Oxidative stress-mediated regulation of proteasome complexes.

Oxidative stress has been implicated in aging and many human diseases, notably neurodegenerative disorders and various cancers. The reactive oxygen species that are generated by aerobic metabolism and environmental stressors can chemically modify proteins and alter their biological functions. Cells possess protein repair pathways to rescue oxidized proteins and restore their functions. If these repair processes fail, oxidized proteins may become cytotoxic. Cell homeostasis and viability are therefore dependent on the removal of oxidatively damaged proteins. Numerous studies have demonstrated that the proteasome plays a pivotal role in the selective recognition and degradation of oxidized proteins. Despite extensive research, oxidative stress-triggered regulation of proteasome complexes remains poorly defined. Better understanding of molecular mechanisms underlying proteasome function in response to oxidative stress will provide a basis for developing new strategies aimed at improving cell viability and recovery as well as attenuating oxidation-induced cytotoxicity associated with aging and disease. Here we highlight recent advances in the understanding of proteasome structure and function during oxidative stress and describe how cells cope with oxidative stress through proteasome-dependent degradation pathways.

Phosphorylation of threonine 3: implications for Huntingtin aggregation and neurotoxicity.

Huntingtin (Htt) is a widely expressed protein that causes tissue-specific degeneration when mutated to contain an expanded polyglutamine (poly(Q)) domain. Although Htt is large, 350 kDa, the appearance of amino-terminal fragments of Htt in extracts of postmortem brain tissue from patients with Huntington disease (HD), and the fact that an amino-terminal fragment, Htt exon 1 protein (Httex1p), is sufficient to cause disease in models of HD, points to the importance of the amino-terminal region of Htt in the disease process. The first exon of Htt encodes 17 amino acids followed by a poly(Q) repeat of variable length and culminating with a proline-rich domain of 50 amino acids. Because modifications to this fragment have the potential to directly affect pathogenesis in several ways, we have surveyed this fragment for potential post-translational modifications that might affect Htt behavior and detected several modifications of Httex1p. Here we report that the most prevalent modifications of Httex1p are NH(2)-terminal acetylation and phosphorylation of threonine 3 (pThr-3). We demonstrate that pThr-3 occurs on full-length Htt in vivo, and that this modification affects the aggregation and pathogenic properties of Htt. Thus, therapeutic strategies that modulate these events could in turn affect Htt pathogenesis.

A cell-based screen for drugs to treat Huntington's disease.

We have developed a medium-throughput cell-based assay to screen drugs for Huntington's disease (HD). The assay measures the ability of drugs to protect cultured neuronal (PC12) cells from death caused by an expanded polyglutamine (poly Q) form of huntingtin exon 1. Using this assay, we have blindly screened a library of 1040 compounds compiled by the NINDS: the NIH Custom Collection (NCC). Each compound was tested at five concentrations for its ability to protect cells against huntingtin-induced cell death as well as for its toxicity. Of the compounds tested, 18 prevented cell death completely, and 51 partially. Some of these also exhibited toxicity at higher doses. The majority of drugs (81%) were ineffective. Caspase inhibitors and cannabinoids showed reproducible protection in our assay. We believe these compounds, and others in our hit list, are appealing candidates for further investigation. Additionally, this assay is amenable to scaling up to screen additional compounds for treating Huntington's disease.