A large number of neurodegenerative diseases in humans result from protein misfolding and aggregation. Protein misfolding is believed to be the primary cause of Alzheimer's disease, Parkinson's disease, Huntington's disease, Creutzfeldt–Jakob disease, cystic fibrosis, Gaucher's disease and many other degenerative and neurodegenerative disorders. Cellular molecular chaperones, which are ubiquitous, stress‐induced proteins, and newly found chemical and pharmacological chaperones have been found to be effective in preventing misfolding of different disease‐causing proteins, essentially reducing the severity of several neurodegenerative disorders and many other protein‐misfolding diseases. In this review, we discuss the probable mechanisms of several protein‐misfolding diseases in humans, as well as therapeutic approaches for countering them. The role of molecular, chemical and pharmacological chaperones in suppressing the effect of protein misfolding‐induced consequences in humans is explained in detail. Functional aspects of the different types of chaperones suggest their uses as potential therapeutic agents against different types of degenerative diseases, including neurodegenerative disorders.

In a newly isolated temperature-sensitive lethal Escherichia coli mutant affecting the chaperonin GroEL, we observed wholesale aggregation of newly translated proteins. After temperature shift, transcription, translation, and growth slowed over two to three generations, accompanied by filamentation and accretion (in Ϸ2% of cells) of paracrystalline arrays containing mutant chaperonin complex. A biochemically isolated inclusion body fraction contained the collective of abundant proteins of the bacterial cytoplasm as determined by SDS͞PAGE and proteolysis͞MS analyses. Pulse-chase experiments revealed that newly made proteins, but not preexistent ones, were recruited to this insoluble fraction. Although aggregation of ''stringent'' GroEL͞GroES-dependent substrates may secondarily produce an ''avalanche'' of aggregation, the observations raise the possibility, supported by in vitro refolding experiments, that the widespread aggregation reflects that GroEL function supports the proper folding of a majority of newly translated polypeptides, not just the limited number indicated by interaction studies and in vitro experiments.chaperone ͉ misfolding ͉ protein folding

The chaperonin GroEL binds nonnative proteins too large to fit inside the productive GroEL-GroES cis cavity, but whether and how it assists their folding has remained unanswered. We have examined yeast mitochondrial aconitase, an 82 kDa monomeric Fe(4)S(4) cluster-containing enzyme, observed to aggregate in chaperonin-deficient mitochondria. We observed that aconitase folding both in vivo and in vitro requires both GroEL and GroES, and proceeds via multiple rounds of binding and release. Unlike the folding of smaller substrates, however, this mechanism does not involve cis encapsulation but, rather, requires GroES binding to the trans ring to release nonnative substrate, which likely folds in solution. Following the phase of ATP/GroES-dependent refolding, GroEL stably bound apoaconitase, releasing active holoenzyme upon Fe(4)S(4) cofactor formation, independent of ATP and GroES.

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Although a cis mechanism of GroEL-mediated protein folding, occurring inside a hydrophilic chamber encapsulated by the co-chaperonin GroES, has been well documented, recently the GroEL±GroES-mediated folding of aconitase, a large protein (82 kDa) that could not be encapsulated, was described. This process required GroES binding to the ring opposite the polypeptide (trans) to drive release and productive folding. Here, we have evaluated this mechanism further using trans-only complexes in which GroES is closely tethered to one of the two GroEL rings, blocking polypeptide binding by that ring. In vitro, trans-only folded aconitase with kinetics identical to GroEL±GroES. Surprisingly, trans-only also folded smaller GroEL± GroES-dependent substrates, Rubisco and malate dehydrogenase, but at rates slower than the cis reaction. Remarkably, in vivo, a plasmid encoding a transonly complex rescued a GroEL-de®cient strain, but the colony size was approximately one-tenth that produced by wild-type GroEL±GroES. We conclude that a trans mechanism, involving rounds of binding to an open ring and direct release into the bulk solution, can be generally productive although, where size permits, cis encapsulation supports more ef®cient folding.

