Chaperonin-mediated Folding Research Articles

Proteomic analysis of the ATP synthase interactome in notothenioids highlights a pathway that inhibits ceruloplasmin production.

Antarctic notothenioids have unique adaptations that allow them to thrive in subzero Antarctic waters. Within the suborder Notothenioidei, species of the family Channichthyidae (icefish) lack hemoglobin and in some instances myoglobin too. In studies of mitochondrial function of notothenioids, few have focused specifically on ATP synthase. In this study, we find that the icefish Champsocephalus gunnari has a significantly higher level of ATP synthase subunit α expression than the red-blooded Notothenia rossii, but a much smaller interactome than the other species. We characterize the interactome of ATP synthase subunit α in two red-blooded species Trematomus bernacchii, N. rossii, and in the icefish Chionodraco rastrospinosus and C. gunnari and find that, in comparison with the other species, reactome enrichment for C. gunnari lacks chaperonin-mediated protein folding, and fewer oxidative-stress-associated proteins are present in the identified interactome of C. gunnari. Reactome enrichment analysis also identifies a transcript-specific translational silencing pathway for the iron oxidase protein ceruloplasmin, which has previously been reported in studies of icefish as distinct from other red-blooded fish and vertebrates in its activity and RNA transcript expression. Ceruloplasmin protein expression is detected by Western blot in the liver of T. bernacchii, but not in N. rossii, C. rastrospinosus, and C. gunnari. We suggest that the translation of ceruloplasmin transcripts is silenced by the identified pathway in icefish notothenioids, which is indicative of altered iron metabolism and Fe(II) detoxification.

1-Deoxydihydroceramide causes anoxic death by impairing chaperonin-mediated protein folding.

Ischaemic heart disease and stroke are the most common causes of death worldwide. Anoxia, defined as the lack of oxygen, is commonly seen in both these pathologies and triggers profound metabolic and cellular changes. Sphingolipids have been implicated in anoxia injury, but the pathomechanism is unknown. Here we show that anoxia-associated injury causes accumulation of the non-canonical sphingolipid 1-deoxydihydroceramide (DoxDHCer). Anoxia causes an imbalance between serine and alanine resulting in a switch from normal serine-derived sphinganine biosynthesis to non-canonical alanine-derived 1-deoxysphinganine. 1-Deoxysphinganine is incorporated into DoxDHCer, which impairs actin folding via the cytosolic chaperonin TRiC, leading to growth arrest in yeast, increased cell death upon anoxia-reoxygenation in worms and ischaemia-reperfusion injury in mouse hearts. Prevention of DoxDHCer accumulation in worms and in mouse hearts resulted in decreased anoxia-induced injury. These findings unravel key metabolic changes during oxygen deprivation and point to novel strategies to avoid tissue damage and death.

Adapting the Chemistry and/or Biology of Proteostasis to Ameliorate Protein Aggregation Diseases

The cellular protein homeostasis, or proteostasis network, regulates proteome function by controlling ribosomal protein synthesis, chaperone and chaperonin mediated protein folding, protein trafficking, protein degradation and related processes. Stress responsive signaling pathways match proteostasis network capacity with demand in each subcellular compartment to maintain or alter cellular homeostasis. The beginning of the seminar will focus on how the proteostasis network can be adapted pharmacologically through unfolded protein response arm‐selective signaling to alleviate the gain‐of‐toxic‐function diseases, including light chain amyloidosis and the transthyretin amyloidosis, where excessive secretion of misfolding and aggregation of proteins leads to a degenerative phenotypes. This strategy will be contrasted with high affinity small molecule binding to the normally folded structural ensemble of an aggregation‐prone protein inside and/or outside of the cell to stabilize the native state, lowering the population of misfolded, misassembly competent states that lead to aggregates, including amyloid fibrils. Lastly, we will cover the progress made to discover autophagy activators. These drug candidates are envisioned to be generally useful for ameliorating multiple neurodegenerative diseases based on human genetic evidence.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional Contributions of Prefoldin to Gene Expression.

