Edited by Velia FowlerCRAF kinase maintains cell viability, growth, and proliferation by participating in the MAPK pathway. Unlike BRAF, CRAF requires continuous chaperoning by Hsp90 to retain MAPK signaling. However, the reason behind the continuous association of Hsp90 with CRAF is still elusive. In this study, we have identified the bipartite role of Hsp90 in chaperoning CRAF kinase. Hsp90 facilitates Ser-621 phosphorylation of CRAF and prevents the kinase from degradation. Co-chaperone Cdc37 assists in this phosphorylation event. However, after folding, the stability of the kinase becomes insensitive to Hsp90 inhibition, although the physical association between Hsp90 and CRAF remains intact. We observed that overexpression of Hsp90 stimulates MAPK signaling by activating CRAF. The interaction between Hsp90 and CRAF is substantially increased under an elevated level of cellular Hsp90 and in the presence of either active Ras (Ras V12 ) or EGF. Surprisingly, enhanced binding of Hsp90 to CRAF occurs prior to the Ras-CRAF association and facilitates actin recruitment to CRAF for efficient Ras-CRAF interaction, which is independent of the ATPase activity of Hsp90. However, monomeric CRAF (CRAF R401H ) shows abrogated interaction with both Hsp90 and actin, thereby affecting Hsp90-dependent CRAF activation. This finding suggests that stringent assemblage of Hsp90 keeps CRAF kinase equipped for participating in the MAPK pathway. Thus, the role of Hsp90 in CRAF maturation and activation acts as a limiting factor to maintain the function of a strong client like CRAF kinase.

The three GxxxG repeating motifs from the C-terminal region of β-amyloid (Aβ) peptide play a significant role in regulating the aggregation kinetics of the peptide. Mutation of these glycine residues to leucine greatly accelerates the fibrillation process but generates a varied toxicity profile. Using an array of biophysical techniques, we demonstrated the uniqueness of the composite glycine residues in these structural repeats. We used solvent relaxation NMR spectroscopy to investigate the role played by the surrounding water molecules in determining the corresponding aggregation pathway. Notably, the conformational changes induced by Gly 33 and Gly 37 mutations result in significantly decreased toxicity in a neuronal cell line. Our results indicate that G 33 xxxG 37 is the primary motif responsible for Aβ neurotoxicity, hence providing a direct structurefunction correlation. Targeting this motif, therefore, can be a promising strategy to prevent neuronal cell death associated with Alzheimer's and other related diseases, such as type II diabetes and Parkinson's. ROESY were 48 and 64, respectively. Data processing and analysis were carried out using Topspin TM v3.2 software (Bruker Biospin, Switzerland) and Sparky (https://www.cgl.ucsf.edu/home/sparky) software, respectively.

A network of chaperones and ubiquitin ligases sustain intracellular proteostasis, and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases: Spinocerebellar ataxia Type 3 (SCA3) and Huntington's disease (HD), we aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. We report here that Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain is diminished when polyQ repeat proteins (Ataxin-3/Huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in transgenic Drosophila model of SCA3. Thus, our findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyglutamine aggregates.

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A network of chaperones and ubiquitin ligases sustain intracellular proteostasis, and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases: Spinocerebellar ataxia Type 3 (SCA3) and Huntington’s disease (HD), we aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. We report here that Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain is diminished when polyQ repeat proteins (Ataxin-3/Huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in transgenic Drosophila model of SCA3. Thus, our findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyglutamine aggregates.