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Open Access Publications from the University of California

Scholarly Works (13 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2006)
My research focuses on understanding the mechanisms of eukaryotic inwardly rectifying potassium channels (Kir), which are responsible for creating membrane potential stability. N- and C-terminal cytoplasmic domains of inwardly rectifying K (Kir) channels control the ion- permeation pathway through diverse interactions with small molecules and protein ligands in the cytoplasm. Two crystal structures of the cytoplasmic domains of Kir2.1 (Kir2.1L) and the G protein-sensitive Kir3.1 (Kir3.1S) channels in the absence of PIP2 show the cytoplasmic ion- permeation pathways occluded by four cytoplasmic loops that form a girdle around the central pore (G-loop). Significant flexibility of the pore-facing G-loop of Kir2.1L and Kir3.1S suggest a possible role as a diffusion barrier between cytoplasmic and transmembrane pores. Consistent with this, mutations of the G-loop disrupted gating or inward rectification. Structure comparison reveals a di-aspartate cluster on the distal end of the cytoplasmic pore of Kir2.1L that is important for modulating inward rectification. The Kir2.1L structural allows us the first structural look at the potential causes of Andersen's Syndrome, which is caused by defects in the inwardly rectifying potassium channel 2.1 (Kir2.1). We have characterized individual Andersen's Syndrome mutants R218Q, G300V, E303K, and [delta]314-315 and have shown that they have multiple affects on the ability of Kir2.1 channel's cytoplasmic domains to assemble into proper tetrameric assemblies. The 2.0 Å x-ray crystal structure of the N- and C-terminal cytoplasmic domains of Kir2.1 R218Q combined with T309K, reveals the G-loop and CD loop disruptions by the R218Q mutation. We show that E303 plays an important role in gating. Moreover, we demonstrate a novel DE loop stabilizing 2-methyl-2,4- pentanediol binding site, and cytoplasmic bound potassium ion, the first identified for this class of K channels. We have also conducted NMR studies on the last 58 residues of Kir2.1 to show the flexibility of this region and binding footprint of PSD95 PDZ domains, which tethers the channels to the cell's cytoskeleton. Taken together, these results demonstrate that cytoplasmic domains of Kir channels are structurally significant components of the channel and undergo structural changes to modulate gating and inward rectification
    Cover page: Molecular gating dynamics of the cytoplasmic domains of inwardly rectifying potassium (Kir) channels
    • Thesis
    • Peer Reviewed
    UC Riverside Electronic Theses and Dissertations (2023)

    Nairoviruses, tick-borne viruses from the Bunyaviridae family, pose persistent and significant threats to human health. While the most notable is Crimean-Congo hemorrhagic fever virus (CCHFV), which possesses a fatality rate pushing 30%, other nairoviruses are establishing themselves in our viromes including Pacific Coast tick nairovirus (PCTN) and Songling virus (SGLV). To thwart host antiviral immune response, specifically reversing the post-translational modifications of interferon-stimulated gene product 15 (ISG15) after infection, nairoviruses encode for and utilize an ovarian tumor domain protease (OTU) capable of deISGylation. The fact that ISG15 structure and sequence is hardly conserved between species, coupled with previous studies demonstrating diverse OTU preferences for certain ISG15s, pinpoints these two as key subjects of disease prevention due to possible variations in interaction. To gain a broader understanding of nairovirus OTU-ISG15 interface and identify possible host carriers, a high-throughput method of obtaining binding kinetic data was developed which employed bio-layer interferometry (BLI). ~72 interactions between vOTUs and ISG15 were examined, which revealed significant differences for nairovirus OTU binding preferences. CCHF and PCTN vOTUs put on robust displays of binding, while SGLV vOTU showcased more specificity for which ISG15s it bound to. These insights serve to be progress and contributions towards building a larger biosurveillance tool ultimately unveiling pivotal animal reservoirs of nairoviruses.

      Cover page: Development of Molecular Host-Centric Methodology Seeking to Pinpoint Reservoirs of Nairoviruses by Interferon-Stimulated Gene Product 15
      • Thesis
      • Peer Reviewed
      UC Riverside Electronic Theses and Dissertations (2023)

      This thesis presents a comprehensive study on the crystallography of a novel SMAC mimetic, (R)-1-((S)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino) propanamido) acetyl) pyrrolidine-2-carboxamido) -2,3-dihydro-1H-inden-4-yl sulfurofluoridate, also known as 142D6, and its covalent adduct formation with XIAP BIR3. The inhibitor, 142D6, was designed to simulate the binding properties of Second Mitochondria-derived Activator of Caspases (SMAC) protein, a critical regulator of apoptosis. By utilizing crystallographic techniques, we aimed to elucidate the structural basis of the interaction between 142D6 and XIAP BIR3. First, a detailed characterization of the crystal structure of 142D6 was performed, revealing its precise and tightly fitted arrangement within the domain subpockets. This structural analysis provided valuable insights into the molecular features of 142D6 that contribute to its binding specificity and potential as an inhibitor. Furthermore, the covalent adduct formation between 142D6 and XIAP BIR3 determined the complex structure, capturing the precise binding interactions between 142D6 and XIAP BIR3. This analysis highlighted a novel finding, the formation of a covalent bond between 142D6 and XIAP BIR3 Lys299 residue, shedding light on the mechanism of inhibition and the potential for irreversible inhibition strategies. By understanding the structural details of this interaction, we can gain valuable insights into the design of more potent and selective SMAC mimetics for potential clinical applications. Overall, this thesis provides a comprehensive crystallographic analysis of the novel SMAC mimetic, 142D6, and its covalent adduct formation with XIAP BIR3 at Lys299 residue. The findings presented here enhance our understanding of the structural basis of inhibition and pave the way for the development of improved apoptotic modulators with potential therapeutic application.

