Insights into chaperonin function from studies on archaeal thermosomes

It is now well understood that, although proteins fold spontaneously (in a thermodynamic sense), many nevertheless require the assistance of helpers called molecular chaperones to reach their correct and active folded state in living cells. This is because the pathways of protein folding are full of traps for the unwary: the forces that drive proteins into their folded states can also drive them into insoluble aggregates, and, particularly when cells are stressed, this can lead, without prevention or correction, to cell death. The chaperonins are a family of molecular chaperones, practically ubiquitous in all living organisms, which possess a remarkable structure and mechanism of action. They act as nanoboxes in which proteins can fold, isolated from their environment and from other partners with which they might, with potentially deleterious consequences, interact. The opening and closing of these boxes is timed by the binding and hydrolysis of ATP. The chaperonins which are found in bacteria are extremely well characterized, and, although those found in archaea (also known as thermosomes) and eukaryotes have received less attention, our understanding of these proteins is constantly improving. This short review will summarize what we know about chaperonin function in the cell from studies on the archaeal chaperonins, and show how recent work is improving our understanding of this essential class of molecular chaperones.     

Insights into the MCM functional mechanism: lessons learned from the archaeal MCM complex

The helicase function of the minichromosome maintenance protein (MCM) is essential for genomic DNA replication in archaea and eukaryotes. There has been rapid progress in studies of the structure and function of MCM proteins from different organisms, leading to better understanding of the MCM helicase mechanism. Because there are a number of excellent reviews on this topic, we will use this review to summarize some of the recent progress, with particular focus on the structural aspects of MCM and their implications for helicase function. Given the hexameric and double hexameric architecture observed by X-ray crystallography and electron microscopy of MCMs from archaeal and eukaryotic cells, we summarize and discuss possible unwinding modes by either a hexameric or a double hexameric helicase. Additionally, our recent crystal structure of a full length archaeal MCM has provided structural information on an intact, multi-domain MCM protein, which includes the salient features of four unusual β-hairpins from each monomer, and the side channels of a hexamer/double hexamer. These new structural data enable a closer examination of the structural basis of the unwinding mechanisms by MCM.     

Insights into peptide and protein function: a convergent approach.

From viruses to multicellular organisms, life is inseparable from the genetic instructions aimed at regulating its maintenance, division, multiplication, differentiation and death (apoptosis). Over the past 15 years, structural studies have begun to resolve the complex reactions involved in these fundamental processes in biology. The three-dimensional representations of the complexes formed with peptides and/or proteins have allowed interpretation of the biochemical data and formulation of novel hypotheses about the control and execution of these processes. Moreover, they have opened the way to rational approaches for designing compounds able to interfere with these crucial events in normal or pathological conditions. Various results obtained in our laboratory in these fields are briefly summarized in this review.     

The archaeal molecular chaperone machine: peculiarities and paradoxes.

A major finding within the field of archaea and molecular chaperones has been the demonstration that, while some species have the stress (heat-shock) gene hsp70(dnaK), others do not. This gene encodes Hsp70(DnaK), an essential molecular chaperone in bacteria and eukaryotes. Due to the physiological importance and the high degree of conservation of this protein, its absence in archaeal organisms has raised intriguing questions pertaining to the evolution of the chaperone machine as a whole and that of its components in particular, namely, Hsp70(DnaK), Hsp40(DnaJ), and GrpE. Another archaeal paradox is that the proteins coded by these genes are very similar to bacterial homologs, as if the genes had been received via lateral transfer from bacteria, whereas the upstream flanking regions have no bacterial markers, but instead have typical archaeal promoters, which are like those of eukaryotes. Furthermore, the chaperonin system in all archaea studied to the present, including those that possess a bacterial-like chaperone machine, is similar to that of the eukaryotic-cell cytosol. Thus, two chaperoning systems that are designed to interact with a compatible partner, e.g., the bacterial chaperone machine physiologically interacts with the bacterial but not with the eucaryal chaperonins, coexist in archaeal cells in spite of their apparent functional incompatibility. It is difficult to understand how these hybrid characteristics of the archaeal chaperoning system became established and work, if one bears in mind the classical ideas learned from studying bacteria and eukaryotes. No doubt, archaea are intriguing organisms that offer an opportunity to find novel molecules and mechanisms that will, most likely, enhance our understanding of the stress response and the protein folding and refolding processes in the three phylogenetic domains.     

