Tipping the delicate balance: defining how proteasome maturation affects the degradation of a substrate for autophagy and endoplasmic reticulum associated degradation (ERAD)

An increasing body of data links endoplasmic reticulum (ER) function to autophagy. Not surprisingly, then, some aberrant proteins in the ER can be destroyed either via ER associated degradation (ERAD), which is proteasome-mediated, or via autophagy. One such substrate is the "Z" variant of the alpha-1 protease inhibitor (A1Pi), variably known as A1Pi-Z or AT-Z ("anti-trypsin, Z variant"). The wild type protein is primarily synthesized in the liver and is secreted. In contrast, AT-Z, like other ERAD substrates, is retro-translocated from the ER and delivered to the proteasome. However, AT-Z can form high molecular weight polymers that are degraded via autophagy, and cells that accumulate AT-Z polymers ultimately succumb, which leads to liver disease. Therefore, identifying genes that have an impact AT-Z turnover represents an active area of research. To this end, a yeast expression system for AT-Z has proven valuable. For example, a recent study using this system indicates that the activity of a proteasome assembly chaperone (PAC) is critical for maximal AT-Z turnover, which suggests a new role for PACs. Because PACs are conserved, it will be critical to analyze whether these dedicated chaperones are implicated in other diseases associated with ERAD and autophagy.     

Tipping the Delicate Balance: Defining How Proteasome Maturation Affects the Degradation of a Substrate for Autophagy and Endoplasmic Reticulum Associated Degradation (ERAD)

An increasing body of data links endoplasmic reticulum (ER) function to autophagy. Not surprisingly, then, some aberrant proteins in the ER can be destroyed either via ER associated degradation (ERAD), which is proteasome-mediated, or via autophagy. One such substrate is the “Z” variant of the alpha-1 protease inhibitor (A1Pi), variably known as A1Pi-Z or AT-Z (“anti-trypsin, Z variant”). The wild type protein is primarily synthesized in the liver and is secreted. In contrast, AT-Z—like other ERAD substrates—is retro-translocated from the ER and delivered to the proteasome. However, AT-Z can form high molecular weight polymers that are degraded via autophagy, and cells that accumulate AT-Z polymers ultimately succumb, which leads to liver disease. Therefore, identifying genes that have an impact AT-Z turnover represents an active area of research. To this end, a yeast expression system for AT-Z has proven valuable. For example, a recent study using this system indicates that the activity of a proteasome assembly chaperone (PAC) is critical for maximal AT-Z turnover, which suggests a new role for PACs. Because PACs are conserved, it will be critical to analyze whether these dedicated chaperones are implicated in other diseases associated with ERAD and autophagy.

Addendum to:

ADD66, a Gene Involved in the Endoplasmic Reticulum Associated Degradation of a1-Antitrypsin-Z in Yeast, Facilitates Proteasome Activity and Assembly

C.M. Scott, K.B. Kruse, B.Z. Schmidt, D.H. Perlmutter, A.A. McCracken and J.L. Brodsky

Mol Biol Cell 2007; In press     

Endoplasmic reticulum-associated degradation mediated by MoHrd1 and MoDer1 is pivotal for appressorium development and pathogenicity of Magnaporthe oryzae

Most secretory proteins are folded and modified in the endoplasmic reticulum (ER); however, protein folding is error-prone, resulting in toxic protein aggregation and cause ER stress. Irreversibly misfolded proteins are subjected to ER-associated degradation (ERAD), modified by ubiquitination, and degraded by the 26S proteasome. The yeast ERAD ubiquitin ligase Hrd1p and multispanning membrane protein Der1p are involved in ubiquitination and transportation of the folding-defective proteins. Here, we performed functional characterization of MoHrd1 and MoDer1 and revealed that both of them are localized to the ER and are pivotal for ERAD substrate degradation and the ER stress response. MoHrd1 and MoDer1 are involved in hyphal growth, asexual reproduction, infection-related morphogenesis, protein secretion and pathogenicity of M. oryzae. Importantly, MoHrd1 and MoDer1 mediated conidial autophagic cell death and subsequent septin ring assembly at the appressorium pore, leading to abnormal appressorium development and loss of pathogenicity. In addition, deletion of MoHrd1 and MoDer1 activated the basal unfolded protein response (UPR) and autophagy, suggesting that crosstalk between ERAD and two other closely related mechanisms in ER quality control system (UPR and autophagy) governs the ER stress response. Our study indicates the importance of ERAD function in fungal development and pathogenesis of M. oryzae.     

