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Abstract
Expansion of the polyglutamine (polyQ) tract in the huntingtin (Htt) protein causes Huntington’s disease (HD), a fatal inherited movement disorder linked to neurodegeneration in the striatum and cortex. S-nitrosylation and S-acylation of cysteine residues regulate many functions of cytosolic proteins. We therefore used a resin-assisted capture approach to identify these modifications in Htt. In contrast to many proteins that have only a single S-nitrosylation or S-acylation site, we identified sites along much of the length of Htt. Moreover, analysis of cells expressing full-length Htt or a large N-terminal fragment of Htt shows that polyQ expansion strongly increases Htt S-nitrosylation. This effect appears to be general since it is also observed in Ataxin-1, which causes spinocerebellar ataxia type 1 (SCA1) when its polyQ tract is expanded. Overexpression of nitric oxide synthase increases the S-nitrosylation of normal Htt and the frequency of conspicuous juxtanuclear inclusions of Htt N-terminal fragments in transfected cells. Taken together with the evidence that S-nitrosylation of Htt is widespread and parallels polyQ expansion, these subcellular changes show that S-nitrosylation affects the biology of this protein in vivo.
Highlights
Huntington’s disease (HD) is an inherited movement disorder characterized by progressive degeneration of medium spiny neurons of the striatum, as well as other neuronal cell types
These experiments employed a “resin-assisted capture” (RAC) protocol that is based on selective retrieval of proteins from cell lysates using Thiopropyl Sepharose beads that covalently bind to the sulfhydryl groups which were exposed upon reduction of S-nitrosylated cysteine residues or S-acylated residues
The bound proteins were eluted with sample buffer containing β-mercaptoethanol and analyzed by Western blotting [52, 53]
Summary
Huntington’s disease (HD) is an inherited movement disorder characterized by progressive degeneration of medium spiny neurons of the striatum, as well as other neuronal cell types. The pathogenic mutations of the HTT gene are (CAG) trinucleotide repeats that exceed 40. The (CAG) repeats encode a polyglutamine tract (polyQ) near the N-terminus of the Htt protein (Fig 1A) [1,2,3,4,5,6]. Full length Htt is conserved among vertebrates and chordates and is widely expressed in nucleated cells [7,8,9]. Expression of Htt is essential for embryogenesis in mouse models [10]; the function(s) of Htt are not well understood. Htt is found primarily in the PLOS ONE | DOI:10.1371/journal.pone.0163359. Htt is found primarily in the PLOS ONE | DOI:10.1371/journal.pone.0163359 September 22, 2016
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