Biomedical subjects

Allan M Weissman

Publications and source records attributed to Allan M Weissman.

At least 19 recordsLinked to original sources

PubMed2006

E6AP mediates regulated proteasomal degradation of the nuclear receptor coactivator amplified in breast cancer 1 in immortalized cells.

The steroid receptor coactivator oncogene, amplified in breast cancer 1 (AIB1; also known as ACTR/RAC-3/TRAM-1/SRC-3/p/CIP), is amplified and overexpressed in a variety of epithelial tumors. AIB1 has been reported to have roles in both steroid-dependent and steroid-independent transcription during tumor progression. In this report, we describe that the cellular levels of AIB1 are controlled through regulated proteasomal degradation. We found that serum withdrawal or growth in high cell density caused rapid degradation of AIB1 protein, but not mRNA, in immortalized cell lines. Proteasome inhibitors prevented this process, and high molecular weight ubiquitylated species of AIB1 were detected. Nuclear export was required for proteasomal degradation of AIB1 and involved the ubiquitin ligase, E6AP. AIB1/E6AP complexes were detected in cellular extracts, and reduction of cellular E6AP levels with E6AP short interfering RNA prevented proteasomal degradation of AIB1. Conversely, overexpression of E6AP promoted AIB1 degradation. The COOH terminus of AIB1 interacted with E6AP in vitro and deletion of this region in AIB1 rendered it resistant to degradation in cells. From our results, we propose a model whereby signals promoted by changes in the cellular milieu initiate E6AP-mediated proteasomal degradation of AIB1 and thus contribute to the control of steady-state levels of this protein.

Breast Neoplasms↗

PubMed2006

Structural basis for ubiquitin recognition and autoubiquitination by Rabex-5.

Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination and contains an intrinsic ubiquitin ligase activity, all of which require an N-terminal A20 zinc finger followed immediately by a helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5-A resolution shows that Rabex-5-ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin-binding domain, an inverted ubiquitin-interacting motif, which binds with approximately 29-microM affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with approximately 22-microM affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc-finger diaromatic patch mediates ubiquitin-ligase activity by directly recruiting a ubiquitin-loaded ubiquitin-conjugating enzyme.

Amino Acid Sequence↗

PubMed2006

The Rab5 guanine nucleotide exchange factor Rabex-5 binds ubiquitin (Ub) and functions as a Ub ligase through an atypical Ub-interacting motif and a zinc finger domain.

Rabex-5, the mammalian orthologue of yeast Vps9p, is a guanine nucleotide exchange factor for Rab5. Rabex-5 forms a tight complex with Rabaptin-5, a multivalent adaptor protein that also binds to Rab4, Rab5, and to domains present in gamma-adaptins and the Golgi-localized, gamma-ear-containing, ARF-binding proteins (GGAs). Rabaptin-5 augments the Rabex-5 exchange activity, thus generating GTP-bound, membrane-associated Rab5 that, in turn, binds Rabaptin-5 and stabilizes the Rabex-5.Rabaptin-5 complex on endosomes. Although the Rabex-5.Rabaptin-5 complex is critical to the regulation of endosomal fusion, the structural determinants of this interaction are unknown. Likewise, the possible binding and covalent attachment of ubiquitin to Rabex-5, two modifications that are critical to the function of yeast Vps9p in endosomal transport, have not been studied. In this study, we identify the 401-462 and 551-661 coiled-coils as the regions in Rabex-5 and Rabaptin-5, respectively, that interact with one another. We also demonstrate that Rabex-5 undergoes ubiquitination and binds ubiquitin, though not via its proposed C-terminal CUE-like domain. Instead, the N-terminal region of Rabex-5 (residues 1-76), comprising an A20-like Cys2/Cys2 zinc finger and an adjacent alpha-helix, is important for ubiquitin binding and ubiquitination. Importantly, we demonstrate that the Rabex-5 zinc finger displays ubiquitin ligase (E3) activity. These observations extend our understanding of the regulation of Rabex-5 by Rabaptin-5. Moreover, the demonstration that Rabex-5 is a ubiquitin ligase that binds ubiquitin and undergoes ubiquitination indicates that its role in endosome fusion may be subject to additional regulation by ubiquitin-dependent modifications.

Adaptor Proteins, Signal Transducing↗

PubMed2006

The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site.

