The Coiled-coil Domain Is the Structural Determinant for Mammalian Homologues of Drosophila Sina-mediated Degradation of Promyelocytic Leukemia Protein and Other Tripartite Motif Proteins by the Proteasome

Mammalian homologues of Drosophila Seven in Absentia (SIAHs) target for proteasome-mediated degradation several factors involved in cell growth and tumorigenesis. Here we show that SIAH-1/2 binds and targets for proteasome-mediated degradation the putative tumor suppressor and tripartite motif (TRIM) family member PML, leading to the loss of its transcriptional co-activating properties and a reduction in the number of endogenous PML nuclear bodies. Association with PML requires the substrate-binding domain (SBD) of SIAH-1/2 through an interacting surface apparently distinct from those predicted by the structural studies, or shown experimentally to mediate binding to SIAH-associated factors. Within PML, the coiled-coil domain is required for Siah- and proteasome-mediated degradation, and deletions of regions critical for the integrity of this region impair the ability of Siah to trigger PML-RAR degradation. Fusion of the coiled-coil domain to heterologous proteins resulted in the capacity of mSiah-2 to target their degradation. All of the TRIM proteins tested were degraded upon mSiah-2 overexpression. Finally, we show that the fusion protein PML-RAR (that retains the coiled-coil domain), which causes acute promyelocytic leukemias, is also a potential substrate of mSiah-2. As a result of mSiah-2 overexpression and subsequent degradation of the fusion protein, the arrest in hematopoietic differentiation because of expression of PML-RAR is partially rescued. These results identify PML and other TRIMs as new factors post-translationally regulated by SIAH and involve the coiled-coil region of PML and of other SIAH substrates as a novel structural determinant for targeted degradation

Regulation of alphaherpesvirus infections by the ICP0 family of proteins

Immediate-early protein ICP0 of herpes simplex virus type 1 (HSV-1) is important for the regulation of lytic and latent viral infection. Like the related proteins expressed by other alphaherpesviruses, ICP0 has a zinc-stabilized RING finger domain that confers E3 ubiquitin ligase activity. This domain is essential for the core functions of ICP0 and its activity leads to the degradation of a number of cellular proteins, some of which are involved in cellular defences that restrict viral infection. The article reviews recent advances in ICP0-related research, with an emphasis on the mechanisms by which ICP0 and related proteins counteract antiviral restriction and the roles in this process of cellular nuclear substructures known as ND10 or PML nuclear bodies. We also summarize recent advances in the understanding of the biochemical aspects of ICP0 activity. These studies highlight the importance of the SUMO conjugation pathway in both intrinsic resistance to HSV-1 infection and in substrate targeting by ICP0. The topics discussed in this review are relevant not only to HSV-1 infection, but also to cellular intrinsic resistance against herpesviruses more generally and the mechanisms by which viruses can evade this restriction

Elucidation of the substrate binding site of Siah ubiquitin ligase

The Siah family of RING proteins function as ubiquitin ligase components, contributing to the degradation of multiple targets involved in cell growth, differentiation, angiogenesis, oncogenesis, and inflammation. Previously, a binding motif (degron) was recognized in many of the Siah degradation targets, suggesting that Siah itself may facilitate substrate recognition. We report the crystal structure of the Siah in complex with a peptide containing the degron motif. Binding is within a groove formed in part by the zinc fingers and the first two ß strands of the TRAF-C domain of Siah. We show that residues in the degron, previously described to facilitate binding to Siah, interact with the protein. Mutagenesis of Siah at sites of interaction also abrogates both in vitro peptide binding and destabilization of a known Siah target

Histone deacetylase inhibitors regulate the proteasomal degradation of oncoproteins

