Friends in need: how chaperonins recognize and remodel proteins that require folding assistance

Chaperonins are biological nanomachines that help newly translated proteins to fold by rescuing them from kinetically trapped misfolded states. Protein folding assistance by the chaperonin machinery is obligatory in vivo for a subset of proteins in the bacterial proteome. Chaperonins are large oligomeric complexes, with unusual seven fold symmetry (group I) or eight/nine fold symmetry (group II), that form double-ring constructs, enclosing a central folding chamber. Dramatic large-scale conformational changes, that take place during ATP-driven cycles, allow chaperonins to bind misfolded proteins, encapsulate them into the expanded cavity and release them back into the cellular environment, regardless of whether they are folded or not. The theory associated with the iterative annealing mechanism, which incorporated the conformational free energy landscape description of protein folding, \textit{quantitatively} explains most, if not all, the available data. Misfolded conformations are associated with low energy minima in a rugged energy landscape. Random disruptions of these low energy conformations result in higher free energy, less folded, conformations that can stochastically partition into the native state. Group I chaperonins (GroEL homologues in eubacteria and endosymbiotic organelles), recognize a large number of misfolded proteins non-specifically and operate through highly coordinated cooperative motions. By contrast, the less well understood group II chaperonins (CCT in Eukarya and thermosome/TF55 in Archaea), assist a selected set of substrate proteins. Chaperonins are implicated in bacterial infection, autoimmune disease, as well as protein aggregation and degradation diseases. Understanding the chaperonin mechanism and their substrates is important not only for the fundamental aspect of cellular protein folding, but also for effective therapeutic strategies.Comment: 26 pages, 4 figures, to be published in Frontiers in Molecular Bioscience

Difference in the distribution pattern of substrate enzymes in the metabolic network of Escherichia coli, according to chaperonin requirement

<p>Abstract</p> <p>Background</p> <p>Chaperonins are important in living systems because they play a role in the folding of proteins. Earlier comprehensive analyses identified substrate proteins for which folding requires the chaperonin GroEL/GroES (GroE) in <it>Escherichia coli</it>, and they revealed that many chaperonin substrates are metabolic enzymes. This result implies the importance of chaperonins in metabolism. However, the relationship between chaperonins and metabolism is still unclear.</p> <p>Results</p> <p>We investigated the distribution of chaperonin substrate enzymes in the metabolic network using network analysis techniques as a first step towards revealing this relationship, and found that as chaperonin requirement increases, substrate enzymes are more laterally distributed in the metabolic. In addition, comparative genome analysis showed that the chaperonin-dependent substrates were less conserved, suggesting that these substrates were acquired later on in evolutionary history.</p> <p>Conclusions</p> <p>This result implies the expansion of metabolic networks due to this chaperonin, and it supports the existing hypothesis of acceleration of evolution by chaperonins. The distribution of chaperonin substrate enzymes in the metabolic network is inexplicable because it does not seem to be associated with individual protein features such as protein abundance, which has been observed characteristically in chaperonin substrates in previous works. However, it becomes clear by considering this expansion process due to chaperonin. This finding provides new insights into metabolic evolution and the roles of chaperonins in living systems.</p

Recognition of the CCT5 di-glu degron by CRL4(DCAF12) is incompatible with TRiC assembly

The molecular processes within all living organisms rely on the correct functioning of proteins, most of which must assemble into multimeric complexes of defined architecture and composition. In eukaryotes, assembly quality control (AQC) E3 ubiquitin ligases target incomplete or incorrectly assembled protein complexes for degradation to ensure protein complex functionality and proteostasis. The CUL4-RBX1-DDB1-DCAF12 (CRL4(DCAF12)) E3 ubiquitin ligase induces the proteasomal degradation of proteins with a C-terminal double glutamate (di-Glu) motif. Putative CRL4(DCAF12) substrates include CCT5, a subunit of the eukaryotic TRiC chaperonin. TRiC is responsible for the folding of around 10% of the human proteome. Its functionality relies on the correct arrangement of its eight subunits, but how TRiC assembly is ensured has not yet been investigated. Furthermore, how DCAF12 recognizes its substrates is unknown. Here the cryo-EM structure of the CCT5-bound DDB1-DCAF12 complex at 2.8 Å resolution is presented. DCAF12 serves as a canonical WD40 DCAF substrate receptor and uses a positively charged pocket at the center of its β-propeller to bind the C-terminus of CCT5. DCAF12 specifically reads out the CCT5 di-Glu side chains, and contacts other visible degron amino acids through weaker Van der Waals interactions, explaining the flexibility in substrate recognition. The CCT5 C-terminus is inaccessible in an assembled TRiC complex, and functional assays demonstrate that CRL4(DCAF12) binds and ubiquitinates monomeric CCT5, but not TRiC. The presented results suggest a previously unanticipated AQC role for the CRL4(DCAF12) E3 ligase towards TRiC, and likely other complexes

GENETIC CHAPERONOPATHIES ASSOCIATED WITH GROUP II CHAPERONIN VARIANTS

Genetic chaperonopathies manifest themselves from very early in life. Chaperonopathies related to neurodegenerative disorders discussed in “Introduction” section are a heterogeneous group of disorders which affect one or more of the various physiological systems, for example, the nervous system. This heterogeneity is due, in particular, to the not fully known molecular activity, which every single molecular chaperone has within a specific tissue. My general questions about them were 1) why a mutation on a molecular chaperone that is expressed by most, if not all cytotypes, seems to affect the functioning of a single physiological system? 2) why do different mutations on the same molecular chaperone cause apparently different pathologies especially in terms of clinical manifestations? This heterogeneity limits the research approach on diseases, which now is conducted towards every single mutation without being able to generalize a unique molecular process. I spent the first 18 months of my Ph.D. project at the SBARRO Department of Temple University in Philadelphia to study the V98I mutation on chaperone Hsp60 causing hereditary spastic paraplegia (SPG13). For a better understanding of the associated diseases, it would be highly beneficial to examine the impact of mutant chaperone genes during development, starting with fertilization and proceeding throughout the entire ontogenetic process. Zebrafish is amenable to such embryonal analysis as well as studies during adulthood. In addition, the zebrafish genome contains a wide range of genes encoding proteins similar to those that form the chaperoning system in humans. Due to the very complex roles played by Hsp60 in cell and tissue homeostasis, the gene is highly conserved during evolution. Nucleotide and amino acid sequences of Hsp60 in zebrafish have 88% of identity with its human orthologous. The first aim of my research was the establishment of a zebrafish model as an innovative approach for the study of the molecular basis of SPG13 and define the role of missense mutations V98I. In the last 18 months of my Ph.D. project, I was at the BIND Department of the University of Palermo and I had the possibility to study a new mutation that occurred in subunit number 5 of CCT complex. This mutation was found in a pediatric patient who is now being treated by the Department Of Sciences For The Promotion Of Health And Childhood "G. D'Alessandro" at the University of Palermo. Thus, I focused my attention on this novel variant. The main aims were 1) understanding, with the help of bioinformatics software, the type of mutation and if it causes some alteration of chaperonin molecular anatomy; 2) define the morphological changes caused by the mutation in skeletal muscle tissue

Experimental Milestones in the Discovery of Molecular Chaperones as Polypeptide Unfolding Enzymes.

Molecular chaperones control the cellular folding, assembly, unfolding, disassembly, translocation, activation, inactivation, disaggregation, and degradation of proteins. In 1989, groundbreaking experiments demonstrated that a purified chaperone can bind and prevent the aggregation of artificially unfolded polypeptides and use ATP to dissociate and convert them into native proteins. A decade later, other chaperones were shown to use ATP hydrolysis to unfold and solubilize stable protein aggregates, leading to their native refolding. Presently, the main conserved chaperone families Hsp70, Hsp104, Hsp90, Hsp60, and small heat-shock proteins (sHsps) apparently act as unfolding nanomachines capable of converting functional alternatively folded or toxic misfolded polypeptides into harmless protease-degradable or biologically active native proteins. Being unfoldases, the chaperones can proofread three-dimensional protein structures and thus control protein quality in the cell. Understanding the mechanisms of the cellular unfoldases is central to the design of new therapies against aging, degenerative protein conformational diseases, and specific cancers

Chaperones convert the energy from ATP into the nonequilibrium stabilization of native proteins.

During and after protein translation, molecular chaperones require ATP hydrolysis to favor the native folding of their substrates and, under stress, to avoid aggregation and revert misfolding. Why do some chaperones need ATP, and what are the consequences of the energy contributed by the ATPase cycle? Here, we used biochemical assays and physical modeling to show that the bacterial chaperones GroEL (Hsp60) and DnaK (Hsp70) both use part of the energy from ATP hydrolysis to restore the native state of their substrates, even under denaturing conditions in which the native state is thermodynamically unstable. Consistently with thermodynamics, upon exhaustion of ATP, the metastable native chaperone products spontaneously revert to their equilibrium non-native states. In the presence of ATPase chaperones, some proteins may thus behave as open ATP-driven, nonequilibrium systems whose fate is only partially determined by equilibrium thermodynamics

Assisted protein folding at low temperature: evolutionaryadaptation of the Antarctic fish chaperonin CCT and its clientproteins

Eukaryotic ectotherms of the Southern Ocean face energetic challenges to protein folding assisted by the cytosolic chaperonin CCT. We hypothesize that CCT and its client proteins (CPs) have co-evolved molecular adaptations that facilitate CCT–CP interaction and the ATP-driven folding cycle at low temperature. To test this hypothesis, we compared the functional and structural properties of CCT–CP systems from testis tissues of an Antarctic fish, Gobionotothen gibberifrons (Lo¨nnberg) (habitat/body T=-1.9 to +2˚C), and of the cow (body T=37˚C). We examined the temperature dependence of the binding of denatured CPs (bactin, b-tubulin) by fish and bovine CCTs, both in homologous and heterologous combinations and at temperatures between 24˚C and 20˚C, in a buffer conducive to binding of the denatured CP to the open conformation of CCT. In homologous combination, the percentage of G. gibberifrons CCT bound to CP declined linearly with increasing temperature, whereas the converse was true for bovine CCT. Binding of CCT to heterologous CPs was low, irrespective of temperature. When reactions were supplemented with ATP, G. gibberifrons CCT catalyzed the folding and release of actin at 2˚C. The ATPase activity of apo-CCT from G. gibberifrons at 4˚C was, 2.5-fold greater than that of apo-bovine CCT, whereas equivalent activities were observed at 20˚C. Based on these results, we conclude that the catalytic folding cycle of CCT from Antarctic fishes is partially compensated at their habitat temperature, probably by means of enhanced CP-binding affinity and increased flexibility of the CCT subunits