Prefoldin is a co-chaperone that evolutionarily originates in archaea, is universally present in all eukaryotes and acts as a co-chaperone by facilitating the supply of unfolded or partially folded substrates to class II chaperonins. Eukaryotic prefoldin is known mainly for its functional relevance in the cytoplasmic folding of actin and tubulin monomers during cytoskeleton assembly. However, the role of prefoldin in chaperonin-mediated folding is not restricted to cytoskeleton components, but extends to both the assembly of other cytoplasmic complexes and the maintenance of functional proteins by avoiding protein aggregation and facilitatingproteolytic degradation. Evolution has favoured the diversification of prefoldin subunits, and has allowed the so-called prefoldin-like complex, with specialised functions, to appear. Subunits of both canonical and prefoldin-like complexes have also been found in the nucleus of yeast and metazoan cells, where they have been functionally connected with different gene expression steps. Plant prefoldin has also been detected in the nucleus and is physically associated with a gene regulator. Here we summarise information available on the functional involvement of prefoldin in gene expression, and discuss the implications of these results for the relationship between prefoldin structure and function.

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.

GroEL actively stimulates folding of the endogenous substrate protein PepQ

Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli. How chaperonins accelerate protein folding remains controversial. Here we test key predictions of both passive and active models of GroELS-stimulated folding, using the endogenous E. coli metalloprotease PepQ. While GroELS increases the folding rate of PepQ by over 15-fold, we demonstrate that slow spontaneous folding of PepQ is not caused by aggregation. Fluorescence measurements suggest that, when folding inside the GroEL-GroES cavity, PepQ populates conformations not observed during spontaneous folding in free solution. Using cryo-electron microscopy, we show that the GroEL C-termini make physical contact with the PepQ folding intermediate and help retain it deep within the GroEL cavity, resulting in reduced compactness of the PepQ monomer. Our findings strongly support an active model of chaperonin-mediated protein folding, where partial unfolding of misfolded intermediates plays a key role.

Functional specialization in regulation and quality control in thermal adaptive evolution

Distinctive survival strategies, specialized in regulation and in quality control, were observed in thermal adaptive evolution with a laboratory Escherichia coli strain. The two specialists carried a single mutation either within rpoH or upstream of groESL, which led to the activated global regulation by sigma factor 32 or an increased amount of GroEL/ES chaperonins, respectively. Although both specialists succeeded in thermal adaptation, the common winner of the evolution was the specialist in quality control, that is, the strategy of chaperonin-mediated protein folding. To understand this evolutionary consequence, multilevel analyses of cellular status, for example, transcriptome, protein and growth fitness, were carried out. The specialist in quality control showed less change in transcriptional reorganization responding to temperature increase, which was consistent with the finding of that the two specialists showed the biased expression of molecular chaperones. Such repressed changes in gene expression seemed to be advantageous for long-term sustainability because a specific increase in chaperonins not only facilitated the folding of essential gene products but also saved cost in gene expression compared with the overall transcriptional increase induced by rpoH regulation. Functional specialization offered two strategies for successful thermal adaptation, whereas the evolutionary advantageous was more at the points of cost-saving in gene expression and the essentiality in protein folding.

A human CCT5 gene mutation causing distal neuropathy impairs hexadecamer assembly in an archaeal model.

Chaperonins mediate protein folding in a cavity formed by multisubunit rings. The human CCT has eight non-identical subunits and the His147Arg mutation in one subunit, CCT5, causes neuropathy. Knowledge is scarce on the impact of this and other mutations upon the chaperone's structure and functions. To make progress, experimental models must be developed. We used an archaeal mutant homolog and demonstrated that the His147Arg mutant has impaired oligomeric assembly, ATPase activity, and defective protein homeostasis functions. These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog. The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective. Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

Chaperonin-mediated Protein Folding

We have been studying chaperonins these past twenty years through an initial discovery of an action in protein folding, analysis of structure, and elucidation of mechanism. Some of the highlights of these studies were presented recently upon sharing the honor of the 2013 Herbert Tabor Award with my early collaborator, Ulrich Hartl, at the annual meeting of the American Society for Biochemistry and Molecular Biology in Boston. Here, some of the major findings are recounted, particularly recognizing my collaborators, describing how I met them and how our great times together propelled our thinking and experiments.

