Tapan K. Chaudhuri

Protein-misfolding diseases and chaperone-based therapeutic approaches.

A large number of neurodegenerative diseases in humans result from protein misfolding and aggregation. Protein misfolding is believed to be the primary cause of Alzheimers disease, Parkinsons disease, Huntingtons disease, Creutzfeldt–Jakob disease, cystic fibrosis, Gauchers 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.

GroEL/GroES-Mediated Folding of a Protein Too Large to Be Encapsulated

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.

Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL

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 ≈2% 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 SDS/PAGE and proteolysis/MS analyses. Pulse–chase experiments revealed that newly made proteins, but not preexistent ones, were recruited to this insoluble fraction. Although aggregation of “stringent” GroEL/GroES-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.

Folding with and without encapsulation by cis- and trans-only GroEL–GroES complexes

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 trans‐only complex rescued a GroEL‐deficient 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 efficient folding.

GroEL assisted folding of large polypeptide substrates in Escherichia coli : Present scenario and assignments for the future

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.

The 69 kDa Escherichia coli maltodextrin glucosidase does not get encapsulated underneath GroES and folds through trans mechanism during GroEL/GroES-assisted folding

Escherichia coli chaperonin GroEL and GroES assist in folding of a wide variety of substrate proteins in the molecular mass range of ~50 kDa, using cis mechanism, but limited information is available on how they assist in folding of larger proteins. Considering that the central cavity of GroEL can accommodate a non‐native protein of ~60 kDa, it is important to study the GroEL‐GroES‐assisted folding of substrate proteins that are large enough for cis encapsulation. In this study, we have reported the mechanism of GroEL/ GroES‐assisted in vivo and in vitro folding of a 69 kDa monomelic E. coli protein maltodextrin glucosidase (MalZ). Coexpression of GroEL and GroES in E. coli causes a 2‐fold enhancement of exogenous MalZ activity in vivo. In vitro, GroEL and GroES in the presence of ATP give rise to a 7‐fold enhancement in MalZ refolding. Neither GroEL nor single ring GroEL (SR1) in the presence or absence of ATP could enhance the in vitro folding of MalZ. GroES could not encapsulate GroEL‐bound MalZ. All these experimental findings suggested that GroEL/GroES‐assisted folding of MalZ followed trans mechanism, whereas denatured MalZ and GroES bound to the opposite rings of a GroEL molecule.—Paul, S., Singh, C., Mishra, S., Chaudhuri, T. K. The 69 kDa Escherichia coli maltodextrin glucosidase does not get encapsulated underneath GroES and folds through trans mechanism during GroEL/GroES‐assisted folding. FASEB J. 21, 2874–2885 (2007)

Liver Uptake of 99mTc-Diphosphonate

A report of intense liver uptake of a bone-imaging agent (99mTc-EHDP) is presented. Millimicron colloid particles may be responsible for such uptake. Previous reports of this finding have not come to the authors attention.

Autoradiographic Studies of Distribution in the Stomach of 99mTc-Pertechnetate

The histological and cytological distribution of 99mTc-pertechnetate in animal stomachs was studied by autoradiography. Unlike halogens, technetium is handled predominantly by the gastric surface mucosal cells. The parietal and chief cells have little or no role in the gastric secretion of Tc-pertechnetate.

Chaperone-assisted refolding of Escherichia coli maltodextrin glucosidase

In vitro refolding of maltodextrin glucosidase, a 69 kDa monomeric Escherichia coli protein, was studied in the presence of glycerol, dimethylsulfoxide, trimethylamine‐N‐oxide, ethylene glycol, trehalose, proline and chaperonins GroEL and GroES. Different osmolytes, namely proline, glycerol, trimethylamine‐N‐oxide and dimethylsulfoxide, also known as chemical chaperones, assist in protein folding through effective inhibition of the aggregation process. In the present study, it was observed that a few chemical chaperones effectively reduced the aggregation process of maltodextrin glucosidase and hence the in vitro refolding was substantially enhanced, with ethylene glycol being the exception. Although, the highest recovery of active maltodextrin glucosidase was achieved through the ATP‐mediated GroEL/GroES‐assisted refolding of denatured protein, the yield of correctly folded protein from glycerol‐ or proline‐assisted spontaneous refolding process was closer to the chaperonin‐assisted refolding. It was also observed that the combined application of chemical chaperones and molecular chaperone was more productive than their individual contribution towards the in vitro refolding of maltodextrin glucosidase. The chemical chaperones, except ethylene glycol, were found to provide different degrees of protection to maltodextrin glucosidase from thermal denaturation, whereas proline caused the highest protection. The observations from the present studies conclusively demonstrate that chemical or molecular chaperones, or the combination of both chaperones, could be used in the efficient refolding of recombinant E. coli maltodextrin glucosidase, which enhances the possibility of identifying or designing suitable small molecules that can act as chemical chaperones in the efficient refolding of various aggregate‐prone proteins of commercial and medical importance.

Factors governing the substrate recognition by GroEL chaperone: a sequence correlation approach

Abstract The chaperonin GroEL binds to a large number of polypeptides, prevents their self-association, and mediates appropriate folding in a GroES and adenosine triphosphate–dependent manner. But how the GroEL molecule actually recognizes the polypeptide and what are the exact GroEL recognition sites in the substrates are still poorly understood. We have examined more than 50 in vivo substrates as well as well-characterized in vitro substrates, for their binding characteristics with GroEL. While addressing the issue, we have been driven by the basic concept that GroES, being the cochaperonin of GroEL, is the best-suited substrate for GroEL, as well as by the fact that polypeptide substrate and GroES occupy the same binding sites on the GroEL apical domain. GroES interacts with GroEL through selective hydrophobic residues present on its mobile loop region, and we have considered the group of residues on the GroES mobile loop as the key element in choosing a substrate for GroEL. Considering the hydrophobic region on the GroES mobile loop as the standard, we have attempted to identify the homologous region on the peptide sequences in the proteins of our interest. Polypeptides have been judged as potential GroEL substrates on the basis of the presence of the GroES mobile loop–like hydrophobic segments in their amino acid sequences. We have observed 1 or more GroES mobile loop–like hydrophobic patches in the peptide sequence of some of the proteins of our interest, and the hydropathy index of most of these patches also seems to be approximately close to that of the standard. It has been proposed that the presence of hydrophobic patches having substantial degree of hydropathy index as compared with the standard segment is a necessary condition for a peptide sequence to be recognized by GroEL molecules. We also observed that the overall hydrophobicity is also close to 30% in these substrates, although this is not the sufficient criterion for a polypeptide to be assigned as a substrate for GroEL. We found that the binding of aconitase, α-lactalbumin, and murine dihydrofolate reductase to GroEL falls in line with our present model and have also predicted the exact regions of their binding to GroEL. On the basis of our GroEL substrate prediction, we have presented a model for the binding of apo form of some proteins to GroEL and the eventual formation of the holo form. Our observation also reveals that in most of the cases, the GroES mobile loop–like hydrophobic patch is present in the unstructured region of the protein molecule, specifically in the loop or β-sheeted region. The outcome of our study would be an essential feature in identifying a potential substrate for GroEL on the basis of the presence of 1 or more GroES mobile loop–like hydrophobic segments in the amino acid sequence of those polypeptides and their location in three-dimensional space.