Efficient targeting of proteins for degradation from the secretory pathway is essential to homeostasis. This occurs through endoplasmic reticulum (ER)-associated degradation (ERAD). In this study, we establish that a human ubiquitin ligase (E3), gp78, and a specific E2, Ube2g2, are both critically important for ERAD of multiple substrates. gp78 exhibits a complex domain structure that, in addition to the RING finger, includes a ubiquitin-binding Cue domain and a specific binding site for Ube2g2. Disruption of either of these domains abolishes gp78-mediated ubiquitylation and protein degradation, resulting in accumulation of substrates in their fully glycosylated forms in the ER. This suggests that gp78-mediated ubiquitylation is an early step in ERAD that precedes dislocation of substrates from the ER. The in vivo requirement for both an E2-binding site distinct from the RING finger and a ubiquitin-binding domain intrinsic to an E3 suggests a previously unappreciated level of complexity in ubiquitin ligase function. These results also provide proof of principle that interrupting a specific E2-E3 interaction can selectively inhibit ERAD.

Amino Acid Motifs↗

PubMed2005

Small molecule inhibitors of HDM2 ubiquitin ligase activity stabilize and activate p53 in cells.

The p53 tumor suppressor protein is regulated by its interaction with HDM2, which serves as a ubiquitin ligase (E3) to target p53 for degradation. We have identified a family of small molecules (HLI98) that inhibits HDM2's E3 activity. These compounds show some specificity for HDM2 in vitro, although at higher concentrations effects on unrelated RING and HECT domain E3s are detectable, which could be due, at least in part, to effects on E2-ubiquitin thiol-ester levels. In cells, the compounds allow the stabilization of p53 and HDM2 and activation of p53-dependent transcription and apoptosis, although other p53-independent toxicity was also observed.

Animals↗

PubMed2005

Expression, purification, and properties of the Ubc4/5 family of E2 enzymes.

Ubiquitin-conjugating enzymes (E2s) play a central role in ubiquitylation. They function to bridge the first, nonspecific step of ubiquitin activation by E1 with the transfer of activated ubiquitin to substrates by substrate-specific E3s. While sharing a common core UBC domain, members of this family exhibit significant specificity in their physical and functional interactions with E3s. Among the families of E2s, members of the yeast Ubc4/5 family are particularly well conserved in higher metazoans. In humans, these are represented by the UbcH5 family. Members of this ubiquitously expressed family show a capacity to interact with a wide range of E3s from both HECT and RING finger families, making them particularly useful tools in the laboratory. Using the UbcH5 family as a prototype, this chapter describes methods for the expression, purification, and characterization of E2 enzymes in vitro and some of the basics for their use in experiments in cells.

Escherichia coli↗

PubMed2005

Expression and evaluation of RING finger proteins.

RING finger proteins represent the largest class of potential ubiquitin ligases. This chapter describes methods used to express and assess the activity of proteins containing RING fingers based on our experience with a number of different family members. In addition to general protocols for assessing activity, specific protocols are provided for evaluating the ubiquitylation of p53 by the RING finger E3 Hdm2/Mdm2. Use of these methods may help identify new E3s, dissect factors involved in ubiquitylation of substrates, and screen for molecules that affect ubiquitylation.

Animals↗

PubMed2005

Multiple roles of Rbx1 in the VBC-Cul2 ubiquitin ligase complex.

The importance of the ubiquitin system largely depends on ubiquitin ligases, E3s, as they determine the specificity of the system. Rbx1/ROC1/Hrt1, a RING finger protein, functions as an important component of the cullin-containing SCF and VBC-Cul2 ligases. Modification of cullins by NEDD8 (NEDDylation), has been shown to be essential for the E3 activity of both SCF and VBC-Cul2, and it was suggested that Rbx1 acts as the E3 for cullin NEDDylation. RING finger is composed of eight cysteine and histidine residues that bind to zinc ions. Rbx1 is a highly evolutionarily conserved protein; however, the eighth coordination residue in its RING finger is aspartate (D97) rather than cysteine. Substitution of D97 with each of the other 19 amino acids demonstrates that aspartate is superior to cysteine in cullin NEDDylation. Interestingly, however, different D97 mutants demonstrate different activities towards 6 cullins tested. Importantly, we were able to discriminate between the NEDDylating activity of Rbx1 and its involvement in the ubiquitylation reaction within the context of VBC-Cul2. Moreover, while Rbx1 is not involved in governing the stability of SCF, Rbx1 mutants destabilize VBC-Cul2. Taken together, these results indicate that various mechanisms regulate both the activities and the stability of cullin-based ligases.

Carrier Proteins↗

PubMed2004

Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import.

Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.

Active Transport, Cell Nucleus↗

PubMed2004

Regulating the p53 system through ubiquitination.

The tumor suppressor p53 is tightly controlled at low levels in cells by constant ubiquitination and proteasomal degradation. In response to stresses, ubiquitination of p53 is inhibited through diverse pathways, depending on the nature of the stimulus and cell type. This leads to the accumulation and activation of p53, which induces cell cycle arrest and/or apoptosis to prevent cells from transformation. Many studies have indicated that defects of the p53 system are present in most, if not all, human tumor cells. Meanwhile, significant progress has been made in understanding the molecular mechanisms of p53 ubiquitination and the regulation of the p53 system. Therefore, it is possible now to consider targeting ubiquitination as a means to regulate and reactivate p53 in tumors. Emerging evidence suggests that inhibiting the E3 activity of Mdm2, blocking the interaction of p53 and Mdm2, and restoring the function of mutated p53 are potential effective strategies to kill certain tumor cells selectively. It is conceivable that new chemotherapeutic agents based on these studies will be generated in the not-so-distant future.

Animals↗

PubMed2003

A conserved catalytic residue in the ubiquitin-conjugating enzyme family.

Ubiquitin (Ub) regulates diverse functions in eukaryotes through its attachment to other proteins. The defining step in this protein modification pathway is the attack of a substrate lysine residue on Ub bound through its C-terminus to the active site cysteine residue of a Ub-conjugating enzyme (E2) or certain Ub ligases (E3s). So far, these E2 and E3 cysteine residues are the only enzyme groups known to participate in the catalysis of conjugation. Here we show that a strictly conserved E2 asparagine residue is critical for catalysis of E2- and E2/RING E3-dependent isopeptide bond formation, but dispensable for upstream and downstream reactions of Ub thiol ester formation. In contrast, the strictly conserved histidine and proline residues immediately upstream of the asparagine are dispensable for catalysis of isopeptide bond formation. We propose that the conserved asparagine side chain stabilizes the oxyanion intermediate formed during lysine attack. The E2 asparagine is the first non-covalent catalytic group to be proposed in any Ub conjugation factor.

Asparagine↗

PubMed2003

WW domain HECT E3s target Cbl RING finger E3s for proteasomal degradation.

Cbl proteins have RING finger-dependent ubiquitin ligase (E3) activity that is essential for down-regulation of tyrosine kinases. Here we establish that two WW domain HECT E3s, Nedd4 and Itch, bind Cbl proteins and target them for proteasomal degradation. This is dependent on the E3 activity of the HECT E3s but not on that of Cbl. Consistent with these observations, in cells expressing the epidermal growth factor receptor, Nedd4 reverses Cbl-b effects on receptor down-regulation, ubiquitylation, and proximal events in signaling. Cbl-b also targets active Src for degradation in cells, and Nedd4 similarly reverses Cbl-mediated Src degradation. These findings establish that RING finger E3s can be substrates, not only for autoubiquitylation but also for ubiquitylation by HECT E3s and suggest an additional level of regulation for Cbl substrates including protein-tyrosine kinases.

Cell Line↗

PubMed2003

BTBD1 and BTBD2 colocalize to cytoplasmic bodies with the RBCC/tripartite motif protein, TRIM5delta.

We previously identified BTBD1 and BTBD2 as novel topoisomerase I-interacting proteins that share 80% amino acid identity. Here we report the characterization of their subcellular localization. In a number of mouse and human cells, BTBD1 and BTBD2 (BTBD1/2) colocalized to punctate or elongated cytoplasmic bodies (< 5 microm long and several per cell) that were larger and more elongated in cancer cell lines than in fibroblasts and myoblasts. A search for potential colocalizing proteins identified TRIM family members that localize to morphologically similar cytoplasmic bodies, which were then tested for colocalization with BTBD1/2. TRIM5delta, expressed as a GFP fusion, colocalized with BTBD1/2 immunostaining and appeared to serve as a scaffold for the assembly of endogenous BTBD1/2 proteins. TRIM family members contain a RING domain, B-box(es), and coiled-coil regions, which have a characteristic order and spacing (RBCC domain). RING-dependent ubiquitin ligase activity and multimerization via the coiled-coil region may be defining properties of the RBCC/TRIM protein family. We found that TRIM5delta with a deleted coiled-coil region or a mutated RING domain failed to colocalize with BTBD1/2. Additionally, TRIM5delta ubiquitylated itself in a RING finger- and UbcH5B-dependent manner. BTBD1/2 each contain a PHR-similarity region, repeated twice on the putative ubiquitin ligases PAM, highwire and RPM-1, which also contain a RING and B-box. Thus, four protein modules found on each of these putative ubiquitin ligases, a RING, a B-box and two PHR repeats, are present on BTBD1/2 and TRIM5delta that are colocalized to cytoplasmic bodies.

Amino Acid Sequence↗

PubMed2003

Inositol 1,4,5-trisphosphate receptor ubiquitination is mediated by mammalian Ubc7, a component of the endoplasmic reticulum-associated degradation pathway, and is inhibited by chelation of intracellular Zn2+.

