Huntingtin Interacts with the Cue Domain of gp78 and Inhibits gp78 Binding to Ubiquitin and p97/VCP

Huntington''s disease (HD) is caused by polyglutamine expansion in huntingtin (htt) protein, but the exact mechanism of HD pathogenesis remains uncertain. Recent evidence suggests that htt proteins with expanded polyglutamine tracts induce endoplasmic reticulum (ER) stress, probably by interfering with ER-associated degradation (ERAD). Here we report that mutant htt interacts and interferes with the function of gp78, an ER membrane-anchored ubiquitin ligase (E3) involved in ERAD. Mapping studies showed that the HEAT repeats 2&3 of htt interact with the cue domain of gp78. The interaction competitively reduces polyubiquitinated protein binding to gp78 and also sterically blocks gp78 interaction of p97/VCP, a molecular chaperone that is essential for ERAD. These effects of htt negatively regulate the function of gp78 in ERAD and are aggravated by polyglutamine expansion. Paradoxically, gp78 is still able to ubiquitinate and facilitate degradation of htt proteins with expanded polyglutamine. The impairment of ERAD by mutant htt proteins is associated with induction of ER stress. Our studies provide a novel molecular mechanism that supports the involvement of ER stress in HD pathogenesis.     

Sequestration of glyceraldehyde-3-phosphate dehydrogenase to aggregates formed by mutant huntingtin

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） has been reported to interact with proteins containing the polyglutamine （polyQ） domain. The present study was undertaken to evaluate the potential contributions of the polyQ and polyproline （polyP） domains to the co-localization of mutant huntingtin （htt） and GAPDH. Overexpression of N-terminal htt （1-969 amino acids） with 100Q and 46Q （httl-969- 100Q and httl-969-46Q, mutant htt） in human mammary gland carcinoma MCF-7 cells formed more htt aggregates than that of httl-969-18Q （wild-type htt）. The co-localization of GAPDH with htt aggregates was found in the cells expressing mutant but not wild-type htt. Deletion of the polyP region in the N-terminal htt had no effect on the co-localization of GAPDH and mutant htt aggregates. These results suggest that the polyQ domain, but not the polyP domain, plays a role in the sequestration of GAPDH to aggregates by mutant htt. This effect might contribute to the dysfunction of neurons caused by mutant htt in Huntington＇s disease.     

ER stress differentially regulates the stabilities of ERAD ubiquitin ligases and their substrates

Endoplasmic reticulum (ER) stress-induced accumulation of misfolded proteins in the ER stimulates the ER-associated degradation (ERAD) process. ERAD in turn eliminates those misfolded proteins. Upregulation of ubiquitination enzymes is an essential mechanism by which ER stress enhances ERAD. However, ectopic overexpression of ubiquitination enzymes often fails to increase, and sometimes, inhibits ERAD. To further understand how ER stress regulates ERAD, we studied the effects of ER stress on ubiquitin ligase (E3) gp78-mediated ERAD and on the stabilities of gp78 and another ERAD E3 Hrd1. The results showed that ER stress-inducing agent tunicamycin significantly enhanced ERAD in cells that either express endogenous or overexpress gp78. Importantly, ER stress could increase ERAD even when new protein synthesis was inhibited by cycloheximide. Surprisingly, tunicamycin treatment stabilized gp78, an established ERAD E3 and an ERAD substrate as well, for up to 8h. By contrast, ER stress had little effects on the stability of another E3 Hrd1 except that it reduced the total ubiquitination level of Hrd1. Our data suggest that ER stress differentially regulates the stabilities of ERAD E3s and their substrates, which may represent a novel mechanism by which ER stress increases ERAD.     