Insights into the mechanism of progressive RNA degradation by the archaeal exosome

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.     

MD-Miner: a network-based approach for personalized drug repositioning

Background

Due to advances in next generation sequencing technologies and corresponding reductions in cost, it is now attainable to investigate genome-wide gene expression and variants at a patient-level, so as to better understand and anticipate heterogeneous responses to therapy. Consequently, it is feasible to inform personalized drug treatment decisions using personal genomics data. However, these efforts are limited due to a lack of reliable computational approaches for predicting effective drugs for individual patients. The reverse gene set enrichment analysis (i.e., connectivity mapping) approach and its variants have been widely and successfully used for drug prediction. However, the performance of these methods is limited by undefined mechanism of action (MoA) of drugs and reliance on cohorts of patients rather than personalized predictions for individual patients.

Results

In this study, we have developed and evaluated a computational approach, known as Mechanism and Drug Miner (MD-Miner), using a network-based computational approach to predict effective drugs and reveal potential drug mechanisms of action at the level of signaling pathways. Specifically, the patient-specific signaling network is constructed by integrating known disease associated genes with patient-derived gene expression profiles. In parallel, a drug mechanism of action network is constructed by integrating drug targets and z-score profiles of drug-induced gene expression (pre vs. post-drug treatment). Potentially effective candidate drugs are prioritized according to the number of common genes between the patient-specific dysfunctional signaling network and drug MoA network. We evaluated the MD-Miner method on the PC-3 prostate cancer cell line, and showed that it significantly improved the success rate of discovering effective drugs compared with the random selection, and could provide insight into potential mechanisms of action.

Conclusions

This work provides a signaling network-based drug repositioning approach. Compared with the reverse gene signature based drug repositioning approaches, the proposed method can provide clues of mechanism of action in terms of signaling transduction networks.

     

Network medicine: a network-based approach to human disease

Given the functional interdependencies between the molecular components in a human cell, a disease is rarely a consequence of an abnormality in a single gene, but reflects the perturbations of the complex intracellular and intercellular network that links tissue and organ systems. The emerging tools of network medicine offer a platform to explore systematically not only the molecular complexity of a particular disease, leading to the identification of disease modules and pathways, but also the molecular relationships among apparently distinct (patho)phenotypes. Advances in this direction are essential for identifying new disease genes, for uncovering the biological significance of disease-associated mutations identified by genome-wide association studies and full-genome sequencing, and for identifying drug targets and biomarkers for complex diseases.     

Insights into Hsp70 chaperone activity from a crystal structure of the yeast Hsp110 Sse1

Classic Hsp70 chaperones assist in diverse processes of protein folding and translocation, and Hsp110s had seemed by sequence to be distant relatives within an Hsp70 superfamily. The 2.4 A resolution structure of Sse1 with ATP shows that Hsp110s are indeed Hsp70 relatives, and it provides insight into allosteric coupling between sites for ATP and polypeptide-substrate binding in Hsp70s. Subdomain structures are similar in intact Sse1(ATP) and in the separate Hsp70 domains, but conformational dispositions are radically different. Interfaces between Sse1 domains are extensive, intimate, and conservative in sequence with Hsp70s. We propose that Sse1(ATP) may be an evolutionary vestige of the Hsp70(ATP) state, and an analysis of 64 mutant variants in Sse1 and three Hsp70 homologs supports this hypothesis. An atomic-level understanding of Hsp70 communication between ATP and substrate-binding domains follows. Requirements on Sse1 for yeast viability are in keeping with the distinct function of Hsp110s as nucleotide exchange factors.     

New insights into the archaeal diversity of a hypersaline microbial mat obtained by a metagenomic approach

A metagenomic approach was carried out in order to study the genetic pool of a hypersaline microbial mat, paying more attention to the archaeal community and, specifically, to the putatively methanogenic members. The main aim of the work was to expand the knowledge of a likely ecologically important archaeal lineage, candidate division MSBL1, which is probably involved in methanogenesis at very high salinities.     

