Michael Kalchman

Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract

Apoptosis has recently been recognized as a mode of cell death in Huntington disease (HD). Apopain, a human counterpart of the nematode cysteine protease death–gene product, CED–3, has a key role in proteolytic events leading to apoptosis. Here we show that apoptotic extracts and apopain itself specifically cleave the HD gene product, huntingtin. The rate of cleavage increases with the length of the huntingtin polyglutamine tract, providing an explanation for the gain–of–function associated with GAG expansion. Our results show that huntingtin is cleaved by cysteine proteases and suggest that HD might be a disorder of inappropriate apoptosis.

HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain

Huntington disease (HD) is associated with the expansion of a polyglutamine tract, greater than 35 repeats, in the HD gene product, huntingtin. Here we describe a novel huntingtin interacting protein, HIP1, which co-localizes with huntingtin and shares sequence homology and biochemical characteristics with Sla2p, a protein essential for function of the cytoskeleton in Saccharomyces cerevisiae. The huntingtin–HIP1 interaction is restricted to the brain and is inversely correlated to the polyglutamine length in huntingtin. This provides the first molecular link between huntingtin and the neuronal cytoskeleton and suggests that, in HD, loss of normal huntingtin–HIP1 interaction may contribute to a defect in membrane-cytoskeletal integrity in the brain.

Huntingtin Is Ubiquitinated and Interacts with a Specific Ubiquitin-conjugating Enzyme

Using the yeast two-hybrid system, we have identified a human ubiquitin-conjugating enzyme (hE2-25K) as a protein that interacts with the gene product for Huntington disease (HD) (Huntingtin). This protein has complete amino acid identity with the bovine E2-25K protein and has striking similarity to the UBC-1, −4 and −5 enzymes of Saccharomyces cerevisiae. This protein is highly expressed in brain and a slightly larger protein recognized by an anti-E2-25K polyclonal antibody is selectively expressed in brain regions affected in HD. The huntingtin-E2-25K interaction is not obviously modulated by CAG length. We also demonstrate that huntingtin is ubiquitinated. These findings have implications for the regulated catabolism of the gene product for HD.