Live cell imaging of protein dislocation from the endoplasmic reticulum

Misfolded proteins in the endoplasmic reticulum (ER) are dislocated to the cytosol to be degraded by the proteasomes. Various plant and bacterial toxins and certain viruses hijack this dislocation pathway to exert their toxicity or to infect cells. In this study, we report a dislocation-dependent reconstituted GFP (drGFP) assay that allows, for the first time, imaging proteins dislocated from the ER lumen to the cytosol in living cells. Our results indicate that both luminal and membrane-spanning ER proteins can be fully dislocated from the ER to the cytosol. By combining the drGFP assay with RNAi or chemical inhibitors of proteins in the Hrd1 ubiquitin ligase complex, we demonstrate that the Sel1L, Hrd1, p97/VCP, and importin β proteins are required for the dislocation of misfolded luminal α-1 antitrypsin. The strategy described in this work is broadly applicable to the study of other types of transmembrane transport of proteins and likely also of viruses and toxins in living cells.     

Determinants of RING-E2 fidelity for Hrd1p, a membrane-anchored ubiquitin ligase

A critical aspect of E3 ubiquitin ligase function is the selection of a particular E2 ubiquitin-conjugating enzyme to accomplish ubiquitination of a substrate. We examined the requirements for correct E2-E3 specificity in the RING-H2 ubiquitin ligase Hrd1p, an ER-localized protein known to use primarily Ubc7p for its function. Versions of Hrd1p containing the RING motif from homologous E3s were unable to carry out Hrd1p function, revealing a requirement for the specific Hrd1p RING motif in vivo. An in vitro assay revealed that these RING motifs were sufficient to function as ubiquitin ligases, but that they did not display the E2 specificity predicted from in vivo results. We further refined the in vitro assay of Hrd1p function by demanding not only ubiquitin ligase activity, but also specific activity that recapitulated both the E2 specificity and RING selectivity observed in vivo. Doing so revealed that correct E2 engagement by Hrd1p required the presence of portions of the Hrd1p soluble cytoplasmic domain outside the RING motif, the placement of the Hrd1p ubiquitin ligase in the ER membrane, and presentation of Ubc7p in the cytosolic context. We confirmed that these conditions supported the ubiquitination of Hrd1p itself, and the transfer of ubiquitin to the prototype substrate Hmg2p-GFP, validating Hrd1p self-ubiquitination as a viable assay of ligase function.     

Targeting of gp78 for ubiquitin-mediated proteasomal degradation by Hrd1: Cross-talk between E3s in the endoplasmic reticulum

There are an increasing number of ubiquitin ligases (E3s) implicated in endoplasmic reticulum (ER)-associated degradation (ERAD) in mammals. The two for which the greatest amount of information exists are the RING finger proteins gp78 and Hrd1, which are the structural orthologs of the yeast ERAD E3 Hrd1p. We now report that Hrd1, also known as synoviolin, targets gp78 for proteasomal degradation independent of the ubiquitin ligase activity of gp78, without evidence of a reciprocal effect. This degradation is observed in mouse embryonic fibroblasts lacking Hrd1, as well as with acute manipulation of Hrd1. The significance of this is underscored by the diminished level of a gp78-specific substrate, Insig-1, when Hrd1 expression is decreased and gp78 levels are consequently increased. These finding demonstrate a previously unappreciated level of complexity of the ubiquitin system in ERAD and have potentially important ramifications for processes where gp78 is implicated including regulation of lipid metabolism, metastasis, cystic fibrosis and neurodegenerative disorders.     

ER stress is the initial response to polyglutamine toxicity in PC12 cells

Persistent endoplasmic reticulum (ER) stress and impairment of the ubiquitin-proteasome system (UPS) cause neuronal cell death. However, the relationship between these two phenomena remains controversial. In our current study, we have utilized an expanded polyglutamine fusion protein (polyQ81) expression system in PC12 cells to further examine the involvement of ER stress and UPS impairment in cell death. The expression of polyQ81-induced ER stress and cell death. PolyQ81 also induced the activation of c-Jun N-terminal kinase (JNK) and caspase-3 and an increase in polyubiquitin immunoreactivity, suggesting UPS impairment. ER stress was induced prior to the accumulation of polyubiquitinated proteins. Low doses of lactacystin had almost similar effects on cell viability and on the activation of JNK and caspase-3 between normal cells and polyQ81-expressing cells. These results suggest that ER stress mediates polyglutamine toxicity prior to UPS impairment during the initial stages of these toxic effects.     

Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p

Misfolded, luminal endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol and degraded by the ubiquitin/proteasome system. This ERAD-L pathway requires a protein complex consisting of the ubiquitin ligase Hrd1p, which spans the ER membrane multiple times, and the membrane proteins Hrd3p, Usa1p, and Der1p. Here, we show that Hrd1p is the central membrane component in ERAD-L; its overexpression bypasses the need for the other components of the Hrd1p complex. Hrd1p function requires its oligomerization, which in wild-type cells is facilitated by Usa1p. Site-specific photocrosslinking indicates that, at early stages of retrotranslocation, Hrd1p interacts with a substrate segment close to the degradation signal. This interaction follows the delivery of substrate through other ERAD components, requires the presence of transmembrane segments of Hrd1p, and depends on both the ubiquitin ligase activity of Hrd1p and the function of the Cdc48p ATPase complex. Our results suggest a model for how Hrd1p promotes polypeptide movement through the ER membrane.     

Polyglutamine expansion in huntingtin increases its insertion into lipid bilayers

An expanded polyglutamine (Q) tract (>37Q) in huntingtin (htt) causes Huntington disease. Htt associates with membranes and polyglutamine expansion in htt may alter membrane function in Huntington disease through a mechanism that is not known. Here we used differential scanning calorimetry to examine the effects of polyQ expansion in htt on its insertion into lipid bilayers. We prepared synthetic lipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine and tested interactions of htt amino acids 1-89 with 20Q, 32Q or 53Q with the vesicles. GST-htt1-89 with 53Q inserted into synthetic lipid vesicles significantly more than GST-htt1-89 with 20Q or 32Q. We speculate that by inserting more into cell membranes, mutant huntingtin could increase disorder within the lipid bilayer and thereby disturb cellular membrane function.     

Aggregate formation and the impairment of long-term synaptic facilitation by ectopic expression of mutant huntingtin in Aplysia neurons

Huntington's disease (HD) is caused by an expansion of a polyglutamine (polyQ) tract within huntingtin (htt) protein. To examine the cytotoxic effects of polyQ-expanded htt, we overexpressed an enhanced green fluorescent protein (EGFP)-tagged N-terminal fragment of htt with 150 glutamine residues (Nhtt150Q-EGFP) in Aplysia neurons. A combined confocal and electron microscopic study showed that Aplysia neurons expressing Nhtt150Q-EGFP displayed numerous abnormal aggregates (diameter 0.5-5 microm) of filamentous structures, which were formed rapidly (approximately 2 h) but which were sustained for at least 18 days in the cytoplasm. Furthermore, the overexpression of Nhtt150Q-EGFP in sensory cells impaired 5-hydroxytryptamine (5-HT)-induced long-term synaptic facilitation in sensori-motor synapses without affecting basal synaptic strength or short-term facilitation. This study demonstrates the stability of polyQ-based aggregates and their specific effects on long-term synaptic plasticity.     

RACK1 modulates polyglutamine-induced neurodegeneration by promoting ERK degradation in Drosophila

