Ubiquitin ligase Hrd1 enhances the degradation and suppresses the toxicity of polyglutamine-expanded huntingtin.
E3 ubiquitin ligases catalyze the conjugation of ubiquitin onto proteins, which acts as a signal for targeting proteins for degradation by the proteasome. Hrd1 is an endoplasmic reticulum (ER) membrane-spanning E3 with its catalytic active RING finger facing the cytosol. We speculated that this topology might allow Hrd1 to ubiquitinate misfolded proteins in the cytosol. We tested this idea by using polyglutamine (polyQ)-containing huntingtin (htt) protein as a model substrate. We found that the protein levels of Hrd1 were increased in cells overexpressing the N-terminal fragment of htt containig an expanded polyQ tract (httN). Forced expression of Hrd1 enhanced the degradation of httN in a RING finger-dependent manner, whereas silencing of endogenous Hrd1 expression by RNA interference stabilized httN. Degradation of httN was found to be p97/VCP-dependent, but independent of Ufd1 and Npl4, all of which are thought to form a complex with Hrd1 during ER-associated degradation. Consistent with its role as an E3 for httN, we demonstrate that Hrd1 interacts with and ubiquitinates httN. Subcellular fractionation and confocal microscopy revealed that Hrd1recruits HttN to the ER and co-localizes with juxtanuclear aggregates of httN in cells. Interaction of Hrd1 with httN was found to be independent of the length of the polyglutamine tract. However, httN with expanded polyglutamine tracts appeared to be a preferred substrate for Hrd1. Functionally, we found that Hrd1 protects cells against the httN-induced cell death. These results suggest that Hrd1 is a novel htt-interacting protein that can target pathogenic httN for degradation and is able to protect cells against httN-induced cell death.
Yang
Hui
H
Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 W. Lombard Street, Baltimore, MD 21201, USA.
Zhong
Xiaoyan
X
Ballar
Petek
P
Luo
Shouqing
S
Shen
Yuxian
Y
Rubinsztein
David C
DC
Monteiro
Mervyn J
MJ
Fang
Shengyun
S
D033942:Adaptor Proteins, Vesicular Transport; D000251:Adenosine Triphosphatases / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D006801:Humans; D000071058:Huntingtin Protein; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism* / Q000502:physiology; D000074405:Valosin Containing Protein
D033942:Adaptor Proteins, Vesicular Transport; D017252:CD3 Complex; C472562:CD3delta antigen; D018797:Cell Cycle Proteins; C086055:HTT protein, human; D000071058:Huntingtin Protein; D047908:Intracellular Signaling Peptides and Proteins; C514112:NPLOC4 protein, human; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011506:Proteins; C491497:UFD1 protein, human; D025801:Ubiquitin; C097188:polyglutamine; C482345:SYVN1 protein, human; D044767:Ubiquitin-Protein Ligases; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein