Myopathy mutations in DNAJB6 slow conformer specific substrate processing that is corrected by NEF modulation

Molecular chaperones, or heat shock proteins (HSPs), protect against the toxic misfolding and aggregation of proteins. As such, mutations or deficiencies within the chaperone network can lead to disease. In fact, dominant mutations in DNAJB6 (Hsp40/Sis1), an Hsp70 co-chaperone, leads to a protein aggregate myopathy termed Limb-Girdle Muscular Dystrophy Type D1 (LGMDD1). DNAJB6 client proteins and co-chaperone interactions in skeletal muscle are not known. Here, we used the yeast prion model client in conjunction with in vitro chaperone activity assays to gain mechanistic insights, and found that LGMDD1 mutants affect Hsp40 functions. Strikingly, the mutants changed the structure of client protein aggregates, as determined by altered distribution of prion strains. They also impair the Hsp70 ATPase cycle, dimerization, and substrate processing and consequently poison the function of wild-type protein. These results define the mechanisms by which LGMDD1 mutations alter chaperone activity and provide avenues for therapeutic intervention.

In Vitro Evaluation of Bis-3-Chloropiperidines as RNA Modulators Targeting TAR and TAR-Protein Interaction

After a long limbo, RNA has gained its credibility as a druggable target, fully earning its deserved role in the next generation of pharmaceutical R&D. We have recently probed the trans-activation response (TAR) element, an RNA stem–bulge–loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabilizing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analytical approaches for the study of multicomponent RNA-based interactions.

Myopathy mutations in DNAJB6 slow conformer specific substrate processing that is rescued by NEF modulation

Molecular chaperones, or heat shock proteins (HSPs), protect against the toxic misfolding and aggregation of proteins. As such, mutations or deficiencies within the chaperone network can lead to disease. In fact, dominant mutations in DNAJB6 (Hsp40/Sis1), an Hsp70 co-chaperone, leads to a protein aggregate myopathy termed Limb-Girdle Muscular Dystrophy Type D1 (LGMDD1). DNAJB6 client proteins and co-chaperone interactions in skeletal muscle are not known. Here, we used the yeast prion model client in conjunction with in vitro chaperone activity assays to gain mechanistic insights, and found that LGMDD1 mutants affect Hsp40 functions. Strikingly, the mutants changed the structure of client protein aggregates, as determined by altered distribution of prion strains. They also impair the Hsp70 ATPase cycle, dimerization, and substrate processing and consequently poison the function of wild-type protein. These results define the mechanisms by which LGMDD1 mutations alter chaperone activity and provide avenues for therapeutic intervention.

In vitro Evaluation of Bis-3-chloropiperidines as RNA Modulators Targeting TAR and TAR-protein Interaction

After a long limbo, RNA has gained its credibility as a druggable target, fully earning its de-served role in the next-generation area of pharmaceutical R&D. We have recently probed the Trans-Activation Response element (TAR), a RNA stem–bulge–loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabiliz-ing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analyti-cal approaches for the study of multicomponent RNA-based interactions.

Phosphorylation activates the yeast small heat shock protein Hsp26 by weakening domain contacts in the oligomer ensemble

Hsp26 is a small heat shock protein (sHsp) from S. cerevisiae. Its chaperone activity is activated by oligomer dissociation at heat shock temperatures. Hsp26 contains 9 phosphorylation sites in different structural elements. Our analysis of phospho-mimetic mutations shows that phosphorylation activates Hsp26 at permissive temperatures. The cryo-EM structure of the Hsp26 40mer revealed contacts between the conserved core domain of Hsp26 and the so-called thermosensor domain in the N-terminal part of the protein, which are targeted by phosphorylation. Furthermore, several phosphorylation sites in the C-terminal extension, which link subunits within the oligomer, are sensitive to the introduction of negative charges. In all cases, the intrinsic inhibition of chaperone activity is relieved and the N-terminal domain becomes accessible for substrate protein binding. The weakening of domain interactions within and between subunits by phosphorylation to activate the chaperone activity in response to proteotoxic stresses independent of heat stress could be a general regulation principle of sHsps.

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated proteins

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), as produced by the host defense, RidAHOCl turns into a potent chaperone-like holdase that can effectively protect the proteome of E. coli during oxidative stress. We previously reported that the activation of RidA's chaperone-like function coincides with the addition of at least seven and up to ten chlorine atoms. These atoms are reversibly added to basic amino acids in RidAHOCl and removal by reducing agents leads to inactivation. Nevertheless, it remains unclear, which residues in particular need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA's chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue (and, coincidentally, two tyrosine residues), while other N-chlorinated residues could not be detected, presumably due to the instability of the modification and its potential interference with a proteolytic digest. Therefore, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and R128 leads to a substantial reduction of RidAHOCl's chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.