Jon M. Huibregtse - US grants

DESCRIPTION (provided by applicant): Cervical cancer is the second-leading cause of cancer-related deaths among women worldwide, and epidemiologic and laboratory studies have shown that the human papillomavirus (HPV) E6 and E7 proteins of the "high-risk" subgroup of sexually transmitted HPVs play a causative role in over 90% of these cancers. The biochemical activity of the E6 proteins most clearly related to carcinogenesis is their ability to stimulate the ubiquitin-mediated degradation of the p53 tumor suppressor protein. This is dependent on the cellular ubiquitin ligase, E6AP. Biochemical analyses indicate that E6 functions to redirect or reprogram the substrate specificity of E6AP so that it ubiquitinates p53, a protein that it does not normally recognize. While many lines of evidence indicate that targeted degradation of p53 is critical to the role of HPVs in carcinogenesis, it has become increasingly clear that high-risk HPV E6 proteins have additional, p53-independent functions related to cellular immortalization. Analysis of E6 mutants suggests that at least some of these activities are dependent on E6AP. We therefore hypothesize that the high-risk HPVE6/E6AP complex recognizes and ubiquitinates a set of cellular proteins, which includes but is not limited to p53, and that targeting of these cellular proteins is linked to HPV-associated carcinogenesis. The first goal of this proposal is to characterize a set of proteins that we have shown to be targeted by the E6/E6AP complex and to determine if degradation of these proteins is relevant to cellular immortalization. These proteins are Scribble, Discs large (DIg), and utrophin. Interestingly, all three of these proteins are linked to architecture of multiprotein complexes formed at the plasma membrane, and work on Scribble and Dlg indicates that these are cooperating neoplastic tumor suppressors in Drosophila. While study of high-risk HPV E6 proteins led to discovery of E6AP and an established model for HECT E3 function, our understanding of the structure-function relationships of the E6 proteins, themselves, remains very limited. Several lines of evidence suggest that this is largely due to the difficulty of expressing properly folded E6 proteins for biochemical analyses. Recent advances in this area form the basis for the second goal of this proposal, characterization of the structure-function relationships of the HPV E6 proteins with respect to their role in facilitating protein ubiquitination. The third goal of this proposal is to further characterize specific aspects of the enzymatic mechanism of E6AP and HECT ubiquitin ligases, in general. Several of the approximately 30 human HECT E3s play critical roles in disease states in addition to cervical cancer, including Liddle's syndrome, Angelman's syndrome, and the life cycle of several types of viruses. A more thorough understanding of HECT E3 mechanism will therefore contribute to our understanding of several important health-related problems.

