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

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

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

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

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

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

SIAH-MEDIATED UPS REGULATION OF THE DEVELOPMENT OF THE VISUAL SYSTEM IN ZEBRAFISH (DANIO RERIO)

The eye is a complex organ responsible for vision that which formation depends on several intricate developmental steps. Vision for humans is responsible for the majority of its sensory interactions with the environment. Eye development can be divided into two critical stages: morphogenesis, which establishes the eye structure culminating with the fusion of the optic fissure, a transient cleft important for eye vascularization, and subsequently specification and differentiation of the retina to form every type of retinal neuronal cell, including photoreceptors, rods and cones. Developmental failure in either of these critical stages can lead to inherited congenital or age-related blinding disorders. Thus, understanding the proper mechanism for eye development is crucial to help avoid social and cognitive impairments commonly associated with visual loss, especially in the most economically challenged parts of the world. The regulation of morphogenesis and retinal formation has been vigorously investigated at the transcriptional level, but little effort has been placed on potential post-translational regulatory mechanisms. The ubiquitin mediated proteasome degradation system plays an important role in regulating protein function in many different developmental stages. The siah family of ubiquitin E3 ligases are homologues to Drosophila Seven In Absentia (SINA), and also known regulators of drosophila photoreceptor development. In order to determine whether Siah mediated ubiquitin-targeting plays a role in vertebrate eye morphogenesis and retinal differentiation, the zebrafish proteome was screened for siah degron motifs and identified 2 potential targets related to eye development, Nlz2 and Cdhr1a. The first is a zinc-finger transcription factor, responsible for negatively regulating Pax2 gene expression, a master regulator of the optic fissure closure. The second, is a photoreceptor’ specific cadherin, known to be associated with cone-rod dystrophies. My dissertation has focused on examining Siah-mediated regulation of these two targets during critical stages of eye development. In Chapter 3 I outline how modulation of Siah activity leads to failure of optic fissure fusion dependent on ubiquitin-mediated proteasomal degradation of Nlz2, outlining a novel, UPS-mediated degradation regulatory pathway involved in optic fissure fusion. In Chapter 4 I provide the first evidence that Cdhr1a is involved in photoreceptor development and that Siah1 is responsible for regulating its protein stability via the UPS. My findings provide new avenues for investigation of the pathogenesis of inherited cone-rod dystrophies. Overall, my work outlines new pathways involved in posttranslational mechanisms of vertebrate retinal development and strengthens efforts to improve our understanding of the predisposition and pathogenesis of Coloboma and inherited cone-rod dystrophy

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

CHARACTERIZATION OF THE E3 UBIQUITIN LIGASE SIAH2 AS AN ANTI-CANCER TARGET

Ph.DDOCTOR OF PHILOSOPH

A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin

AbstractThe SCF (Skp1–Cullin–F-box) E3 ubiquitin ligase family was discovered through genetic requirements for cell cycle progression in budding yeast. In these multisubunit enzymes, an invariant core complex, composed of the Skp1 linker protein, the Cdc53/Cul1 scaffold protein and the Rbx1/Roc1/Hrt1 RING domain protein, engages one of a suite of substrate adaptors called F-box proteins that in turn recruit substrates for ubiquitination by an associated E2 enzyme. The cullin–RING domain–adaptor architecture has diversified through evolution, such that in total many hundreds of distinct SCF and SCF-like complexes enable degradation of myriad substrates. Substrate recognition by adaptors often depends on posttranslational modification of the substrate, which thus places substrate stability under dynamic regulation by intracellular signaling events. SCF complexes control cell proliferation through degradation of critical regulators such as cyclins, CDK inhibitors and transcription factors. A plethora of other processes in development and disease are controlled by other SCF-like complexes, including those based on Cul2–SOCS-box adaptor protein and Cul3–BTB domain adaptor protein combinations. Recent structural insights into SCF-like complexes have begun to illuminate aspects of substrate recognition and catalytic reaction mechanisms