Proteins generally must fold into precise three-dimensional conformations to fulfill their biological functions. In the cell, this fundamental process is aided by molecular chaperones, which act in preventing protein misfolding and aggregation. How this machinery assists newly synthesized polypeptide chains in navigating the complex folding energy landscape is now being understood in considerable detail. The mechanisms that ensure the maintenance of a functional proteome under normal and stress conditions are also of great medical relevance, as the aggregation of proteins that escape the cellular quality control underlies a range of debilitating diseases, including many age-of-onset neurodegenerative disorders.
When exposed to proteotoxic environmental conditions, mammalian cells activate the cytosolic stress response in order to restore protein homeostasis. A key feature of this response is the heat shock transcription factor 1 (HSF1)-dependent expression of molecular chaperones. Here, we describe the results of an RNA interference screen in HeLa cells to identify modulators of stress response induction and attenuation. The modulator proteins are localized in multiple cellular compartments, with chromatin modifiers and nuclear protein quality control playing a central regulatory role. We find that the acetyltransferase, EP300, controls the cellular level of activatable HSF1. This involves acetylation of HSF1 at multiple lysines not required for function and results in stabilization of HSF1 against proteasomal turnover. Acetylation of functionally critical lysines during stress serves to fine-tune HSF1 activation. Finally, the nuclear proteasome system functions in attenuating the stress response by degrading activated HSF1 in a manner linked with the clearance of misfolded proteins.
Expansion of polymorphic glutamine (Q) numbers present at the protein Huntingtin (Htt) beyond 36Q results in its misfolding and aggregation, and the aggregates recruit several other proteins. Here we show that HYPK, initially identified as an Htt-interacting partner by yeast two-hybrid assay, physically interacts with N-terminal Htt in Neuro2A cells and alters the numbers and distribution of aggregates formed by N-terminal Htt with 40Q. HYPK also alters the kinetics of mutated N-terminal Htt-mediated aggregate formation. Fluorescence recovery after photobleaching studies reveal that over-expression of HYPK results in the appearance of Htt poly Q aggregates, which upon bleaching recovers approximately 80% of initial fluorescence intensity within 6 min. Fluorescence loss in photobleaching studies indicate loss off fluorescence intensity of the aggregates with time in presence of HYPK. Over-expression of this protein reduces poly Q-mediated caspase-2, caspase-3 and caspase-8 activations, whereas gamma ray-induced activations of these enzymes are not affected. In vitro and in vivo studies demonstrate that HYPK possesses a novel chaperone-like activity. We conclude that HYPK, without having any sequence similarity with known chaperones, plays an effective role in protecting neuronal cells against apoptosis induced by mutated N-terminal Htt by modulating the aggregate formation.
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