Leukemogenesis is often linked to fusion proteins generated by chromosomal translocation products. Examples are AML1-ETO and PML-RARα which contribute to the pathogenesis of acute myeloid leukemia (AML). The work presented here reveals the novel insight that the turnover of both, AML1-ETO and PML-RARα, depends on the HDACi-inducible ubiquitin conjugase UBCH8 and the ubiquitin ligase SIAH1. Beyond showing that HDACi promote the degradation of oncoproteins, this work reveals that the ubiquitin ligase RLIM equally is a substrate for SIAH1. Thus, a formerly unknown hierarchical order of ubiquitin ligases affects the ubiquitin-proteasome system. Since constitutively activated mutant FMS-like tyrosine kinase 3 (FLT3-ITD) causally contributes to leukemic transformation and is frequently found in conjunction with AML1-ETO and PML-RARα in AML patients, it was also tested whether HDACi attenuate FLT3-ITD. Indeed, UBCH8 together with SIAH1 interact in a tyrosine phosphorylation-dependent way with FLT3-ITD and promote its proteasomal degradation. Accordingly, unstimulated wild-type FLT3 is hardly affected by HDACi. Thus, UBCH8, which has been implicated primarily in nuclear processes, could be identified as a novel important HDACi-inducible modulator of FLT3-ITD stability and leukemic cell survival. In summary, I could demonstrate in various AML cell lines and heterologous expression systems that UBCH8 and SIAH1 physically interact with and target FLT3-ITD, AML1-ETO, PML-RARα, and RLIM for proteasomal degradation. This work furthermore provides a deeper understanding on how enzymes promoting proteasomal degradation are regulated and how they interact with each other as well as with their cancer-relevant substrates. Conclusions presented here reveal novel biochemical mechanisms and molecular networks. In addition, they have implications for translational research

Components of the Ubiquitin Proteasome System are Required for the Nonapoptotic Death of the C. Elegans Linker Cell

Cell death is a major cell fate that promotes tissue sculpting and morphogenesis during animal development. Many developmental cell-culling events cannot be accounted for solely by caspase-dependent apoptosis, yet, alternate pathways are poorly understood. Direct evidence that caspase-independent non-apoptotic cell death pathways operate during animal development is provided by studies of the C. elegans linker cell. Genetic studies of linker cell death have led to the identification of genes that promote this process, including pqn-41, which encodes a glutamine-rich protein, as well as tir-1/TIRdomain and sek-1/MAPKK, which may function in the same pathway as pqn-41. The let- 7 microRNA and its indirect target, the Zn-finger transcription factor LIN-29, also promote linker cell death, and may act early in the process. Our work suggests that components of the ubiquitin proteasome system (UPS) act to promote linker cell death. We show that LET-70, an E2 ubiquitin-conjugating enzyme, is required cell-autonomously for linker cell death. LET-70 levels, as well as those of ubiquitin and some proteasome components, increase just before linker cell death initiation. This rise is dependent on an MLL-type histone methyltransferase complex and a MAPK cascade, whose activities are required for linker cell death. The E3 ligase components SIAH-1, RBX-1, and CUL-3 are also required for linker cell death and appear to act in the same pathway as let-70. We also identify the PLZF transcription factor EOR-1, and its accessory protein, EOR-2 as major regulators of linker cell death. Our studies suggest that EOR-1/2, as well as all known regulators of the linker cell death pathway may act upstream of UPS components to promote cell death. Our studies reveal that activation of the ubiquitin proteasome system is an important event promoting linker cell death. Given the morphological similarities between linker cell death and non-apoptotic developmental and pathological cell death in vertebrates, we raise the possibility that the proteasome may be a key mediator of vertebrate cell death

Functional Studies of Deubiquitinating Enzymes

The attachment of ubiquitin to substrate proteins is a key process in regulating cellular events such as cell cycle progression, signal transduction, differentiation, apoptosis, and the clearance of misfolded or aberrant proteins. Like other post-translational modifications, ubiquitination is also reversible. Deconjugation is performed by a family of cysteine- or metallo-proteases collectively known as deubiquitinating enzymes (DUBs). Approximately 100 putative DUBs have been identified in the human genome but only a minority of them has been functionally characterized. The aim of this thesis has been to study the function of selected DUBs in disease-relevant cellular pathways. Screen of the canonical Wnt-signaling pathway with an RNA interference (RNAi) library targeting the human DUBs identified the ubiquitin-specific protease (USP)-4 as a negative regulator. USP4 interacts with two known components in the pathway: the Nemo like kinase (Nlk) and the T-cell factor 4 (TCF4). NLK promotes nuclear accumulation of USP4 where a subpopulation of TCF4 is a substrate of USP4-dependent deubiquitination. Using a yeast-2 hybrid strategy to search for relevant interactions, we identified the proteasome as a binding partner of USP4. USP4 interacts with the S9 subunit of the 19S regulatory particle (RP) through an N-terminal ubiquitin-like (UBL) domain that resembles, but is functionally distinct from, the UBLs of hHR23a/b and Ubiquilin-1. S9 is as an essential proteasome subunit that may regulate the structural integrity of the 26S complex. Thus, USP4 may play a role in the dynamics of ubiquitination at the proteasome. A bioinformatics strategy was used to search for membrane-associated DUBs. We found that a putative transmembrane domain targets USP19 to the endoplasmic reticulum (ER). USP19 is a target of the unfolded protein response and rescues ERAD substrate from proteasomal degradation. Moreover, USP19 interacts with the E3 ligases seven in absentia homolog (SIAH) 1 and SIAH2 that mediate USP19 ubiquitination and degradation by the proteasome. Bioinformatics and biochemical analysis revealed the presence in USP19 of a SIAH-interacting motif that is found in a subset of SIAH targets and may function as a degradation signal. A non-enzymatic role of USP19 in the regulation of the reposed to hypoxia was suggested by the finding that wild-type and catalytic mutant USP19 interact with the hypoxia-inducible factor-1α (HIF-1α). In the absence of USP19, cells fail to mount a proper response to hypoxia

