HYPK, a Huntingtin interacting protein, reduces aggregates and apoptosis induced by N-terminal Huntingtin with 40 glutamines in Neuro2a cells and exhibits chaperone-like activity

Abstract

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.

Huntingtin protein interactions altered by polyglutamine expansion as determined by quantitative proteomic analysis

“…be enriched for intrinsically disordered (naturally unfolded) proteins, since intrinsically disordered proteins are involved in several kinds of diseases, especially neurodegenerative diseases, including HD. 26,27,33 We found that 76% of 50 randomly selected preferential interactors of Htt-50Q contain regions of disorder of ≥ 30 residues, as predicted by at least three out of four PONDR predictors used, compared with 47% in the total pool of eukaryotic proteins in SwissProt database. 27 To identify proteins most significantly changed in abundance between normal and expanded Htt complexes, we used statistical analysis of variance (ANOVA) approach, which considers both biologic and experimental sources of variability.…”

Micro RNA -214,-150,-146a and-125b targetHuntingtingene

“…Expression of fluorescent-tagged mutated exon1 of HTT (mHTT) in cultured cells led to the formation of m-HTT aggregates as described by many authors including us. 25,26 To study the effect of miRNAs on aggregate forming propensity of the m-HTT, we engineered HTT exon1. We ligated HTT exon-1 (that codes 83 Glu for mutated HTT and 16 Glu for wild-type HTT) with the full length 3'-UTR of mouse Htt gene.…”

The Chaperone-Like Protein HYPK Acts Together with NatA in Cotranslational N-Terminal Acetylation and Prevention of Huntingtin Aggregation

“…Here we present data identifying the Huntingtin (Htt) yeast two-hybrid protein K (HYPK) as a novel factor involved in cotranslational NatA acetylation. HYPK, originally identified in a yeast two-hybrid screen during a search for potential interaction partners for the Huntingtin protein (19), was recently found to reduce Htt polyglutamine (polyQ) aggregation upon overexpression (36). However, the role of the endogenous HYPK protein has yet to be revealed.…”