Mutational analysis of mammalian ubiquitin

Abstract

Much of what is known about the ubiquitin/proteasome pathway has been deduced from mutational analysis performed in the yeast model system. From the high level of conservation between yeast and mammalian ubiquitin it would be expected that expression of analogous ubiquitin isoforms in higher eukaryotes would result in similar phenotypes. A site directed mutagenesis approach was employed to investigate the phenotypes of expression of mutant ubiquitin in higher eukaryotes and in the in vivo setting of novel ubiquitin transgenic mice. It was found that Ub-EGFP fusion proteins are efficiently recognized and processed by ubiquitin specific proteases both in mammalian cells and in transgenic mice; the transgene-derived ubiquitin moiety was found to substitute for endogenous ubiquitin in poly-Ub chain assembly and ubiquitinated conjugates were recovered using standard purification methodologies. The expression of chain-terminating ubiquitin derivatives (K48R and K63R) predisposed cells to the toxic effects of misfolded proteins and sensitized cells to DNA damaging agents. In transgenic mice, the expression of K48R mutant ubiquitin was found to confer protective effects and delay the deterioration of Purkinje neurons in a mouse model of SCA-1. The neuroprotective effect of K48R mutant ubiquitin may be mediated though stabilization of key transcription factors whose loss figured in the normal course of the SCA1 disease. The expression of C-terminal variants in yeast has been proposed to have profound effects on ubiquitin metabolism. A mechanistically related mechanism has been proposed to contribute to the pathogenesis of Alzheimer's disease wherein transcriptional frameshifting of the ubiquitin B mRNA generates an aberrant ubiquitin protein (termed UBB+1) with an altered C-terminus. To investigate the constraints with regard to processing/conjugation and recycling of ubiquitin in higher eukaryotes a plethora of C-terminal ubiquitin variants were generated and introduced in mammalian cells as linear fusions with EGFP. Mutations that inactivate yeast ubiquitin did not abolish the function of ubiquitin in higher eukaryotes; C-terminal ubiquitin variants were processed by deubiquitinating enzymes and in some cases were found to conjugate to cellular proteins. The tolerance of mammalian cells to mutant ubiquitin may be attributable to loosened constraints that exist at the C-terminus due to mechanisms that couple deubiquitination, targeting and destruction of Ub-EGFP fusion proteins. Preliminary data suggest that prolonged exposure of cells of neuronal lineage to C-terminal ubiquitin variant as assessed in transgenic mice may result in perturbed ubiquitin homeostasis, a feature observed in the pathogenesis of Alzheimer's disease.