Insights into the Clp/HSP100 chaperone system from chloroplasts of Arabidopsis thaliana

HSP100 proteins are molecular chaperones involved in protein quality control. They assist in protein (un)folding, prevent aggregation, and are thought to participate in precursor translocation across membranes. Caseinolytic proteins ClpC and ClpD from plant chloroplasts belong to the HSP100 family. Their role has hitherto been investigated by means of physiological studies and reverse genetics. In the present work, we employed an in vitro approach to delve into the structural and functional characteristics of ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). They were expressed in Escherichia coli and purified to near-homogeneity. The proteins were detected mainly as dimers in solution, and, upon addition of ATP, the formation of hexamers was observed. Both proteins exhibited basal ATPase activity (K(m), 1.42 mm, V(max), 0.62 nmol/(min × μg) for AtClpC2 and K(m) ～19.80 mm, V(max) ～0.19 nmol/(min × μg) for AtClpD). They were able to reactivate the activity of heat-denatured luciferase (～40% for AtClpC2 and ～20% for AtClpD). The Clp proteins tightly bound a fusion protein containing a model transit peptide. This interaction was detected by binding assays, where the chaperones were selectively trapped by the transit peptide-containing fusion, immobilized on glutathione-agarose beads. Association of HSP100 proteins to import complexes with a bound transit peptide-containing fusion was also observed in intact chloroplasts. The presented data are useful to understand protein quality control and protein import into chloroplasts in plants.     

Insights into a mummy: a paleoradiological analysis

The aim of the study was to analyze possible human skeletal remains within the wrappings of a mummy from the Archaeological Museum, Zagreb, Croatia through the use of the multidetector CT (MDCT) technology. Plain X-ray films and MDCT images of the mummy were taken in both frontal and lateral views. In a single volumetric acquisition of the whole body by MDCT 0.75 mm axial slices were obtained and combined with sagittal and coronal reformatting and three-dimensional (3D) reconstruction. Sex and age was assessed visually using standard anthropological methods. The results suggest that the mummy was of an adult female, most likely over 40 years of age at death. Pathologies observed included degenerative changes on the vertebral column and healed fractures of the lower right arm. Damage of the ethmoid bone at the roof of the nasal cavity was most likely caused by mortuary brain removal practice. Remnants of a resin and an unusual object were found inside the cranial cavity. An elongated metal object and additional three metal "belts" can be seen on the lower portion of the body. All internal organs were removed and thoracic and abdominal cavities were filled with various substances, most likely mud and pieces of linen cloth. Our results show that the MDCT is a very useful technique for assessing the human remains in archeological samples, especially in comparison to the use of plain film (X-ray), where important details are obscured and 3D imaging impossible.     

Insight into initiator-DNA interactions: a lesson from the archaeal ORC

Although initiation of DNA replication is considered to be highly coordinated through multiple protein-DNA and protein-protein interactions, it is poorly understood how particular locations within the eukaryotic chromosome are selected as origins of DNA replication. Here, we discuss recent reports that present structural information on the interaction characteristics of the archaeal orthologues of the eukaryotic origin recognition complex with their cognate binding sequences. Since the archaeal replication system is postulated as a simplified version of the one in eukaryotes, by analogy, these works provide insights into the functions of the eukaryotic initiator proteins.     

Transient interactions of a slow-folding protein with the Hsp70 chaperone machinery

Most known proteins have at least one local Hsp70 chaperone binding site. Does this mean that all proteins interact with Hsp70 as they fold? This study makes an initial step to address the above question by examining the interaction of the E.coli Hsp70 chaperone (known as DnaK) and its co-chaperones DnaJ and GrpE with a slow-folding E.coli substrate, RNase H^D. Importantly, this protein is a nonobligatory client, and it is able to fold in vitro even in the absence of chaperones. We employ stopped-flow mixing, chromatography, and activity assays to analyze the kinetic perturbations induced by DnaK/DnaJ/GrpE (K/J/E) on the folding of RNase H^D. We find that K/J/E slows down RNase H^D''s apparent folding, consistent with the presence of transient chaperone-substrate interactions. However, kinetic retardation is moderate for this slow-folding client and it is expected to be even smaller for faster-folding substrates. Given that the interaction of folding-competent substrates such as RNase H^D with the K/J/E chaperones is relatively short-lived, it does not significantly interfere with the timely production of folded biologically active substrate. The above mode of action is important because it preserves K/J/E bioavailability, enabling this chaperone system to act primarily by assisting the folding of other misfolded and (or) aggregation-prone cellular proteins that are unable to fold independently. When refolding is carried out in the presence of K/J and absence of the nucleotide exchange factor GrpE, some of the substrate population becomes trapped as a chaperone-bound partially unfolded state.     

Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group

The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the 'Thaumarchaeota' and 'Korarchaeota'. Here, we show the genome sequence of Candidatus 'Caldiarchaeum subterraneum' that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea.     

MtGimC, a novel archaeal chaperone related to the eukaryotic chaperonin cofactor GimC/prefoldin

Group II chaperonins in the eukaryotic and archaeal cytosol assist in protein folding independently of the GroES-like cofactors of eubacterial group I chaperonins. Recently, the eukaryotic chaperonin was shown to cooperate with the hetero-oligomeric protein complex GimC (prefoldin) in folding actin and tubulins. Here we report the characterization of the first archaeal homologue of GimC, from Methanobacterium thermoautotrophicum. MtGimC is a hexamer of 87 kDa, consisting of two alpha and four beta subunits of high alpha-helical content that are predicted to contain extended coiled coils and represent two evolutionarily conserved classes of Gim subunits. Reconstitution experiments with MtGimC suggest that two subunits of the alpha class (archaeal Gimalpha and eukaryotic Gim2 and 5) form a dimer onto which four subunits of the beta class (archaeal Gimbeta and eukaryotic Gim1, 3, 4 and 6) assemble. MtGimalpha and beta can form hetero-complexes with yeast Gim subunits and MtGimbeta partially complements yeast strains lacking Gim1 and 4. MtGimC is a molecular chaperone capable of stabilizing a range of non-native proteins and releasing them for subsequent chaperonin-assisted folding. In light of the absence of Hsp70 chaperones in many archaea, GimC may fulfil an ATP-independent, Hsp70-like function in archaeal de novo protein folding.     

The Hsp90 chaperone machinery: conformational dynamics and regulation by co-chaperones

Hsp90 is a dimeric molecular chaperone required for the activation and stabilization of numerous client proteins many of which are involved in essential cellular processes like signal transduction pathways. This activation process is regulated by ATP-induced large conformational changes, co-chaperones and posttranslational modifications. For some co-chaperones, a detailed picture on their structures and functions exists, for others their contributions to the Hsp90 system is still unclear. Recent progress on the conformational dynamics of Hsp90 and how co-chaperones affect the Hsp90 chaperone cycle significantly increased our understanding of the gearings of this complex molecular machinery. This article is part of a Special Issue entitled: Heat Shock Protein 90 (Hsp90).     

Structural analysis of protein folding by the long-chain archaeal chaperone FKBP26

In the cell, protein folding is mediated by folding catalysts and chaperones. The two functions are often linked, especially when the catalytic module forms part of a multidomain protein, as in Methanococcus jannaschii peptidyl-prolyl cis/trans isomerase FKBP26. Here, we show that FKBP26 chaperone activity requires both a 50-residue insertion in the catalytic FKBP domain, also called ‘Insert-in-Flap’ or IF domain, and an 80-residue C-terminal domain. We determined FKBP26 structures from four crystal forms and analyzed chaperone domains in light of their ability to mediate protein-protein interactions. FKBP26 is a crescent-shaped homodimer. We reason that folding proteins are bound inside the large crescent cleft, thus enabling their access to inward-facing peptidyl-prolyl cis/trans isomerase catalytic sites and ipsilateral chaperone domain surfaces. As these chaperone surfaces participate extensively in crystal lattice contacts, we speculate that the observed lattice contacts reflect a proclivity for protein associations and represent substrate interactions by FKBP26 chaperone domains. Finally, we find that FKBP26 is an exceptionally flexible molecule, suggesting a mechanism for nonspecific substrate recognition.