16 December 2003. Misfolded and unwanted proteins are usually quickly cleared from the cell. But when this process goes awry, the proteins can end up in intracellular aggregates or inclusions, like the neurofibrillary tangles and Lewy bodies found in Alzheimer's and Parkinson's diseases, respectively. Reports in the 12 December issue of Cell reveal that post-translational modification of proteins may play a crucial role in two key steps required for protein removal—proteasomal degradation and transportation. The findings may lead to new therapeutic strategies for combating disorders of protein toxicity.
In the first paper, Jeffrey Kudlow and colleagues at the University of Alabama, Birmingham, and The Salk Institute, La Jolla, California, reveal that the proteasome, part of the cellular machinery for recycling proteins, and which is implicated in the pathogenesis of a variety of neurodegenerative diseases (see ARF related news story and ARF news story), can be regulated by glycosylation.
First author Fengxue Zhang and colleagues discovered this by incubating proteasome-containing nuclear extracts from mammalian cells with the enzyme OGT, or O-linked N-acetylglucosamine transferase. OGT catalyses the esterification of amino acid hydroxyl groups with N-acetylglucosamine (GlcNAc). When Zhang included OGT in the extracts, proteasome-catalyzed degradation of the transcription factor Sp1 was dramatically reduced.
To test if this loss of activity is directly attributable to modification of one or more proteasome subunits, Zhang separated OGT-treated 26S proteasome subunits by two-dimensional electrophoresis, and then probed them with a monoclonal antibody that recognizes O-GlcNAc. The antibody labeled Rpt2, one of the ATPases that make up the proteasome regulatory cap, a multimeric complex that sits on top of the catalytic core of the complex.
The data suggest that the proteasome is regulated post-translationally in vivo, a conclusion that was supported by treating cells with forskolin, which stimulates degradation of Sp1. Zhang found that less Rpt2 reacts with the O-GlcNAc antibody in forskolin-treated cells than in cells treated with glucosamine, suggesting that the proteasome has been activated by removing the glucosamine moiety.
In the second paper, Tso-Pang Yao and colleagues at Duke University, Durham, North Carolina, report that acetylation may play a crucial role in the formation of the aggresome, a repository for toxic misfolded protein that has striking similarity to Lewy bodies. The protein motor dynein is known to play a key role in aggresome formation by shunting both ubiquitinated and non-ubiquitinated proteins. How dynein recognizes such proteins is unclear, but Yao and colleagues, noting that histone deacetylase 6 (HDAC6) associates with both poly-ubiquitinated proteins (see Hook et al., 2002) and p150glued, a component of the dynein motor (see Hubbert et al., 2002), decided to investigate its potential role in the formation of aggresomes.
The first hint that HDAC6 may play a key role came when first author Yoshiharu Kawaguchi treated cells with tunicamycin, DTT, or thapsigargin, agents that cause protein misfolding. In these cells, HDAC6 was relocalized from the cytoplasm to an aggresome-like structure. By transfecting cells with a poly-ubiquitin-prone cystic fibrosis transmembrane regulator (CFTR-deltaF508) or a GFP variant (GFG-250) that does not get ubiquitinated, the authors were able to show that HDAC primarily localizes with the former, suggesting an affinity for ubiquitinated proteins. This is perhaps not surprising, because HDAC6 contains a ubiquitin-binding BUZ finger motif.
But what is the role of the deacetylase in aggresomes? The authors hypothesized that it may link ubiquitinated proteins to dynein, given that HDAC6 has affinity for both. To address this, Kawaguchi and colleagues used immunoprecipitation to determine if all three components interact. In cells treated with the proteasome blocker MG132, which causes an accumulation of aggresomal proteins, the authors found that dynein antibodies also precipitated poly-ubiquitinated protein. However, when HDAC6 was silenced with siRNA in these same cells, co-precipitation of poly-ubiquitinated proteins was dramatically reduced.
The authors put forth a model whereby HDAC6 recruits ubiquitinated, misfolded or toxic proteins to the dynein motor, which then transports them along microtubules to the aggresome; the latter is normally found in close proximity to the microtubule organization center (MTOC). In knockdown experiments, Kawaguchi found that HDAC6 was essential for aggresome formation, and by immunostaining, the authors also found that the deacetylase is concentrated in the Lewy bodies in brain samples taken from Parkinson's disease patients.
As for the deacetylation activity of HDAC, the authors find that this is a key property of the system. In the HDAC6 knockdown cells, normal deacetylase rescued aggresome formation, but a deacetylase inactive mutant failed the same test, suggesting that a deacetylation event is essential for aggresomes to form. It is worth noting that deacetylation of histones has also been implicated in the pathogenesis of Huntington's disease (see ARF related news story).—Tom Fagan.
Zhang F, Su K, Yang X, Bowe DB, Paterson AJ, Kudlow JE. O-GlcNAc modification is an endogenous inhibitor of the proteasome. Cell 2003 December 12;115:715-725. Abstract
Kawaguchi Y, Kovacs JJ, McLaurin A, Vance JM, Ito A, Yao T-P. The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell 2003 December 12;115:727-738. Abstract