12 Apr 2004

Anyone who has minced beef knows that the grinder occasionally gets clogged by a particularly tough bit of meat or gristle. Polyglutamine (polyQ) stretches, it turns out, may have a similar effect on our intracellular protein grinder, the proteasome. So conclude Harvard Medical School’s Alfred Goldberg and colleagues, writing in the April 9 Molecular Cell. The findings may explain why expanded polyglutamine repeats, which cause a variety of neurodegenerative disorders including Huntington’s disease, accumulate to toxic levels in the cell.

The grinder-shaped proteasome sucks proteins in at one end and spits small peptides out the other, thanks to the cutting ability of six proteolytic enzyme sites in the interior of the complex. The proteasome is known to be the major site for degradation of polyglutamine-expanded proteins (see ARF related news story). Nevertheless, in Huntington’s and other polyQ disorders, polyglutamine ends up in intracellular inclusion bodies. So how good is the proteasome at grinding polyglutamine? To answer this question, Goldberg, together with colleagues at the University of Tennessee Medical Center, Knoxville, and RIKEN Brain Science Institute, Wako, Japan, plied isolated mammalian proteasomes with proteins containing various lengths of polyglutamine.

Lead author Prasanna Venkatraman first tried a small peptide with 10 glutamines flanked by two lysine residues on each end (KK(Q10)KK). The only cut the proteasome made was after the first glutamine, suggesting that it may have difficulty cutting up long glutamine stretches. This was confirmed using other constructs. A peptide containing 20 glutamines, for example, was ground down to peptides containing 19 and one glutamine. Addition of 15 mixed amino acids to the N-terminus had no effect on cleavage within the polyQ stretch, while with a (Q)20RRGRR construct, the proteasomes failed to cut anywhere within the polyQ.

These results suggest that while proteasomes process polyQ proteins, the polyQ stretch itself may come out almost fully intact. To address this, Venkatraman used a polyQ35-myoglobin fusion protein. In this protein, a 35Q repeat, together with 15 flanking residues from the polyQ protein ataxin-3, was inserted between amino acids 45 and 48 of myoglobin. This chimeric protein aggregated slowly over time, but not in the presence of proteasomes. Instead, results suggest that the myoglobin part of the chimera was mostly degraded, as judged by reaction with a polyclonal antibody, while the complete polyQ stretch, accompanied by some myoglobin flanking residues, was left intact.

These findings raise a number of questions. For example: How are polyQ repeats degraded? “They must be rapidly digested to free amino acids by cytosolic peptidases,” suggest the authors. But perhaps more importantly: How do large, 100Q or even 300Q polyglutamine repeats pass through the relatively narrow exit of the proteasome? The opening at the bottom of the grinder is not large enough to accommodate such molecules. “Consequently, it is quite unclear how extended Q fragments can exit from the proteasome,” the authors write. “Failure to exit efficiently should occur more frequently as polyQ sequences increase in length beyond 35Qs, which may explain why the age of onset of these diseases is highly dependent on the repeat length,” they conclude.

Meanwhile, another report in the same journal suggests that during apoptosis, caspases can inhibit the proteasome. The paper is by Gerald Cohen and colleagues from the University of Leicester, England, from Humboldt University, Berlin, Germany, and the Technion-Israel Institute of Technology, Haifa.

Is this a case of apoptosis getting revenge? The proteasome, by degrading many proapoptotic factors, has been implicated in preventing the apoptotic cascade from taking off. Now, it seems that the proteasome may sometimes bite off more than it can chew.

First author Xiao-Ming Sun and colleagues show that caspases—members of the notorious proteolytic death squads of the apoptotic machinery—target several key subunits of the protease, including those that recognize polyubiquitinated protein and those that keep the lid and base on the protein grinder. The result is that the proteasome gets crippled, fails to degrade either ubiquitinated or nonubiquitinated substrates, and leaves proapoptotic molecules, such as Smac, with free reign.—Tom Fagan

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References

News Citations

1. Protein Aggregates Block Disposal Mechanism 22 Nov 2002

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