Proteasomes cleave at multiple sites within polyglutamine tracts: activation by PA28gamma(K188E).
Eukaryotic proteasomes have been reported to cleave only once within polyglutamine tracts and then only after the N-terminal glutamine (Venkatraman, P., Wetzel, R., Tanaka, M., Nukina, N., and Goldberg, A. L. (2004) Mol. Cell 14, 95-104). We have obtained results that directly conflict with that report. In the presence of the proteasome activator PA28gamma(K188E) human red cell proteasomes progressively degraded fluorescein-GGQ(10)RR or fluorescein-HPHQ(10)RR into small fragments as shown by size exclusion chromatography and mass spectrometry. MALDI-TOF mass spectrometry revealed that proteolytic products arose from cleavage after every glutamine in fluorescein-HPHQ(10)RR, and mass accuracy rules out deamidation of glutamine to glutamic acid as an explanation for peptide degradation. Moreover, degradation cannot be attributed to a contaminating protease because peptide hydrolysis was completely blocked by the proteasome-specific inhibitors, lactacystin and epoxomicin. We conclude that proteasomes cleave repetitively anywhere within a stretch of ten glutamine residues. Thus our results cast doubt on the idea that mammalian proteasomes cannot degrade glutamine-expanded regions within pathogenic polyQ-expanded proteins, such as Huntingtin.
Pratt
Gregory
G
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Rechsteiner
Martin
M
D004789:Enzyme Activation; D006801:Humans; D007477:Ions / Q000737:chemistry; D013058:Mass Spectrometry; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*
D007477:Ions; D010455:Peptides; C097188:polyglutamine; D046988:Proteasome Endopeptidase Complex