ATP Dependent Rotational Motion of Group II Chaperonin Observed by X-ray Single Molecule Tracking

Group II chaperonins play important roles in protein homeostasis in the eukaryotic cytosol and in Archaea. These proteins assist in the folding of nascent polypeptides and also refold unfolded proteins in an ATP-dependent manner. Chaperonin-mediated protein folding is dependent on the closure and opening of a built-in lid, which is controlled by the ATP hydrolysis cycle. Recent structural studies suggest that the ring structure of the chaperonin twists to seal off the central cavity. In this study, we demonstrate ATP-dependent dynamics of a group II chaperonin at the single-molecule level with highly accurate rotational axes views by diffracted X-ray tracking (DXT). A UV light-triggered DXT study with caged-ATP and stopped-flow fluorometry revealed that the lid partially closed within 1 s of ATP binding, the closed ring subsequently twisted counterclockwise within 2–6 s, as viewed from the top to bottom of the chaperonin, and the twisted ring reverted to the original open-state with a clockwise motion. Our analyses clearly demonstrate that the biphasic lid-closure process occurs with unsynchronized closure and a synchronized counterclockwise twisting motion.

Enzymatic characterization of a lysin encoded by bacteriophage EL

The bacteriophage EL is a virus that specifically attacks the human pathogen Pseudomonas aeruginosa. This phage carries a large genome that encodes for its own chaperonin which presumably facilitates the proper folding of phage proteins independently of the host chaperonin system. EL also encodes a lysin enzyme, a critical component of the lytic cycle that is responsible for digesting the peptidoglycan layer of the host cell wall. Previously, this lysin was believed to be a substrate of the chaperonin encoded by phage EL. In order to characterize the activity of the EL lysin, and to determine whether lysin activity is contingent on chaperonin-mediated folding, a series of peptidoglycan hydrolysis activity assays were performed. Results indicate that the EL-encoded lysin has similar enzymatic activity to that of the Gallus gallus lysozyme and that the EL lysin folds into a functional enzyme in the absence of phage chaperonin and should not be considered a substrate.

P. falciparum cpn20 Is a Bona Fide Co-Chaperonin That Can Replace GroES in E. coli

Human malaria is among the most ubiquitous and destructive tropical, parasitic diseases in the world today. The causative agent, Plasmodium falciparum, contains an unusual, essential organelle known as the apicoplast. Inhibition of this degenerate chloroplast results in second generation death of the parasite and is the mechanism by which antibiotics function in treating malaria. In order to better understand the biochemistry of this organelle, we have cloned a putative, 20 kDa, co-chaperonin protein, Pf-cpn20, which localizes to the apicoplast. Although this protein is homologous to the cpn20 that is found in plant chloroplasts, its ability to function as a co-chaperonin was questioned in the past. In the present study, we carried out a structural analysis of Pf-cpn20 using circular dichroism and analytical ultracentrifugation and then used two different approaches to investigate the ability of this protein to function as a co-chaperonin. In the first approach, we purified recombinant Pf-cpn20 and tested its ability to act as a co-chaperonin for GroEL in vitro, while in the second, we examined the ability of Pf-cpn20 to complement an E. coli depletion of the essential bacterial co-chaperonin GroES. Our results demonstrate that Pf-cpn20 is fully functional as a co-chaperonin in vitro. Moreover, the parasitic co-chaperonin is able to replace GroES in E. coli at both normal and heat-shock temperatures. Thus, Pf-cpn20 functions as a co-chaperonin in chaperonin-mediated protein folding. The ability of the malarial protein to function in E. coli suggests that this simple system can be used as a tool for further analyses of Pf-cpn20 and perhaps other chaperone proteins from P. falciparum.