Polyglutamine diseases are neurodegenerative diseases caused by the expansion of polyglutamine (polyQ) tracts within different proteins. Although multiple pathways have been found to modulate aggregation of the expanded polyQ proteins, the mechanisms by which polyQ tracts induced neuronal cell death remain unknown. We conducted a genome-wide genetic screen to identify genes that suppress polyQ-induced neurodegeneration when mutated. Loss of the scaffold protein RACK1 alleviated cell death associated with the expression of polyQ tracts alone, as well as in models of Machado-Joseph disease (MJD) and Huntington’s disease (HD), without affecting proteostasis of polyQ proteins. A genome-wide RNAi screen for modifiers of this rack1 suppression phenotype revealed that knockdown of the E3 ubiquitin ligase, POE (Purity of essence), further suppressed polyQ-induced cell death, resulting in nearly wild-type looking eyes. Biochemical analyses demonstrated that RACK1 interacts with POE and ERK to promote ERK degradation. These results suggest that RACK1 plays a key role in polyQ pathogenesis by promoting POE-dependent degradation of ERK, and implicate RACK1/POE/ERK as potent drug targets for treatment of polyQ diseases.     

Human HRD1 protects against ER stress-induced apoptosis through ER-associated degradation

Stresses that impair the function of the endoplasmic reticulum (ER) lead to an accumulation of unfolded protein in the ER. Under these conditions, the expression of a variety of genes involved in preventing the accumulation of the unfolded proteins is induced. Yeast Hrd1p is an ER stress-inducible ER membrane protein that acts as a ubiquitin ligase (E3) with a RING finger motif and plays a role in the ubiquitination of proteins in the ER. We report here the identification and characterization of a human homolog to yeast Hrd1p. The predicted structures are highly conserved from yeast to humans. Indeed, human HRD1 was localized to the ER and ubiquitinated its substrates. Furthermore, it was found that human HRD1 was up-regulated by ER stress via IRE1 and ATF6, which are ER stress transducers. Interestingly, 293 cells stably expressing wild-type HRD1, but not the C329S mutant, afforded resistance to ER stress-induced apoptosis. These results suggest that the production of HRD1 is up-regulated to protect against ER stress-induced apoptosis by degrading unfolded proteins accumulated in the ER.     

ER signaling in unfolded protein response

Abnormally folded proteins are susceptible to aggregation and accumulation in cells, ultimately leading to cell death. To protect cells against such dangers, expression of various genes including molecular chaperones can be induced and ER-associated protein degradation (ERAD) activated in response to the accumulation of unfolded protein in the endoplasmic reticulum (ER). This is known as the unfolded protein response (UPR). ERAD requires retrograde transport of unfolded proteins from the ER back to the cytosol via the translocon for degradation by the ubiquitin-proteasome system. Hrd1p is a UPR-induced ER membrane protein that acts as a ubiquitin ligase (E3) in the ERAD system. Hrd3p interacts with and stabilizes Hrd1p. We have isolated and identified human homologs (HRD1 and SEL1/HRD3) of Saccharomyces cerevisiae Hrd1p and Hrd3p. Human HRD1 and SEL1 were up-regulated in response to ER stress and overexpression of human IRE1 and ATF6, which are ER stress-sensor molecules in the ER. HEK293T cells overexpressing HRD1 showed resistance to ER stress-induced cell death. These results suggest that HRD1 and SEL1 are up-regulated by the UPR and contribute to protection against the ER stress-induced cell death by degrading unfolded proteins accumulated in the ER.     

Inhibition of 26S proteasome activity by huntingtin filaments but not inclusion bodies isolated from mouse and human brain