[unreadable] DESCRIPTION (provided by applicant): Conjugation systems for ubiquitin and Ubiquitin-like proteins (Ubls) are generally organized into enzyme cascades involving E1, E2, and, E3 enzymes. Among the several classes of ubiquitin E3s, the HECT E3s are mechanistically unique in that they form a covalent enzyme-ubiquitin intermediate and participate directly in the chemistry of protein ubiquitination. HECT E3s play important roles in several disease states (cervical cancer, Angelman syndrome, Liddle's syndrome), disease-related pathways (TGF-beta signaling, Notch signaling, trafficking of membrane proteins), and are commandeered by several types of viruses (human papillomaviruses, EBV, retroviruses, Ebola). In addition, a single HECT E3, HercS, is critical for conjugation of ISG15, a type 1 interferon-induced Ubl that plays a role in the innate immune response to viral and microbial infections. These examples highlight the importance of understanding the mechanism, function, and regulation of HECT E3s. We have made important contributions toward understanding HECT E3 functions and mechanisms and propose to extend recent discoveries through three major lines of investigation. The first line of investigation, driven by our investigations on yeast Rsp5, will focus on the regulation of Rsp5 and its human homologs by physically associated deubiquitinating enzymes (DUBs). This represents a novel form of E3 regulation that may represent a point of intervention in altering HECT E3 function in human disease. The second line of investigation will address the mechanistic basis for the distinction between HECT E3s that catalyze K63-linked polyubiquitination and those that catalyze K48- linked polyubiquitination. This is an important problem because the ultimate consequences of these two types of ubiquitination are distinct. The third line of investigation will focus on the mechanism and regulation of ISG15 conjugation system by the HercS HECT E3. We will identify the complete set of factors required for HercS-dependent ISG15 conjugation and determine how this single E3 directs conjugation to perhaps hundreds of ISG15 target proteins. A thorough understanding of the biochemistry of ISG15 conjugation is essential for understanding the function of ISG15 in innate immune responses. Public health relevance: Protein modification by ubiquitin and Ubls serves to modify the stability, activity, or localization of many key cell regulatory proteins, and disruptions or alterations in these processes are seen in many disease states, particularly cancer. The elucidation of the biochemistry of the HECT family of ubiquitin ligases will impact our understanding of mechanisms of carcinogenesis, signaling pathways, and the response to viral and microbial infections. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The type 1-interferon (IFN) response is the first line of defense against viral infections. Hundreds of proteins are induced in IFN-stimulated cells, and these proteins mediate a wide spectrum of anti-viral effects. ISG15 was one of the first IFN-induced proteins to be identified and the first ubiquitin-like protein (Ubl) to be discovered, however its biochemical function and the basis of its antiviral activities have remained largely uncharacterized for many years. ISG15 has anti-viral activity against a wide range of human viruses, including influenza, retroviruses, sindbis, ebola, and the ability of ISG15 to be conjugated to other proteins is likely to be essential for its anti-viral activity in all cases. ISG15 is conjugated to hundreds of cellular proteins in IFN-stimulated cells, and a single IFN-induced ligase, Herc5, mediates conjugation to nearly all of these target proteins. Based on extensive preliminary data, we hypothesize that Herc5 is ribosome-associated and ISGylates proteins in a co-translational manner, with little target protein specificity. We further hypothesize that in the context of an interferon response, newly translated viral proteins, rather than cellular proteins are the primary targets of this system, and that ISGylation is an attempt to inactivate the function of viral proteins. The aims of this proposal will test both of these hypotheses. The interferon response is a mutli-faceted defensive shield that protects cells against a wide range of infectious agents, yet only a small number of IFN-induced proteins have been characterized in molecular detail. In addition, interferon is used therapeutically in treating viral infections (e.g., hepatitis B and C and papillomaviruses), as well as certain types of cancers and multiple sclerosis, yet it is not known which IFN-induced proteins mediate useful therapeutic effects and which mediate the notorious side effects of these therapies. A further understanding of the mechanism and function of ISG15 conjugation will present opportunities for either up- or down-modulating ISGylation activity in these clinical settings. PUBLIC HEALTH RELEVANCE: ISG15 conjugation is now recognized to be an important aspect of the innate immune response against a wide range of human viral infections, and interferon therapies are approved for many disease states. It is essential to characterize the many facets of the interferon response system, including the ISG15 pathway, in order to improve and modulate anti-viral and interferon therapies.

DESCRIPTION (provided by applicant): Ubiquitin is a protein modifier that plays a central role in cellular regulation and disease processes. It is covalently attached to proteins through an enzymatic cascade involving three classes of enzymes (E1, E2, E3 enzymes), with the E3s conferring target protein specificity. Among the several classes of E3 enzymes, HECT E3s are unique in that they function via a covalent enzyme-ubiquitin intermediate and participate directly in catalysis of ubiquitin conjugation. First discovered through studies on HPV-associated cervical cancer, HECT E3s also play important roles in other types of cancer, neurologic disease, various signaling pathways, and intracellular protein trafficking. One human HECT E3, Herc5, catalyzes conjugation of an interferon-induced ubiquitin-like modifier called ISG15, which is important in anti-viral responses. These diverse health-related functions highlight the importance of understanding the mechanism, function, and regulation of HECT E3s. Aim 1 of this renewal application will focus on critical aspects of the biochemical mechanism of HECT E3s. A complete understanding of enzyme mechanism is critical for development of small molecules (drugs) that can either up- or down-regulate HECT E3 function in specific disease states. Importantly, very few natural targets of HECT E3s have been identified, and this has hampered our understanding of their biological functions. In Aim 2, we will develop and implement a completely novel approach for HECT E3 substrate identification, based on the unique mechanism and structure of the catalytic domain. Aim 3 represents a new research direction, where we have applied approaches developed in our ISG15 studies to analyze Co-Translational Ubiquitination (CTU), a phenomenon that has been linked to protein quality control, protein folding, and protein aggregation diseases. We will address the function of CTU through identification of target proteins and characterization of the enzymes of the pathway. We anticipate that these studies will have a strong and sustained impact on the many aspects of human health that are affected by the ubiquitin system, including cancer biology, cell signaling pathways, protein folding diseases, and infectious diseases. PUBLIC HEALTH RELEVANCE: Ubiquitin is a protein modifier that plays a central role in virtually all cellular processes and pathways, as well as an incredible array of disease processes, including cancer, neurologic diseases, and infectious diseases. It is essential to characterize the biochemistry of the enzymes that selectively conjugate ubiquitin to target proteins in order to develop small molecules that can be used to modulate the system for therapeutic purposes.

