Mechanisms of protein homeostasis in health, aging and disease.

When emerging from the ribosomes, new polypeptides need to fold properly, eventually translocate, and then assemble into stable, yet functionally flexible complexes. During their lifetime, native proteins are often exposed to stresses that can partially unfold and convert them into stably misfolded and aggregated species, which can in turn cause cellular damage and propagate to other cells. In animal cells, especially in aged neurons, toxic aggregates may accumulate, induce cell death and lead to tissue degeneration via different mechanisms, such as apoptosis as in Parkinson's and Alzheimer's diseases and aging in general. The main cellular mechanisms effectively controlling protein homeostasis in youth and healthy adulthood are: (1) the molecular chaperones, acting as aggregate unfolding and refolding enzymes, (2) the chaperone-gated proteases, acting as aggregate unfolding and degrading enzymes, (3) the aggresomes, acting as aggregate compacting machineries, and (4) the autophagosomes, acting as aggregate degrading organelles. For unclear reasons, these cellular defences become gradually incapacitated with age, leading to the onset of degenerative diseases. Understanding these mechanisms and the reasons for their incapacitation in late adulthood is key to the design of new therapies against the progression of aging, degenerative diseases and cancers

Molecular chaperones are nanomachines that catalytically unfold misfolded and alternatively folded proteins.

By virtue of their general ability to bind (hold) translocating or unfolding polypeptides otherwise doomed to aggregate, molecular chaperones are commonly dubbed "holdases". Yet, chaperones also carry physiological functions that do not necessitate prevention of aggregation, such as altering the native states of proteins, as in the disassembly of SNARE complexes and clathrin coats. To carry such physiological functions, major members of the Hsp70, Hsp110, Hsp100, and Hsp60/CCT chaperone families act as catalytic unfolding enzymes or unfoldases that drive iterative cycles of protein binding, unfolding/pulling, and release. One unfoldase chaperone may thus successively convert many misfolded or alternatively folded polypeptide substrates into transiently unfolded intermediates, which, once released, can spontaneously refold into low-affinity native products. Whereas during stress, a large excess of non-catalytic chaperones in holding mode may optimally prevent protein aggregation, after the stress, catalytic disaggregases and unfoldases may act as nanomachines that use the energy of ATP hydrolysis to repair proteins with compromised conformations. Thus, holding and catalytic unfolding chaperones can act as primary cellular defenses against the formation of early misfolded and aggregated proteotoxic conformers in order to avert or retard the onset of degenerative protein conformational diseases

ZnJ2 Is a Member of a Large Chaperone Family in the Chloroplast of Photosynthetic Organisms that Features a DnaJ-Like Zn-Finger Domain.

Photosynthesis is performed by large complexes, composed of subunits encoded by the nuclear and chloroplast genomes. Assembly is assisted by general and target-specific chaperones, but their mode of action is yet unclear. We formerly showed that ZnJ2 is an algal chaperone resembling BSD2 from land plants. In algae, it co-migrates with the rbcL transcript on chloroplast polysomes, suggesting it contributes to the de-novo synthesis of RbcL (Doron et al., 2014). ZnJ2 contains four CXXCXGXG motifs, comprising a canonical domain typical also of DnaJ-type I (DNAJA). It contributes to the binding of protein substrates to DnaK and promotes an independent oxidoreductase activity (Mattoo et al., 2014). To examine whether ZnJ2 has oxidoreductase activity, we used the RNaseA assay, which measures the oxidation-dependent reactivation of reduced-denatured RNaseA. Although ZnJ2 assisted the native refolding of reduced-denatured RNaseA, its activity was restricted to an oxidizing environment. Thus, ZnJ2 did not carry the exclusive responsibility for the formation of disulfide bridges, but contributed to the stabilization of its target polypeptides, until they reached their native state. A ZnJ2 cysteine deficient mutant maintained a similar holding chaperone activity as the wild-type and did not induce the formation of disulfide bonds. ZnJ2 is devoid of a J-domain. It thus does not belong to the J-domain co-chaperones that target protein substrates to DnaK. As expected, in vitro, its aggregation-prevention activity was not synergic to the ATP-fueled action of DnaK/DnaJ/GrpE in assisting the native refolding of denatured malate dehydrogenase, nor did it show an independent refolding activity. A phylogenetic analysis showed that ZnJ2 and BSD2 from land plants, are two different proteins belonging to a larger group containing a cysteine-rich domain, that also includes the DNAJAs. Members of this family are apparently involved in specific assembly of photosynthetic complexes in the chloroplast