In Huntington's disease (HD), as in the rest of CAG triplet-repeat disorders, the expanded polyglutamine (polyQ)-containing proteins form intraneuronal fibrillar aggregates that are gathered into inclusion bodies (IBs). Since IBs contain ubiquitin and proteasome subunits, it was proposed that inhibition of proteasome activity might underlie pathogenesis of polyQ disorders. Recent in vitro enzymatic studies revealed the inability of eukaryotic proteasomes to digest expanded polyQ, thus suggesting that occasional failure of polyQ to exit the proteasome may interfere with its proteolytic function. However, it has also recently been found that in vitro assembled aggregates made of synthetic polyQ fail to inhibit proteasome activity. Because synthetic polyQ aggregates lack the post-translational modifications found inside affected neurons, such as poly ubiquitylation, we decided to study the effect of mutant huntingtin (htt) aggregates isolated from the Tet/HD94 mouse model and from human HD brain tissue. Here, we show that isolated ubiquitylated filamentous htt aggregates, extracted from IBs by a previously reported method, selectively inhibited the in vitro peptidase activity of the 26S but not of the 20S proteasome in a non-competitive manner. In good agreement, immuno-electron microscopy revealed a direct interaction of htt filaments with the 19S ubiquitin-interacting regulatory caps of the 26S proteasome. Here, we also report a new method for isolation of IBs based on magnetic sorting. Interestingly, isolated IBs did not modify proteasome activity. Our results therefore show that mutant htt filamentous aggregates can inhibit proteasome activity, but only when not recruited into IBs, thus strengthening the notion that IB formation is protective by neutralizing toxicity of dispersed filamentous htt aggregates.     

Membrane topology and function of Der3/Hrd1p as a ubiquitin-protein ligase (E3) involved in endoplasmic reticulum degradation

The endoplasmic reticulum contains a protein quality control system that discovers malfolded or unassembled secretory proteins and subjects them to degradation in the cytosol. This requires retrograde transport of the respective proteins from the endoplasmic reticulum back to the cytosol via the Sec61 translocon. In addition, a fully competent ubiquitination machinery and the 26 S proteasome are necessary for retrotranslocation and degradation. Ubiquitination of mutated and malfolded proteins of the endoplasmic reticulum is dependent mainly on the ubiquitin-conjugating enzyme Ubc7p. In addition, several new membrane components of the endoplasmic reticulum are required for degradation. Here we present the topology of the previously discovered RING-H2 finger protein Der3/Hrd1p, one of the new components of the endoplasmic reticulum membrane. The protein spans the membrane six times. The amino terminus and the carboxyl terminus containing the RING finger domain face the cytoplasm. Altogether, RING finger-dependent ubiquitination of malfolded carboxypeptidase yscY in vivo, as well as of Der3/Hrd1p itself in vitro and RING finger-dependent binding of Ubc7p, uncovers Der3/Hrd1p as the ubiquitin-protein ligase (E3) of the endoplasmic reticulum-associated protein degradation process.     

Mutant huntingtin promotes the fibrillogenesis of wild-type huntingtin: a potential mechanism for loss of huntingtin function in Huntington's disease

Aggregation of huntingtin (htt) in neuronal inclusions is associated with the development of Huntington's disease (HD). Previously, we have shown that mutant htt fragments with polyglutamine (polyQ) tracts in the pathological range (>37 glutamines) form SDS-resistant aggregates with a fibrillar morphology, whereas wild-type htt fragments with normal polyQ domains do not aggregate. In this study we have investigated the co-aggregation of mutant and wild-type htt fragments. We found that mutant htt promotes the aggregation of wild-type htt, causing the formation of SDS-resistant co-aggregates with a fibrillar morphology. Conversely, mutant htt does not promote the fibrillogenesis of the polyQ-containing protein NOCT3 or the polyQ-binding protein PQBP1, although these proteins are recruited into inclusions containing mutant htt aggregates in mammalian cells. The formation of mixed htt fibrils is a highly selective process that not only depends on polyQ tract length but also on the surrounding amino acid sequence. Our data suggest that mutant and wild-type htt fragments may also co-aggregate in neurons of HD patients and that a loss of wild-type htt function may contribute to HD pathogenesis.     

Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner

Accumulation of aberrant proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response pathway that helps the cell to survive under these stress conditions. Herp is a mammalian ubiquitin domain protein, which is strongly induced by the unfolded protein response. It is involved in ER-associated protein degradation (ERAD) and interacts directly with the ubiquitin ligase Hrd1, which is found in high molecular mass complexes of the ER membrane. Here we present the first evidence that Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like (UBL) domain-dependent manner. We found that upon exposure of cells to ER stress, elevation of Herp steady state levels is accompanied by an enhanced association of Herp with pre-existing Hrd1. Hrd1-associated Herp is rapidly degraded and substituted by de novo synthesized Herp, suggesting a continuous turnover of the protein at Hrd1 complexes. Further analysis revealed the presence of multiple Hrd1 copies in a single complex enabling binding of a variable number of Herp molecules. Efficient ubiquitylation of the Hrd1-specific ERAD substrate α1-antitrypsin null Hong Kong (NHK) required the presence of the Herp UBL domain, which was also necessary for NHK degradation. In summary, we propose that binding of Herp to Hrd1-containing ERAD complexes positively regulates the ubiquitylation activity of these complexes, thus permitting survival of the cell during ER stress.     

Phylogenetic comparison of huntingtin homologues reveals the appearance of a primitive polyQ in sea urchin

Huntingtin is a completely soluble 3,144 amino acid (aa) proteincharacterized by the presence of an amino-terminal polymorphicpolyglutamine (polyQ) tract, whose aberrant expansion causesthe progressively neurodegenerative Huntington's disease (HD).Biological evidence indicates that huntingtin (htt) is beneficialto cells (particularly to brain neurons) and that loss of itsneuronal function may contribute to HD. The exact protein domainsinvolved in its neuroprotective function are unknown. Evolutionaryanalyses of htt primary aa have so far been limited to a fewspecies, but its thorough assessment may help to clarify thefunctions emerging during evolution. We made an extensive comparativeanalysis of the available htt protein homologues from differentorganisms along the metazoan phylogenetic tree and defined thepresence of 3 different conservative blocks corresponding tohuman htt aa 1–386 (htt1), 683–1,586 (htt2), and2,437–3,078 (htt3), in which HEAT (Huntingtin, Elongatorfactor3, the regulatory A subunit of protein phosphatase 2A,and TOR1) repeats are well conserved. We also describe the cloningand sequencing of sea urchin htt mRNA, the oldest deuterostomehomologue so far available. Multiple alignment shows the firstappearance of a primitive polyQ in sea urchin, which predatesan ancestral polyQ sequence in a nonchordate environment anddefines the polyQ characteristic as being typical of the deuterostomebranch. The fact that glutamines have conserved positions indeuterostomes and the polyQ size increases during evolutionsuggests that the protein has a possibly Q-dependent role. Finally,we report an evident relaxing constraint of the N-terminal blockin Ciona and drosophilids that correlates with the absence ofpolyQ and which may indicate that the N-terminal portion ofhtt has evolved different functions in Ciona and protostomes.     

Reprint of "Improved spectral resolution in COSY (1)H NMR spectra of proteins via double quantum filtering"

Polyglutamine (polyQ) diseases are inherited neurodegenerative diseases characterized by the aggregation of proteins containing expanded polyQ tract. It has been shown that expanded polyQ tract-containing proteins impair the functions of other cellular proteins. However, quantitative changes of cellular proteins in cells expressing expanded polyQ tract-containing proteins have not been performed. Here, we performed proteomic analysis of cells expressing expanded polyQ tract-containing proteins, and showed that GRP78, the endoplasmic reticulum (ER) chaperone, was significantly decreased in the cells expressing enhanced green fluorescent protein with a pathological-length polyQ tract (EGFP-polyQ97), but not with a non-pathological-length polyQ tract (EGFP-polyQ24). In addition, we revealed that down-regulation of GRP78 expression resulted in increase of the aggregation of EGFP-polyQ97. Conversely, the aggregation of EGFP-polyQ97 was suppressed by the overexpression of GRP78 in the cells. Furthermore, it seemed that the decreased GRP78 expression in the cells expressing EGFP-polyQ97 was due to the enhanced protein degradation of GRP78 through the ubiquitin-proteasome pathway. These findings indicated that GRP78, which has an inhibitory effect on the aggregation of proteins containing expanded polyQ tract, may be an effective target for the treatment of polyQ diseases.