Cysteine Oxidation within N-terminal Mutant Huntingtin Promotes Oligomerization and Delays Clearance of Soluble Protein

Abstract

Huntington disease (HD) is a progressive neurodegenerative disorder caused by expression of polyglutamine-expanded mutant huntingtin protein (mhtt). Most evidence indicates that soluble mhtt species, rather than insoluble aggregates, are the important mediators of HD pathogenesis. However, the differential roles of soluble monomeric and oligomeric mhtt species in HD and the mechanisms of oligomer formation are not yet understood. We have shown previously that copper interacts with and oxidizes the polyglutamine-containing N171 fragment of huntingtin. In this study we report that oxidation-dependent oligomers of huntingtin form spontaneously in cell and mouse HD models. Levels of these species are modulated by copper, hydrogen peroxide, and glutathione. Mutagenesis of all cysteine residues within N171 blocks the formation of these oligomers. In cells, levels of oligomerization-blocked mutant N171 were decreased compared with native N171. We further show that a subset of the oligomerization-blocked form of glutamine-expanded N171 huntingtin is rapidly depleted from the soluble pool compared with "native " mutant N171. Taken together, our data indicate that huntingtin is subject to specific oxidations that are involved in the formation of stable oligomers and that also delay removal from the soluble pool. These findings show that inhibiting formation of oxidation-dependent huntingtin oligomers, or promoting their dissolution, may have protective effects in HD by decreasing the burden of soluble mutant huntingtin. Huntington disease (HD)2 is a progressive neurodegenerative disorder caused by a glutamine-encoding CAG expansion in the huntingtin gene (1). Mutant huntingtin protein (mhtt) is cleaved into a number of polyglutamine containing N-terminal fragments that misfold and form soluble monomeric and oligomeric proteins as well as insoluble aggregates. Several publications have shown that soluble N-terminal fragments of mhtt are important toxic factors that drive HD (2-4). Further, in HD and other neurodegenerative diseases, there is evidence that soluble oligomers may be particularly important (2,(5)(6)(7)(8).Soluble oligomers of mhtt may form or be stabilized through a number of mechanisms including noncovalent polyglutamine-dependent interactions (5) and possibly transglutaminase-mediated covalent cross-links (9). In addition, oxidation of huntingtin could also promote oligomerization (10). In general, cysteine oxidation is an important mechanism for forming intermolecular cross-links that may stabilize protein oligomers (11, 12). Site-specific cysteine oxidation may regulate protein function by affecting structure including oligomerization state (13-15). There is growing evidence that a variety of protein residues up and downstream of the mhtt polyglutamine tract impact HD progression through providing sites of post-translational modification that affect huntingtin degradation, cleavage, or cell location (16 -19). The N terminus of huntingtin contains several cysteine residues, the most C-terminal of which are ...

HTRF analysis of soluble huntingtin in PHAROS PBMCs

“…One possibility is that systemic influences, such as oxidative stress or inflammation, could concordantly modulate brain pathology and cause peripheral changes such as Htt oligomerization and aggregation. 30 The increase in plasma levels of the DNA damage marker 8-OH2dG that occurs as clinical symptoms develop is consistent with this possibility. 31,32 We have also described gene expression changes in leukocytes that indicate progressive influences on them from HD.…”

Human eukaryotic elongation factor 1A forms oligomers through specific cysteine residues

“…Previous studies have demonstrated that disulfide bridges formed by active cysteines contribute to oligomerization of some proteins including disease-related Huntingtin [21], membrane-bound transcription factor [22], and glucan phosphatase family members [23]. eEF1A1 contains six well-conserved cysteine residues located in different domains ( Fig.…”

Post-aggregation Oxidation of Mutant Huntingtin Controls the Interactions between Aggregates

“…For that purpose, an endoproteinase, Glu-C, was used for a limited proteolysis of HTT EX1 , which can produce a relatively small proteolytic fragment (Lys 6 -Glu 12 ) that contains Met 8 but not a polyQ tract. To test whether digestion of HTT EX1 with Glu-C produces a Lys 6 -Glu 12 peptide, in vitro aggregates of HTT EX1 (42Q) before Met 8 oxidation were resolubilized with TFA, incubated with Glu-C and then analyzed with MALDI-TOF mass spectrometry (see "Experimental Procedures"). As shown in Fig.…”

“…A copper ion functions as a pro-oxidant and is suggested to oxi-dize cysteine residues to form disulfide-linked HTT oligomers (12). Given that almost all of mutant HTT proteins are already aggregated at the disease end stage, however, the aggregation model triggered by oxidative modification on soluble HTT proteins seems questionable.…”