FBG1 is a promiscuous ubiquitin ligase that sequesters APC2 and causes S-phase arrest

During cell proliferation, protein degradation is strictly regulated by the cell cycle and involves two complementary ubiquitin ligase complexes, the SCF (Skp, Cullin, F-box) and APC/C (Anaphase Promoting Complex/Cyclosome) ubiquitin ligases. SCF ligases are constitutively active and generally target only proteins after they have been selected for degradation, usually by phosphorylation. In contrast, APC/C complexes are themselves activated by phosphorylation and their substrates contain a targeting signal known as degron, a consensus amino acid sequence such as a D-Box. SCF complexes degrade proteins during the G1 phase. However, as DNA synthesis begins, the SCF complexes are degraded and APC/C complexes are activated. APC-2, a protein crucial to cell division, initiates anaphase by triggering the degradation of multiple proteins. This study explores an unexpected interaction between APC-2 and SCF^FBG1. We found that FBG1 is a promiscuous ubiquitin ligase with many partners. Immunoprecipitation experiments demonstrate that FBG1 and APC2 interact directly. Mutagenesis-based experiments show that this interaction requires a D-Box found within the FBG1 F-box domain. Unexpectedly, we demonstrate that co-expression with FBG1 increases total APC2 levels. However, free APC2 is decreased, inhibiting cell proliferation. Finally, FACS analysis of cell populations expressing different forms of FBG1 demonstrate that this ubiquitin ligase induces S-phase arrest, illustrating the functional consequences of the interaction described. In summary, we have discovered a novel APC2 inhibitory activity of FBG1 independent from its function as ubiquitin ligase, providing the basis for future studies of FBG1 in aging and cancer.     

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.     

TRIM13 regulates ER stress induced autophagy and clonogenic ability of the cells

Autophagy is one of the cellular adaptive processes that provide protection against many pathological conditions like infection, cancer, neurodegeneration, and aging. Recent evidences suggest that ubiquitination plays an important role in degradation of proteins or defective organelle either through proteasome or autophagy. In this study, we describe the role of TRIM13, ER resident ubiquitin E3 ligase in induction of autophagy and its role during ER stress. The ectopic expression of TRIM13 in HEK-293 cells induces autophagy. Domain mapping showed that coiled-coil (CC) domain is required for induction of autophagy. TRIM13 is stabilized during ER stress, interacts with p62/SQSTM1 and co-localizes with DFCP1. TRIM13 regulates initiation of autophagy during ER stress and decreases the clonogenic ability of the cells. This study for the first time demonstrates the role of TRIM13 in induction of autophagy which may play an important role in regulation of ER stress and may act as tumor suppressor.     

Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins

Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3′-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane.     

The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.

The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.     

Insights into links between autophagy and the ubiquitin system from the structure of LC3B bound to the LIR motif from the E3 ligase NEDD4

Members of the LC3/GABARAP family of ubiquitin‐like proteins function during autophagy by serving as membrane linked protein‐binding platforms. Their C‐termini are physically attached to membranes through covalent linkage to primary amines on lipids such as phosphatidylethanolamine, while their ubiquitin‐like fold domains bind “LIR” (LC3‐Interacting Region) sequences found within an extraordinarily diverse array of proteins including regulators of autophagy, adaptors that recruit ubiquitinated cargoes to be degraded, and even proteins controlling processes at membranes that are not associated with autophagy. Recently, LC3/GABARAP proteins were found to bind the ubiquitin E3 ligase NEDD4 to influence ubiquitination associated with autophagy in human cell lines. Here, we use purified recombinant proteins to define LC3B interactions with a specific LIR sequence from NEDD4, present a crystal structure showing atomic details of the interaction, and show that LC3B‐binding can steer intrinsic NEDD4 E3 ligase activity. The data provide detailed molecular insights underlying recruitment of an E3 ubiquitin ligase to phagophores during autophagy.     

Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRΔF508, by specifically promoting ubiquitylation of CFTRΔF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRΔF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRΔF508 and catalyzes further polyubiquitylation of CFTRΔF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRΔF508.     

Estradiol promotes rapid degradation of HER3 in ER-positive breast cancer cell line MCF-7

HER3, a member of the receptor tyrosine kinase super family, is overexpressed in a number of cancers, and is associated with malignant phenotypes. Control of the protein stability of the membrane, as well as nuclear receptors, has been known to be an important process affecting tumor cells; however, their relationships have yet to be elucidated. In this study, we demonstrate that estradiol promotes rapid degradation of HER3 via the proteasome pathway in ER-positive breast cancer, MCF-7. ER prevented HER3 degradation, and knockdown of ER expression by si-RNA promoted rapid degradation of HER3. Breakdown of HER3 and ER were regulated by a ubiquitin ligase Nedd4-1 in the presence of estradiol stimulation. We speculate that estradiol quickly degrades ER, making HER3 accessible by Nedd4-1, and leads to the rapid degradation of HER3. In addition, knockdown of ubiquitin ligase Nedd4-1 enhances estradiol induced cell proliferation. These results indicate that HER3 and Nedd4-1 in ER-positive breast cancers might be an important therapeutic target.     

