Proteasomes Cleave at Multiple Sites within Polyglutamine Tracts

Abstract

We have obtained results that directly conflict with that report. In the presence of the proteasome activator PA28␥(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.

Mimicking proteasomal release of polyglutamine peptides initiates aggregation and toxicity

“…However, their conclusion was also based on other experiments such as western blot analysis of polyQ protein products generated by proteasomes, and are in line with the conclusions by Holmberg and colleagues (Holmberg et al, 2004). The experiments of Pratt and Rechsteiner (Pratt and Rechsteiner, 2008) were done in the presence of a mutated PA28γ subunit, which alters proteasomal access and specificity to peptides. In addition, although isolated proteasomes may be able to cleave short polyQ peptides, our observation that Q65 and Q112 peptides readily aggregate suggests that the proteasome cannot efficiently degrade expanded polyQ peptides and thus cannot prevent their accumulation.…”

“…Thus, the inability to detect any Q16 peptides in cells expressing GFP-Ub-Q16 is most probably due to rapid and efficient degradation of non-expanded polyQ peptides. During the preparation of this article, work has been published that suggests that isolated proteasomes are able to cleave multiple times within a short polyQcontaining peptide (Pratt and Rechsteiner, 2008). They argued that Venkatraman and collegues (Venkatraman et al, 2004) underestimated the amount of cleaved polyQ fragments as a consequence of their mass-spectrometry methods.…”

Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation

“…However, it remains unclear whether these nuclear structures are storage depots for misfolded proteins or are actively engaged in degrading misfolded proteins. Previous studies reported that N/Q-rich proteins cannot be degraded efficiently by the proteasome (74)(75)(76), unless proteasomal activity is increased (77). Therefore, we speculate that D. discoideum has evolved mechanisms to efficiently degrade and prevent the aggregation of prion-like proteins.…”

The role of protein clearance mechanisms in organismal ageing and age-related diseases

“…Accordingly, incubation of proteasomes with mutant huntingtin exerts an inhibitory effect on the proteasome by directly impeding the entrance of other substrates 119 . However, other studies found that huntingtin aggregates do not affect proteasome activity suggesting that proteasomal dysfunction may be a consequence of a general proteostasis collapse 120,121 .…”