D.A. Gray, M. Tsirigotis, J. Brun, M. Tang, M. Zhang, M. Beyers, J. Woulfe

The ubiquitin/proteasome pathway (UPP) is responsible for elimination of damaged and misfolded proteins. In yeast it has been established that mutant isoforms of ubiquitin unable to participate in ubiquitin chain assembly exert a dominant negative effect on proteolysis (in the case of a K48R substitution) or on DNA repair (in the case of a K63R substitution). To explore the consequences of analogous mutations in the context of mammalian cells in vivo, we have created transgenic mouse strains in which wild type or mutant ubiquitin is expressed at high levels in most tissues. These animals are viable and fertile. Homozygous individuals from all strains have reached three years of age, arguing against any dramatic reduction in lifespan. The mice have been tested for sensitivity to various insults likely to present a burden of misfolded or aberrant protein. Remarkably, mice expressing ubiquitin bearing a K48R substitution were found to be resistant to diverse stressors including the amino acid analogue canavanine, a neuropathogenic retrovirus encoding a misfolded envelope protein, and a genetically encoded expanded polyglutamine protein. Such resistance was not observed in mice expressing K63R mutant ubiquitin.

The apparent paradox presented by our in vivo results clearly arises from an unexpected consequence of the impaired UPP in the K48R mice. We are currently investigating several systems in which ubiquitin plays a role, systems possibly perturbed in these mice. Our present interests include regulation of transcription, endoplasmic reticulum associated degradation (ERAD), and endocytosis.

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