Impact of Mdm2-p53 on the proteasome assembly and disassembly Role of the ubiquitination of some 19S subunits

The ubiquitin proteasome system is a fundamental actor for the proteolysis of cellular proteins. My results demonstrated that the E3 ligase Mdm2 and its substrate p53 might be implied in the formation of the fully-assembled proteasome. Mdm2 promotes also the ubiquitination of some 19S subunits of the proteasome via a non-conventional chain and it is not a signal for self-degradation. Moreover, this ubiqitination seemed to play a role in the formation of the proteasome

The Ubiquitin System and Kaposi’s Sarcoma-Associated Herpesvirus

Ubiquitination is a post-translational modification in which one or more ubiquitin molecules are covalently linked to lysine residues of target proteins. The ubiquitin system plays a key role in the regulation of protein degradation, which contributes to cell signaling, vesicular trafficking, apoptosis, and immune regulation. Bacterial and viral pathogens exploit the cellular ubiquitin system by encoding their own proteins to serve their survival and replication in infected cells. Recent studies have revealed that Kaposi’s sarcoma-associated herpesvirus (KSHV) manipulates the ubiquitin system of infected cells to facilitate cell proliferation, anti-apoptosis, and evasion from immunity. This review summarizes recent developments in our understanding of the molecular mechanisms used by KSHV to interact with the cellular ubiquitin machinery

Ubiquitinylation of α-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

Parkinson's disease (PD) is a common neurodegenerative condition in which abnormalities in protein homeostasis, or proteostasis, may lead to accumulation of the protein α-synuclein (α-syn). Mutations within or multiplications of the gene encoding α-syn are known to cause genetic forms of PD and polymorphisms in the gene are recently established risk factors for idiopathic PD. α-syn is a major component of Lewy bodies, the intracellular proteinaceous inclusions which are pathological hallmarks of most forms of PD. Recent evidence demonstrates that α-syn can self associate into soluble oligomeric species and implicates these α-syn oligomers in cell death. We have previously shown that carboxyl terminus of Hsp70-interacting protein (CHIP), a co-chaperone molecule with E3 ubiquitin ligase activity, may reduce the levels of toxic α-syn oligomers. Here we demonstrate that α-syn is ubiquitinylated by CHIP both in vitro and in cells. We find that the products from ubiquitinylation by CHIP include both monoubiquitinylated and polyubiquitinylated forms of α-syn. We also demonstrate that CHIP and α-syn exist within a protein complex with the co-chaperone bcl-2-associated athanogene 5 (BAG5) in brain. The interaction of CHIP with BAG5 is mediated by Hsp70 which binds to the tetratricopeptide repeat domain of CHIP and the BAG domains of BAG5. The Hsp70-mediated association of BAG5 with CHIP results in inhibition of CHIP E3 ubiquitin ligase activity and subsequently reduces α-syn ubiquitinylation. Furthermore, we use a luciferase-based protein-fragment complementation assay of α-syn oligomerization to investigate regulation of α-syn oligomers by CHIP in living cells. We demonstrate that BAG5 mitigates the ability of CHIP to reduce α-syn oligomerization and that non-ubiquitinylated α-syn has an increased propensity for oligomerization. Thus, our results identify CHIP as an E3 ubiquitin ligase of α-syn and suggest a novel function for BAG5 as a modulator of CHIP E3 ubiquitin ligase activity with implications for CHIP-mediated regulation of α-syn oligomerization

Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed