, Rubisco is a remarkably inefficient enzyme and must be made by plants in large quantities. However, efforts to enhance Rubisco performance by bioengineering have been hampered by its extensive reliance on molecular chaperones and auxiliary factors for biogenesis, metabolic repair, and packaging into membraneless microcompartments. Here, we review recent advances in understanding these complex machineries and discuss their implications for improving Rubisco carboxylase activity with the goal to increase crop yields.

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.

Chaperonins in cancer: Expression, function, and migration in extracellular vesicles.

The chaperonins CCT and Hsp60 are molecular chaperones, members of the chaperone system (CS). Chaperones are cytoprotective but if abnormal in quantity or quality they may cause diseases, the chaperonopathies. Here, recent advances in the understanding of CCT and Hsp60 in cancerology are briefly discussed, focusing on breast and brain cancers. CCT subunits, particularly CCT2, were increased in breast cancer cells and this correlated with tumor progression. Experimental induction of CCT2 increase was accompanied by an increase of CCT3, 4, and 5, providing another evidence for the interconnection between the members of the CS and the difficulties expected while manipulating one member with therapeutic purposes. Another in silico study demonstrated a direct correlation between the increase in the tumor tissue of the mRNA levels of all CCT subunits, except CCTB6, with bad prognosis. Studies with glioblastomas demonstrated an increase in the CCT subunits in the tumor tissue and in extracellular vesicles (EVs) derived from them. Expression levels of CCT1, 2, 6A, and 7 were the most increased and markers of bad prognosis, particularly CCT6A. A method for measuring Hsp60 and related miRNA in exosomes from blood of patients with glioblastomas or other brain tumors was discussed, and the results indicate that the triad Hsp60-related miRNAs-exosomes has potential regarding diagnosis and patient monitoring. All these data provide a strong foundation for future studies on the role played by chaperonins in carcinogenesis and for fully developing their theranostics applications along with exosomes.

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.

The Mycobacterium tuberculosis Pup-proteasome system regulates nitrate metabolism through an essential protein quality control pathway.

The human pathogen Mycobacterium tuberculosis encodes a proteasome that carries out regulated degradation of bacterial proteins. It has been proposed that the proteasome contributes to nitrogen metabolism in M. tuberculosis, although this hypothesis had not been tested. Upon assessing M. tuberculosis growth in several nitrogen sources, we found that a mutant strain lacking the Mycobacterium proteasomal activator Mpa was unable to use nitrate as a sole nitrogen source due to a specific failure in the pathway of nitrate reduction to ammonium. We found that the robust activity of the nitrite reductase complex NirBD depended on expression of the groEL/groES chaperonin genes, which are regulated by the repressor HrcA. We identified HrcA as a likely proteasome substrate, and propose that the degradation of HrcA is required for the full expression of chaperonin genes. Furthermore, our data suggest that degradation of HrcA, along with numerous other proteasome substrates, is enhanced during growth in nitrate to facilitate the derepression of the chaperonin genes. Importantly, growth in nitrate is an example of a specific condition that reduces the steady-state levels of numerous proteasome substrates in M. tuberculosis.

Regulation by Different Types of Chaperones of Amyloid Transformation of Proteins Involved in the Development of Neurodegenerative Diseases.

The review highlights various aspects of the influence of chaperones on amyloid proteins associated with the development of neurodegenerative diseases and includes studies conducted in our laboratory. Different sections of the article are devoted to the role of chaperones in the pathological transformation of alpha-synuclein and the prion protein. Information about the interaction of the chaperonins GroE and TRiC as well as polymer-based artificial chaperones with amyloidogenic proteins is summarized. Particular attention is paid to the effect of blocking chaperones by misfolded and amyloidogenic proteins. It was noted that the accumulation of functionally inactive chaperones blocked by misfolded proteins might cause the formation of amyloid aggregates and prevent the disassembly of fibrillar structures. Moreover, the blocking of chaperones by various forms of amyloid proteins might lead to pathological changes in the vital activity of cells due to the impaired folding of newly synthesized proteins and their subsequent processing. The final section of the article discusses both the little data on the role of gut microbiota in the propagation of synucleinopathies and prion diseases and the possible involvement of the bacterial chaperone GroE in these processes.

Molecular mechanisms in chaperonopathies: clues to understanding the histopathological abnormalities and developing novel therapies.

Molecular chaperones, many of which are heat shock proteins (Hsps), are components of the chaperoning system and when defective can cause disease, the chaperonopathies. Chaperone-gene variants cause genetic chaperonopathies, whereas in the acquired chaperonopathies the genes are normal, but their protein products are not, due to aberrant post-transcriptional mechanisms, e.g. post-translational modifications (PTMs). Since the chaperoning system is widespread in the body, chaperonopathies affect various tissues and organs, making these diseases of interest to a wide range of medical specialties. Genetic chaperonopathies are uncommon but the acquired ones are frequent, encompassing various types of cancer, and inflammatory and autoimmune disorders. The clinical picture of chaperonopathies is known. Much less is known on the impact that pathogenic mutations and PTMs have on the properties and functions of chaperone molecules. Elucidation of these molecular alterations is necessary for understanding the mechanisms underpinning the tissue and organ abnormalities occurring in patients. To illustrate this issue, we discuss structural-functional alterations caused by mutation in the chaperones CCT5 and HSPA9, and PTM effects on Hsp60. The data provide insights into what may happen when CCT5 and HSPA9 malfunction in patients, e.g. accumulation of cytotoxic protein aggregates with tissue destruction; or for Hsp60 with aberrant PTM, degradation and/or secretion of the chaperonin with mitochondrial damage. These and other possibilities are now open for investigation. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Different roles of two groEL homologues in methylotrophic utiliser of dichloromethane Methylorubrum extorquens DM4.

The genome of methylotrophic bacteria Methylorubrum extorquens DM4 contains two homologous groESL operons encoding the 60-kDa and 10-kDa subunits of GroE heat shock chaperones with highly similar amino acid sequences. To test a possible functional redundancy of corresponding GroEL proteins we attempted to disrupt the groEL1 and groEL2 genes. Despite the large number of recombinants analysed and the gentle culture conditions the groEL1-lacking mutant was not constructed suggesting that the loss of GroEL1 was lethal for cells. At the same time the ∆groEL2 strain was viable and varied from the wild-type by increased sensitivity to acid, salt and desiccation stresses as well as by the impaired growth with a toxic halogenated compound-dichloromethane (DCM). The evaluation of activity of putative PgroE1 and PgroE2 promoters using the reporter gene of green fluorescent protein (GFP) showed that the expression of groESL1 operon greatly prevails (about two orders of magnitude) over those of groESL2 under all tested conditions. However the above promoters demonstrated differential regulation in response to stresses. The expression from PgroE1 was heat-inducible, while the activity of PgroE2 was upregulated upon acid shock and cultivation with DCM. Based on these results we conclude that the highly conservative groESL1 operon (old locus tags METDI5839-5840) encodes the housekeeping chaperone essential for fundamental cellular processes. On the contrary the second pair of paralogues (METDI4129-4130) is dispensable, but corresponding GroE2 chaperone promotes the tolerance to acid and salt stresses, in particular, during the growth with DCM.

Sequential allosteric mechanism of ATP hydrolysis by the CCT/TRiC chaperone is revealed through Arrhenius analysis.

Knowing the mechanism of allosteric switching is important for understanding how molecular machines work. The CCT/TRiC chaperonin nanomachine undergoes ATP-driven conformational changes that are crucial for its folding function. Here, we demonstrate that insight into its allosteric mechanism of ATP hydrolysis can be achieved by Arrhenius analysis. Our results show that ATP hydrolysis triggers sequential ?conformational waves." They also suggest that these waves start from subunits CCT6 and CCT8 (or CCT3 and CCT6) and proceed clockwise and counterclockwise, respectively.

A Novel CCT5 Missense Variant Associated with Early Onset Motor Neuropathy.