In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP3Rs), which are located in the endoplasmic reticulum, can be rapidly ubiquitinated and then degraded by the proteasome. Ubiquitination is mediated by the concerted action of ubiquitin-conjugating enzymes (Ubcs or E2s) and ubiquitin-protein ligases (E3s). In the present study we have examined the enzymology of ubiquitination of endogenous InsP3Rs in muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, focusing our attention on two mammalian E2s, MmUbc6 and MmUbc7, that have been implicated in endoplasmic reticulum-associated degradation (ERAD) and are homologous to the yeast ERAD E2s, Ubc6p and Ubc7p. Analysis of SH-SY5Y cells stably expressing these enzymes and their dominant-negative mutants revealed that MmUbc7 mediates InsP3R ubiquitination and down-regulation, but that MmUbc6 does not. These data indicate that InsP3Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP3Rs, that are subject to regulated ERAD. Additional studies showed that the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP3R ubiquitination, suggesting that a RING finger domain-containing E3 is also involved in this process. Finally, muscarinic agonist-induced InsP3R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH-SY5Y cells reflect a physiological process.

Animals↗

PubMed2003

CAIR-1/BAG-3 abrogates heat shock protein-70 chaperone complex-mediated protein degradation: accumulation of poly-ubiquitinated Hsp90 client proteins.

BAG family proteins are regulatory co-chaperones for heat shock protein (Hsp) 70. Hsp70 facilitates the removal of injured proteins by ubiquitin-mediated proteasomal degradation. This process can be driven by geldanamycin, an irreversible blocker of Hsp90. We hypothesize that CAIR-1/BAG-3 inhibits Hsp-mediated proteasomal degradation. Human breast cancer cells were engineered to overexpress either full-length CAIR-1 (FL), which binds Hsp70, or a BAG domain-deletion mutant (dBAG) that cannot bind Hsp70. FL overexpression prevented geldanamycin-mediated loss of total and phospho-Akt and other Hsp client proteins. dBAG provided no protection, indicating a requirement for Hsp70 binding. Ubiquitinated Akt accumulated in FL-expressing cells, mimicking the effect of lactacystin proteasomal inhibition, indicating that CAIR-1 inhibits proteasomal degradation distal to protein ubiquitination in a BAG domain-dependent manner. Protein protection in FL cells was generalizable to downstream Akt targets, GSK3beta, P70S6 kinase, CREB, and other Hsp client proteins, including Raf-1, cyclin-dependent kinase 4, and epidermal growth factor receptor. These findings suggest that Hsp70 is a chaperone driving a multiprotein degradation complex and that the inhibitory co-chaperone CAIR-1 functions distal to client ubiquitination. Furthermore, poly-ubiquitination is not sufficient for efficient proteasomal targeting of Hsp client proteins.

Acetylcysteine↗

PubMed2003

Overexpression of the tumor autocrine motility factor receptor Gp78, a ubiquitin protein ligase, results in increased ubiquitinylation and decreased secretion of apolipoprotein B100 in HepG2 cells.

Apolipoprotein B100 (apoB) is a large (520-kDa) complex secretory protein; its secretion is regulated posttranscriptionally by several degradation pathways. The best described of these degradative processes is co-translational ubiquitinylation and proteasomal degradation of nascent apoB, involving the 70- and 90-kDa heat shock proteins and the multiple components of the proteasomal pathway. Ubiquitinylation involves several proteins, including ligases called E3s, that coordinate the covalent binding of ubiquitin to target proteins. The recent discovery that tumor autocrine motility factor receptor, also known as gp78, is an endoplasmic reticulum (ER)-associated E3, raised the possibility that this E3 might be involved in the ER-associated degradation of nascent apoB. In a series of experiments in HepG2 cells, we demonstrated that overexpression of gp78 was sufficient for increased ubiquitinylation and proteasomal degradation of apoB, with reduced secretion of apoB-lipoproteins. This action of gp78 was specific: overexpression of the protein did not affect secretion of either albumin or apolipoprotein AI. Furthermore, overexpression of a cytosolic E3, Itch, had no effect on apoB secretion. Finally, using an in vitro translation system, we demonstrated that gp78 led to increased ubiquitinylation and proteasomal degradation of apoB48. Together, these results indicate that an ER-associated protein, gp78, is a bona fide E3 ligase in the apoB ER-associated degradation pathway.

Apolipoprotein B-100↗

PubMed2003

RING finger ubiquitin protein ligases: implications for tumorigenesis, metastasis and for molecular targets in cancer.

Covalent modification of proteins with ubiquitin regulates almost all aspects of eukaryotic cellular function. Ubiquitin protein ligases (E3s) play central regulatory roles in that they provide substrate specificity to this process and therefore, represent attractive molecular targets for disease therapy. We summarize recent advances in our understanding of RING finger and RING finger-related E3s with emphasis on BRCA1 and the tumor autocrine motility factor receptor (gp78), as well as discuss the potential for components of the ubiquitin pathway for proteasomal degradation as molecular targets.

Amino Acid Motifs↗

PubMed2003

Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta.

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.

Animals↗