Role of HERP and a HERP-related Protein in HRD1-dependent Protein Degradation at the Endoplasmic Reticulum

Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated to the cytosol and degraded by the proteasome via a process termed ER-associated degradation (ERAD). The precise mechanism of retrotranslocation is unclear. Here, we use several lumenal ERAD substrates targeted for degradation by the ubiquitin ligase HRD1 including SHH (sonic hedgehog) and NHK (null Hong Kong α1-antitrypsin) to study the geometry, organization, and regulation of the HRD1-containing ERAD machinery. We report a new HRD1-associated membrane protein named HERP2, which is homologous to the previously identified HRD1 partner HERP1. Despite sequence homology, HERP2 is constitutively expressed in cells, whereas HERP1 is highly induced by ER stress. We find that these proteins are required for efficient degradation of both glycosylated and nonglycosylated SHH proteins as well as NHK. In cells depleted of HERPs, SHH proteins are largely trapped inside the ER with a fraction of the stabilized SHH protein bound to the HRD1-SEL1L ligase complex. Ubiquitination of SHH is significantly attenuated in the absence of HERPs, suggesting a defect in retrotranslocation. Both HERP proteins interact with HRD1 through a region located in the cytosol. However, unlike its homolog in Saccharomyces cerevisiae, HERPs do not regulate HRD1 stability or oligomerization status. Instead, they help recruit DERL2 to the HRD1-SEL1L complex. Additionally, the UBL domain of HERP1 also seems to have a function independent of DERL2 recruitment in ERAD. Our studies have revealed a critical scaffolding function for mammalian HERP proteins that is required for forming an active retrotranslocation complex containing HRD1, SEL1L, and DERL2.     

植物泛素/26S蛋白酶体途径研究进展

泛素/26S蛋白酶体途径是最重要的,有高度选择性的蛋白质降解途径,由泛素激活酶、泛素结合酶、泛素蛋白连接酶和26S蛋白酶体组成,参与调控植物生长发育的多个方面。泛素蛋白酶体途径参与植物体内的众多生理过程,如植物激素信号,光形态建成、自交不亲和反应和细胞周期等。本文就泛素/26S蛋白酶体途径以及在植物生长发育中的作用的研究近况做一综述。     

The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway

To eliminate misfolded proteins that accumulate in the endoplasmic reticulum (ER) the cell mainly relies on ubiquitin-proteasome dependent ER-associated protein degradation (ERAD). Proteolysis of ERAD substrates by the proteasome requires their ubiquitylation and retro-translocation from the ER to the cytoplasm. Here we describe a high molecular mass protein complex associated with the ER membrane, which facilitates ERAD. It contains the ubiquitin domain protein (UDP) HERP, the ubiquitin protein ligase HRD1, as well as the retro-translocation factors p97, Derlin-1 and VIMP. Our data on the structural arrangement of these ERAD proteins suggest that p97 interacts directly with membrane-resident components of the complex including Derlin-1 and HRD1, while HERP binds directly to HRD1. We propose that ubiquitylation, as well as retro-translocation of proteins from the ER are performed by this modular protein complex, which permits the close coordination of these consecutive steps within ERAD.     

APP and APLP1 are degraded through autophagy in response to proteasome inhibition in neuronal cells

Amyloid beta (Aβ) precursor protein (APP) is a key protein in the pathogenesis of Alzheimer’s disease (AD). Both APP and its paralogue APLP1 (amyloid beta precursor-like protein 1) have multiple functions in cell adhesion and proliferation. Previously it was thought that autophagy is a novel beta-amyloid peptide (Aβ)-generating pathway activated in AD. However, the protein proteolysis of APLP1 is still largely unknown. The present study shows that APLP1 is rapidly degraded in neuronal cells in response to stresses, such as proteasome inhibition. Activation of the endoplasmic reticulum (ER) stress by proteasome inhibitors induces autophagy, causing reduction of mature APLP1/APP. Blocking autophagy or JNK stress kinase rescues the protein expression for both APP and APLP1. Therefore, our results suggest that APP/APLP1 is degraded through autophagy and the APLP1 proteolysis is mainly mediated by autophagy-lysosome pathway.     

Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum

The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys(48)-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-alpha and CD3-delta.