Chaperonin-mediated protein folding 1

Chaperonin-mediated protein folding 1

Chaperonin-mediated protein folding 2

Chaperonin-mediated protein folding 2

Chaperonin-Ring's Twist is Critical for Folding Activity of Group II Chaperonin

Group II chaperonin is an indispensable protein in archaea or in the eukaryotic cytosol that captures an unfolded protein and refolds it to the correct conformation in an ATP dependent manner. Chaperonin-mediated protein folding is achieved on the closure and opening of a built-in lid under ATP cycle. Recent study with cryo-electron microscopy and single particle analysis suggested that the ring structure of chaperonin twisted to seal off the central cavity. However such dynamics could not be traced experimentally since the motion was expected to be too small to detect by visible light technology.Here we show that ATP dependent dynamic motion of group II chaperonin from Thermococcus strain KS-1 at single molecular level with high accurate rotational view by diffracted X-ray tracking (DXT). DXT has been considered as a powerful technique in biological science for detecting subtle dynamic motion of the target protein at single molecular level. The dynamics of a single protein can be monitored through trajectory of the Laue spot from the nanocrystal which was labeled on the objective protein immobilized on the substrate surface. UV-light triggered DXT using caged ATP revealed that the chaperonin’s lid closed partially within one second after ATP binding, the closed ring twisted counter clock-wisely from top to bottom view of chaperonin with 130 m rad/sec in angular speed within 2 to 6 seconds, and the twisted ring turned back to the original open state with 80 m rad/sec clockwise twisting motion. Our analyses with precise rotational and macroscopic views for chaperonin’s dynamics show that there are distinct two modes in lid-closure process, in-cooperative closure and cooperative counter-clockwise twisting motion. Moreover, we found that the ring’s twisting motion correlated to the folding activity of group II chaperonin.

Mechanism of nucleotide sensing in group II chaperonins

Group II chaperonins mediate protein folding in an ATP-dependent manner in eukaryotes and archaea. The binding of ATP and subsequent hydrolysis promotes the closure of the multi-subunit rings where protein folding occurs. The mechanism by which local changes in the nucleotide-binding site are communicated between individual subunits is unknown. The crystal structure of the archaeal chaperonin from Methanococcus maripaludis in several nucleotides bound states reveals the local conformational changes associated with ATP hydrolysis. Residue Lys-161, which is extremely conserved among group II chaperonins, forms interactions with the γ-phosphate of ATP but shows a different orientation in the presence of ADP. The loss of the ATP γ-phosphate interaction with Lys-161 in the ADP state promotes a significant rearrangement of a loop consisting of residues 160-169. We propose that Lys-161 functions as an ATP sensor and that 160-169 constitutes a nucleotide-sensing loop (NSL) that monitors the presence of the γ-phosphate. Functional analysis using NSL mutants shows a significant decrease in ATPase activity, suggesting that the NSL is involved in timing of the protein folding cycle.

Gly-345 plays an essential role in Pyrococcus furiosus chaperonin function

Compared to the group I chaperonins, such as Escherichia coli GroEL, which facilitate protein folding, many aspects of the functional mechanism of archaeal group II chaperonins are unclear. Sequence homology between the chaperonin from Pyrococcus furiosus (PfCPN) and other group II chaperonins, together with the homo-oligomeric nature of PfCPN, suggest that PfCPN may serve as a model to clarify the role of the homologous position Gly-345 in the chaperonin-mediated protein folding. Here, we show that the purified chaperonin mutant in which the conserved residue Gly-345 is replaced by Asp (G345D) displays only about 25% ATP/ADP hydrolysis activities of the wild-type in the presence of Co(2+) and has a reduced capacity to promote folding of denatured malate dehydrogenase in vitro. This may be a reflection that Gly-345 plays an essential role in conformational change and protein refolding by archaeal group II chaperonins.

