The 26S proteasome is a large, ∼2.5 MDa, multi-catalytic ATP-dependent protease complex that serves as the degrading arm of the ubiquitin system, which is the major pathway for regulated degradation of cytosolic, nuclear and membrane proteins in all eukaryotic organisms.

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bAcross evolution, N-glycosylation involves oligosaccharyltransferases that transfer lipid-linked glycans to selected Asn residues of target proteins. While these enzymes catalyze similar reactions in each domain, differences exist in terms of the chemical composition, length and degree of phosphorylation of the lipid glycan carrier, the sugar linking the glycan to the lipid carrier, and the composition and structure of the transferred glycan. To gain insight into how oligosaccharyltransferases cope with such substrate diversity, the present study analyzed the archaeal oligosaccharyltransferase AglB from four haloarchaeal species. Accordingly, it was shown that despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloarcula marismortui, Halobacterium salinarum, and Haloferax mediterranei could readily replace their counterpart from Haloferax volcanii when introduced into Hfx. volcanii cells deleted of aglB. As the four enzymes show significant sequence and apparently structural homology, it appears that the functional similarity of the four AglB proteins reflects the relaxed substrate specificity of these enzymes. Such demonstration of AglB substrate promiscuity is important not only for better understanding of N-glycosylation in Archaea and elsewhere but also for efforts aimed at transforming Hfx. volcanii into a glycoengineering platform. N-glycosylation is a posttranslational modification that occurs in all three domains of life. In each case, a core or fully assembled glycan is assembled on a phosphorylated polyprenol lipid carrier, namely, dolichol in Eukarya and Archaea and undecaprenol in Bacteria, and transferred to select Asn residues of a target protein by the actions of an oligosaccharyltransferase (OST) (1-4). In higher Eukarya, the OST is a multisubunit complex, with the Stt3 (staurosporine-and temperature-sensitive) protein serving as the catalytic subunit (5, 6). In Bacteria and Archaea, the OST comprises a single subunit, namely, the Stt3 homologues PglB and AglB, respectively (7,8).While members of all three domains perform N-glycosylation, the diversity presented by the N-linked glycans added to target proteins varies greatly across evolution. In particular, the Nlinked glycans that decorate archaeal glycoproteins offer a degree of architectural and compositional variability not seen in either their bacterial or eukaryal counterparts (4, 9). Archaeal N-glycosylation also presents diversity at the level of the glycan-charged lipid carrier, with both dolichol phosphate (DolP) and dolichol pyrophosphate (DolPP) reportedly serving this role (10-15). Moreover, archaeal N-glycosylation relies on several different sugars to provide the link between the oligosaccharide and either the lipid carrier or target protein Asn residues (4). As such, the archaeal OST must cope with a degree of substrate variety not encountered by either its eukaryal or bacterial homologues. Finally, while the presence of a sequon (the motif Asn-X-Ser/Thr, where X is any residue but proline) is requi...

Huntington’s disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a cytosine–adenine–guanine triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine stretch in the protein product which becomes aggregation prone. The mutant Htt (mHtt) aggregates are associated with components of the ubiquitin–proteasome system, suggesting that mHtt is marked for proteasomal degradation and that, for reasons still debated, are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and subsequent cellular responses. We identified two lysine residues, 6 and 9, in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Proteomic analysis indicated that cellular reactions to mHtt expression were weaker in cells expressing the lysineless protein, possibly implying a reduced capacity to cope with the proteotoxic stress. Taken together, the findings suggest a novel role for ubiquitination—attenuation of the pathogenic effect of mHtt.