Diseases associated with acquired or genetic defects in members of the chaperoning system (CS) are increasingly found and have been collectively termed chaperonopathies. Illustrative instances of genetic chaperonopathies involve the genes for chaperonins of Groups I (e.g., Heat shock protein 60, Hsp60) and II (e.g., Chaperonin Containing T-Complex polypeptide 1, CCT). Examples of the former are hypomyelinating leukodystrophy 4 (HLD4 or MitCHAP60) and hereditary spastic paraplegia (SPG13). A distal sensory mutilating neuropathy has been linked to a mutation [p.(His147Arg)] in subunit 5 of the CCT5 gene. Here, we describe a new possibly pathogenic variant [p.(Leu224Val)] of the same subunit but with a different phenotype. This yet undescribed disease affects a girl with early onset demyelinating neuropathy and a severe motor disability. By whole exome sequencing (WES), we identified a homozygous CCT5 c.670C>G p.(Leu224Val) variant in the CCT5 gene. In silico 3D-structure analysis and bioinformatics indicated that this variant could undergo abnormal conformation and could be pathogenic. We compared the patient's clinical, neurophysiological and laboratory data with those from patients carrying p.(His147Arg) in the equatorial domain. Our patient presented signs and symptoms absent in the p.(His147Arg) cases. Molecular dynamics simulation and modelling showed that the Leu224Val mutation that occurs in the CCT5 intermediate domain near the apical domain induces a conformational change in the latter. Noteworthy is the striking difference between the phenotypes putatively linked to mutations in the same CCT subunit but located in different structural domains, offering a unique opportunity for elucidating their distinctive roles in health and disease.

Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR.

The prototypical chaperonin GroEL assists protein folding through an ATP-dependent encapsulation mechanism. The details of how GroEL folds proteins remain elusive, particularly because encapsulation is not an absolute requirement for successful re/folding. Here we make use of a metastable model protein substrate, comprising a triple mutant of Fyn SH3, to directly demonstrate, by simultaneous analysis of three complementary NMR-based relaxation experiments (lifetime line broadening, dark state exchange saturation transfer, and Carr-Purcell-Meinboom-Gill relaxation dispersion), that apo GroEL accelerates the overall interconversion rate between the native state and a well-defined folding intermediate by about 20-fold, under conditions where the "invisible" GroEL-bound states have occupancies below 1%. This is largely achieved through a 500-fold acceleration in the folded-to-intermediate transition of the protein substrate. Catalysis is modulated by a kinetic deuterium isotope effect that reduces the overall interconversion rate between the GroEL-bound species by about 3-fold, indicative of a significant hydrophobic contribution. The location of the GroEL binding site on the folding intermediate, mapped from (15)N, (1)HN, and (13)Cmethyl relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.

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.

A combination of luxR1 and luxR2 genes activates Pr-promoters of psychrophilic Aliivibrio logei lux-operon independently of chaperonin GroEL/ES and protease Lon at high concentrations of autoinducer.

UNLABELLED: Lux-operon of psychrophilic bacteria Aliivibrio logei contains two copies of luxR and is regulated by Type I quorum sensing (QS). Activation of lux-operon of psychrophilic bacteria A. logei by LuxR1 requires about 100 times higher concentrations of autoinducer (AI) than the activation by LuxR2. On the other hand, LuxR1 does not require GroEL/ES chaperonin for its folding and cannot be degraded by protease Lon, while LuxR2 sensitive to Lon and requires GroEL/ES. Here we show that at 10(-5) - 10(-4)М concentrations of AI a combination of luxR1 and luxR2 products is capable of activating the Pr-promoters of A. logei lux-operon in Escherichia coli independently of GroEL/ES and protease Lon. The presence of LuxR1 assists LuxR2 in gro(-) cells when AI was added at high concentration, while at low concentration of AI in a cell LuxR1 decreases the LuxR2 activity. These observations may be explained by the formation of LuxR1/LuxR2 heterodimers that act in complex with AI independently from GroEL/ES and protease Lon. IMPORTANCE: This study expands current understanding of QS regulation in A. logei as it implies cooperative regulation of lux-operon by LuxR1 and LuxR2 proteins.

GroE chaperonins assisted functional expression of bacterial enzymes in Saccharomyces cerevisiae.