SUMMARY Human ISG15 is a ubiquitin-like protein (Ubl) that functions in innate immune responses, and it is remarkable for possessing three distinct biochemical activities. As a ubiquitin-like modifier it is conjugated to hundreds of cellular and viral proteins. The E1, E2, E3, and de-conjugating enzymes for ISG15 (Ube1L/Uba7, UbcH8/Ube2L6, Herc5, and Usp18, respectively), are, like ISG15, induced at the transcriptional level by Type I interferon (IFN-?/?) signaling. A second function of ISG15 is as an extracellular signaling protein. ISG15 is released into the extracellular space from many cell types and signals to Natural Killer and T cells to secrete IFN-?, which plays a major role in the response to pathogen infections. The cell surface receptor for extracellular SG15 is LFA-1, an immune cell-specific integrin. A third function of human ISG15 is that it negatively regulates Type I IFN signaling, as revealed by the Type I interferonopathy that occurs in some ISG15-deficient patients; this function of human ISG15 is not shared with mouse ISG15. A challenge in the field is to understand how the activities of ISG15 are regulated and temporally controlled and which activities are most closely associated with responses to specific pathogens, including Mycobacterium tuberculosis and SARS-CoV-2. To further our understanding of ISG15 in innate immune responses to these and other pathogens, this proposal will focus on the basis of substrate and lysine selectivity of the Herc5/Herc6 family of ISG15 ligases, the ISG15-induced signaling pathway downstream of LFA-1 that leads to cytokine secretion, and the mechanism by which ISG15 released into the extracellular space.

Project Summary/Abstract Protein quality control (QC) mechanisms exist in eukaryotic cells to monitor and clear misfolded, damaged, or aggregated proteins, and defects in QC systems are recognized to be causative in several disease states, particularly neurodegenerative disorders, cancer, cardiovascular, metabolic diseases, aging. Many protein QC systems function at the earliest stages of the life of a protein, ensuring that newly synthesized proteins reach a mature and functional state. While the ubiquitin-proteasome system (UPS) has long been recognized to play an important role in protein QC, this was generally assumed to occur after unsuccessful attempts at correctly folding newly synthesized proteins. In contrast, we made the surprising discovery that ubiquitylation of nascent polypeptides also occurs within active translation complexes, essentially marking proteins for destruction before their synthesis is even complete. Co- translation ubiquitylation (CTU) is a surprisingly robust process in human cells, with approximately 10% of nascent chains being ubiquitylated within active translation complexes. The incorporation of amino acid analogs into nascent chains, as well as Hsp70 inhibitors, leads to sharp increases in CTU, strongly suggesting that CTU reflects a protein QC pathway that targets aberrant or misfolded nascent polypeptides. Importantly, CTU is distinct from ubiquitylation of nascent chains that occurs in the context of defective and disassembled ribosome complexes (the ?RQC? Ribosome Quality Control pathway, dependent on the Ltn1 ubiquitin ligase). The goals of this proposal are to characterize the CTU pathway with respect to the ubiquitin ligases, the targets, the factors that influence and potentially regulate CTU, and to assess the biological importance of CTU. The specific aims are to 1) identify the CTU ubiquitin ligases, in both yeast and human cells, through biochemical and genetic approaches, 2) characterize the preferential targets of CTU by proteomic approaches in order to determine the features of nascent polypeptides that are recognized by this QC system, and 3) determine the factors that influence and regulate CTU. This will significantly impact our understanding of the relationship between protein synthesis and protein quality control, with implications for disease states where proteostasis is particularly critical.