Chaperonin-Mediated Protein Folding

Chaperonins are remarkable ring assemblies that provide kinetic assistance to protein folding to the native state. They function through the action of the central cavities of their rings. The best studied such system is bacterial GroEL and its cooperating co-chaperonin GroES, which mediate productive folding of a large number of proteins in the presence of ATP. I will recount the early functional studies of chaperonins in mitochondria, carried out collaboratively with Ulrich Hartl, and describe our subsequent X-ray, EM, and biochemical studies of GroEL leading to an understanding of the structure and reaction cycle, studies carried out with Paul Sigler and Helen Saibil. The structure and mechanism studies revealed that an open ring of GroEL exposes hydrophobic surfaces that capture non-native proteins and prevent them from aggregation. In the presence of ATP, which binds cooperatively in the seven equatorial sites of an open ring, the cochaperonin ring GroES associates, producing large rigid body movements of the apical and intermediate domains of the bound, so-called cis ring, releasing substrate protein into a now-encapsulated central cavity that has hydrophilic walls. Folding occurs in this space in what is the longest-lived state of the reaction cycle. ATP hydrolysis gates rapid entry of ATP into the opposite ring, which both allosterically ejects the cis ligands and sets up a new folding active ring. I will present recent cryoEM studies of the allosteric movements driven by ATP binding, which both facilitate substrate protein binding and enable initial contact of GroES with GroEL, carried out with Helen Saibil, as well as discuss cis folding studies, carried out in part with Kurt Wüthrich.

Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber

Group II chaperonins are ATP-dependent ring-shaped complexes that bind nonnative polypeptides and facilitate protein folding in archaea and eukaryotes. A built-in lid encapsulates substrate proteins within the central chaperonin chamber. Here, we describe the fate of the substrate during the nucleotide cycle of group II chaperonins. The chaperonin substrate-binding sites are exposed, and the lid is open in both the ATP-free and ATP-bound prehydrolysis states. ATP hydrolysis has a dual function in the folding cycle, triggering both lid closure and substrate release into the central chamber. Notably, substrate release can occur in the absence of a lid, and lid closure can occur without substrate release. However, productive folding requires both events, so that the polypeptide is released into the confined space of the closed chamber where it folds. Our results show that ATP hydrolysis coordinates the structural and functional determinants that trigger productive folding.

Mechanism of folding chamber closure in a group II chaperonin

Group II chaperonins are essential mediators of cellular protein folding in eukaryotes and archaea. These oligomeric protein machines, ~1MDa, consist of two back-to-back rings encompassing a central cavity that accommodates polypeptide substrates1,2,3. Chaperonin-mediated protein folding is critically dependent on the closure of a built-in lid4,5, which is triggered by ATP hydrolysis6. The structural rearrangements and molecular events leading to lid closure are still unknown. Here, we report four single particle cryo-EM structures of Mm-cpn, an archaeal group II chaperonin5,7, in the nucleotide-free (open) and nucleotide-induced (closed) states. The 4.3 Å resolution of the closed conformation allowed building of the first ever atomic model directly from the cryo-EM density map, in which we were able to visualize the nucleotide and over 70% of the sidechains. The model of the open conformation was obtained by using the deformable elastic network modeling with the 8 Å resolution open state cryo-EM density restraints. Together, the open and closed structures reveal how local conformational changes triggered by ATP hydrolysis lead to an alteration of intersubunit contacts within and across the rings, ultimately causing a rocking motion that closes the ring. Our analysis reveals an intricate and unforeseen set of interactions controlling allosteric communication and inter-ring signaling driving the conformational cycle of group II chaperonins. Beyond this, we anticipate our methodology of combining single particle cryo-EM and computational modeling will become a powerful tool in the determination of atomic details involved in the dynamic processes of macromolecular machines in solution.