Rapid advances in the capabilities of reading and writing DNA along with increasing understanding of microbial metabolism at the systems-level have paved an incredible path for metabolic engineering. Despite these advances, post-translational tools facilitating functional expression of heterologous enzymes in model hosts have not been developed well. Some bacterial enzymes, such as Escherichia coli xylose isomerase (XI) and arabinose isomerase (AI) which are essential for utilizing cellulosic sugars, cannot be functionally expressed in Saccharomyces cerevisiae. We hypothesized and demonstrated that the mismatching of the HSP60 chaperone systems between bacterial and eukaryotic cells might be the reason these bacterial enzymes cannot be functionally expressed in yeast. The results showed that the co-expression of E. coli GroE can facilitate the functional expression of E. coli XI and AI, as well as the Agrobacterium tumefaciens D-psicose epimerase in S. cerevisiae. The co-expression of bacterial chaperonins in S. cerevisiae is a promising post-translational strategy for the functional expression of bacterial enzymes in yeast. Biotechnol. Bioeng. 2016;113: 2149-2155. © 2016 Wiley Periodicals, Inc.

Expression and purification of active, stabilized trimethyllysine hydroxylase.

Trimethyllysine hydroxylase (TMLH) catalyses the first step in carnitine biosynthesis - the conversion of N6,N6,N6-trimethyl-l-lysine to 3-hydroxy-N6,N6,N6-trimethyl-l-lysine. By changing carnitine availability it is possible to optimise cardiac energy metabolism, that is beneficial under certain ischemic conditions. Previous efforts have been devoted towards the inhibition of gamma-butyrobetaine dioxygenase, which catalyses the last step in carnitine biosynthesis. However, the effects of TMLH activity regulation are currently unexplored. To facilitate the development of specific ligands of TMLH, large quantities of recombinant protein are necessary for downstream binding and structural studies. Here, we describe an efficient system for expressing and purifying active and stable TMLH as a maltose-binding protein fusion in Escherichiacoli.

Synonymous and non-synonymous codon substitutions can alleviate dependence on GroEL for folding.

The Escherichia coli GroEL/ES chaperonin system facilitates protein folding in an ATP-driven manner. There are <100 obligate clients of this system in E. coli although GroEL can interact and assist the folding of a multitude of proteins in vitro. It has remained unclear, however, which features distinguish obligate clients from all the other proteins in an E. coli cell. To address this question, we established a system for selecting mutations in mouse dihydrofolate reductase (mDHFR), a GroEL interactor, that diminish its dependence on GroEL for folding. Strikingly, both synonymous and non-synonymous codon substitutions were found to reduce mDHFR's dependence on GroEL. The non-synonymous substitutions increase the rate of spontaneous folding whereas computational analysis indicates that the synonymous substitutions appear to affect translation rates at specific sites.

Protein aggregation and biomolecular condensation in hypoxic environments (Review).

Due to molecular forces, biomacromolecules assemble into liquid condensates or solid aggregates, and their corresponding formation and dissolution processes are controlled. Protein homeostasis is disrupted by increasing age or environmental stress, leading to irreversible protein aggregation. Hypoxic pressure is an important factor in this process, and uncontrolled protein aggregation has been widely observed in hypoxia­related conditions such as neurodegenerative disease, cardiovascular disease, hypoxic brain injury and cancer. Biomolecular condensates are also high­order complexes assembled from macromolecules. Although they exist in different phase from protein aggregates, they are in dynamic balance under certain conditions, and their activation or assembly are considered as important regulatory processes in cell survival with hypoxic pressure. Therefore, a better understanding of the relationship between hypoxic stress, protein aggregation and biomolecular condensation will bring marked benefits in the clinical treatment of various diseases. The aim of the present review was to summarize the underlying mechanisms of aggregate assembly and dissolution induced by hypoxic conditions, and address recent breakthroughs in understanding the role of aggregates in hypoxic­related diseases, given the hypotheses that hypoxia induces macromolecular assemblage changes from a liquid to a solid phase, and that adenosine triphosphate depletion and ATP­driven inactivation of multiple protein chaperones play important roles among the process. Moreover, it is anticipated that an improved understanding of the adaptation in hypoxic environments could extend the overall survival of patients and provide new strategies for hypoxic­related diseases.