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

Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones.

Members of the HSP70/HSP110 family (HSP70s) form a central hub of the chaperone network controlling all aspects of proteostasis in bacteria and the ATP-containing compartments of eukaryotic cells. The heat-inducible form HSP70 (HSPA1A) and its major cognates, cytosolic HSC70 (HSPA8), endoplasmic reticulum BIP (HSPA5), mitochondrial mHSP70 (HSPA9) and related HSP110s (HSPHs), contribute about 3% of the total protein mass of human cells. The HSP70s carry out a plethora of housekeeping cellular functions, such as assisting proper de novo folding, assembly and disassembly of protein complexes, pulling polypeptides out of the ribosome and across membrane pores, activating and inactivating signaling proteins and controlling their degradation. The HSP70s can induce structural changes in alternatively folded protein conformers, such as clathrin cages, hormone receptors and transcription factors, thereby regulating vesicular trafficking, hormone signaling and cell differentiation in development and cancer. To carry so diverse cellular housekeeping and stress-related functions, the HSP70s act as ATP-fuelled unfolding nanomachines capable of switching polypeptides between different folded states. During stress, the HSP70s can bind (hold) and prevent the aggregation of misfolding proteins and thereafter act alone or in collaboration with other unfolding chaperones to solubilize protein aggregates. Here, we discuss the common ATP-dependent mechanisms of holding, unfolding-by-clamping and unfolding-by-entropic pulling, by which the HSP70s can apparently convert various alternatively folded and misfolded polypeptides into differently active conformers. Understanding how HSP70s can prevent the formation of cytotoxic protein aggregates, pull, unfold, and solubilize them into harmless species is central to the design of therapies against protein conformational diseases

Misfolding and aggregation of nascent proteins: a novel mode of toxic cadmium action in vivo.

Cadmium is a highly poisonous metal and a human carcinogen, but the molecular mechanisms underlying its cellular toxicity are not fully understood. Recent findings in yeast cells indicate that cadmium exerts its deleterious effects by inducing widespread misfolding and aggregation of nascent proteins. Here, we discuss this novel mode of toxic heavy metal action and propose a mechanism by which molecular chaperones may reduce the damaging effects of heavy metal ions on protein structures

Quantitative proteomics of rat livers shows that unrestricted feeding is stressful for proteostasis with implications on life span.

Studies in young mammals on the molecular effects of food restriction leading to prolong adult life are scares. Here, we used high-throughput quantitative proteomic analysis of whole rat livers to address the molecular basis for growth arrest and the apparent life-prolonging phenotype of the food restriction regimen. Over 1800 common proteins were significantly quantified in livers of ad libitum, restriction- and re-fed rats, which summed up into 92% of the total protein mass of the cells. Compared to restriction, ad libitum cells contained significantly less mitochondrial catabolic enzymes and more cytosolic and ER HSP90 and HSP70 chaperones, which are hallmarks of heat- and chemically-stressed tissues. Following re-feeding, levels of HSPs nearly reached ad libitum levels. The quantitative and qualitative protein values indicated that the restriction regimen was a least stressful condition that used minimal amounts of HSP-chaperones to maintain optimal protein homeostasis and sustain optimal life span. In contrast, the elevated levels of HSP-chaperones in ad libitum tissues were characteristic of a chronic stress, which in the long term could lead to early aging and shorter life span