Escherichia coli chaperonins GroEL and GroES are indispensable for survival and growth of the cell since they provide essential assistance to the folding of many newly translated proteins in the cell. Recent studies indicate that a substantial portion of the proteins involved in the host pathways are completely dependent on GroEL-GroES for their folding and hence providing some explanation for why GroEL is essential for cell growth. Many proteins either small-single domain or large multidomains require assistance from GroEL-ES during their lifetime. Proteins of size up to approximately 70kDa can fold via the cis mechanism during GroEL-ES assisted pathway, but other proteins (>70kDa) that cannot be pushed inside the cavity of GroEL-ATP complex upon binding of GroES fold by an evolved mechanism called trans. In recent years, much work has been done on revealing facts about the cis mechanism involving the GroEL assisted folding of small proteins whereas the trans mechanism with larger polypeptide substrates still remains under cover. In order to disentangle the role of chaperonin GroEL-GroES in the folding of large E. coli proteins, this review discusses a number of issues like the range of large polypeptide substrates acted on by GroEL. Do all these substrates need the complete chaperonin system along with ATP for their folding? Does GroEL act as foldase or holdase during the process? We conclude with a discussion of the various queries that need to be resolved in the future for an extensive understanding of the mechanism of GroEL mediated folding of large substrate proteins in E. coli cytosol.

Resistin, a cysteine-rich adipocytokine, proposed as a link between obesity and diabetes in mice, was shown as a proinflammatory molecule in humans. We earlier reported that human resistin (hRes), a trimer, was resistant to heat and urea denaturation, existed in an oligomeric polydispersed state, and showed a concentrationdependent conformational change. These properties and an intimate correlation of hRes expression with cellular stress prompted us to investigate hRes as a possible chaperone. Here, we show that recombinant human resistin was able to protect the heat-labile enzymes citrate synthase and Nde1 from thermal aggregation and inactivation and was able to refold and restore their enzymatic activities after heat/guanidinium chloride denaturation. Furthermore, recombinant human resistin could bind misfolded proteins only. Molecular dynamics-based association-dissociation kinetics of hRes subunits pointed to resistin being a molecular chaperone. Bis-ANS, which blocks surface hydrophobicity, abrogated the chaperone activity of hRes, establishing the importance of surface hydrophobicity for chaperone activity. Replacement of Phe49 with Tyr (F49YhRes), a critical residue within the hydrophobic patch of hRes, although it could prevent thermal aggregation of citrate synthase and Nde1, was unable to refold and restore their activities. Treatment of U937 cells with tunicamycin/thapsigargin resulted in reduced hRes secretion and concomitant localization in the endoplasmic reticulum. Escherichia coli transformants expressing hRes could be rescued from thermal stress, pointing to hRes's chaperone-like function in vivo. HeLa cells transfected with hRes showed protection from thapsigargin-induced apoptosis. In conclusion, hRes, an inflammatory protein, additionally exhibited chaperone-like properties, suggesting a possible link between inflammation and cellular stress.protein folding | chaperokine R esistin, a small cysteine-rich secreted protein, is predominantly produced in human macrophages (1, 2). Resistin levels in human serum could neither be associated with obesity nor linked with insulin resistance (3), pointing to possible other role(s) for this hormone. We, and later others, showed that human resistin (hRes) is a proinflammatory molecule that stimulates the synthesis and secretion of TNF-α and IL-12 from macrophages through an NF-κB-activated pathway (4, 5). hRes mRNA levels are strongly induced by TNF-α and IL-6 in human peripheral blood mononuclear cells (6, 7). Although human and mouse resistin share 64.4 and 59% sequence homology at mRNA and amino acids levels, respectively, they differ considerably in terms of their structural organization (8). We earlier reported, based on extensive biophysical analyses, that recombinant human resistin (rhRes) is a highly stable molecule that exists in oligomeric states as a function of concentration with no major loss in helicity and displays slightly altered tertiary structure with an increase in temperature (9, 10). The variable oligomeric states and poly-dispersity...

Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function.