        Cover page: X-Ray Crystallography of Novel Aryl-Fluorosulfate Lysine Covalent Inhibitor of Apoptosis Protein (IAP) Antagonist
        • Article
        • Peer Reviewed
        UC Santa Cruz Previously Published Works (2019)
        Interferon-stimulated gene product 15 (ISG15) is a key component of host responses to microbial infection. Despite having been known for four decades, grasping the functions and features of ISG15 has been a slow and elusive process. Substantial work over the past two decades has greatly enhanced this understanding, revealing the complex and variable nature of this protein. This has unveiled multiple mechanisms of action that are only now beginning to be understood. In addition, it has uncovered diversity not only between how ISG15 affects different pathogens but also between the function and structure of ISG15 itself between different host species. Here we review the complexity of ISG15 within the context of viral infection, focusing primarily on its antiviral function and the mechanisms viruses employ to thwart its effects. We highlight what is known regarding the impact of ISG15 sequence and structural diversity on these interactions and discuss the aspects presenting the next frontier toward elucidating a more complete picture of ISG15 function.
          Cover page: ISG15: Its Complicated.
          • Article
          • Peer Reviewed
          UC Santa Cruz Previously Published Works (2019)
          Bats have long been observed to be the hosts and the origin of numerous human diseases. Bats, like all mammals, rely on a number of innate immune mechanisms to combat invading pathogens, including the interferon type I, II and III responses. Ubiquitin-like interferon-stimulated gene product 15 (ISG15) is a key modulator of these interferon responses. Within these pathways, ISG15 can serve to stabilize host proteins modulating innate immune responses and act as a cytokine. Post-translational modifications of viral proteins introduced by ISG15 have also been observed to directly affect the function of numerous viral proteins. Unlike ubiquitin, which is virtually identical across all animals, comparison of ISG15s across species reveals that they are relatively divergent, with sequence identity dropping to as low as ∼58% among mammals. In addition to serving as an obstacle to the zoonotic transmission of influenza, these ISG15 species-species differences have also long been shown to have an impact on the function of viral deISGylases. Recently, the structure of the first nonhuman ISG15, originating from mouse, suggested that the structures of human ISG15 may not be reflective of other species. Here, the structure of ISG15 from the bat species Myotis davidii solved to 1.37 Å resolution is reported. Comparison of this ISG15 structure with those from human and mouse not only underscores the structural impact of ISG15 species-species differences, but also highlights a conserved hydrophobic motif formed between the two domains of ISG15. Using the papain-like deISGylase from Severe acute respiratory syndrome coronavirus as a probe, the biochemical importance of this motif in ISG15-protein engagements was illuminated.
            Cover page: Structure of interferon-stimulated gene product 15 (ISG15) from the bat species Myotis davidii and the impact of interdomain ISG15 interactions on viral protein engagement.
            • Article
            • Peer Reviewed
            UC Santa Cruz Previously Published Works (2019)
            Tick-borne nairoviruses (order Bunyavirales) encode an ovarian tumor domain protease (OTU) that suppresses the innate immune response by reversing the post-translational modification of proteins by ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15). Ub is highly conserved across eukaryotes, whereas ISG15 is only present in vertebrates and shows substantial sequence diversity. Prior attempts to address the effect of ISG15 diversity on viral protein-ISG15 interactions have focused on only a single species ISG15 or a limited selection of nairovirus OTUs. To gain a more complete perspective of OTU-ISG15 interactions, we biochemically assessed the relative activities of 14 diverse nairovirus OTUs for 12 species ISG15 and found that ISG15 activity is predominantly restricted to particular nairovirus lineages reflecting, in general, known virus-host associations. To uncover the underlying molecular factors driving OTUs affinity for ISG15, X-ray crystal structures of Kupe virus and Ganjam virus OTUs bound to sheep ISG15 were solved and compared to complexes of Crimean-Congo hemorrhagic fever virus and Erve virus OTUs bound to human and mouse ISG15, respectively. Through mutational and structural analysis seven residues in ISG15 were identified that predominantly influence ISG15 species specificity among nairovirus OTUs. Additionally, OTU residues were identified that influence ISG15 preference, suggesting the potential for viral OTUs to adapt to different host ISG15s. These findings provide a foundation to further develop research methods to trace nairovirus-host relationships and delineate the full impact of ISG15 diversity on nairovirus infection.
              Cover page: Determining the molecular drivers of species-specific interferon-stimulated gene product 15 interactions with nairovirus ovarian tumor domain proteases.
              • Article
              • Peer Reviewed
              UC Santa Cruz Previously Published Works (2019)
              Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes.
                Cover page: Probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (vOTU) structure and deubiquitinase activity.
                • Article
                • Peer Reviewed
                UC Santa Cruz Previously Published Works (2017)
                Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) encode multifunctional papain-like proteases (PLPs) that have the ability to process the viral polyprotein to facilitate RNA replication and antagonize the host innate immune response. The latter function involves reversing the post-translational modification of cellular proteins conjugated with either ubiquitin (Ub) or Ub-like interferon-stimulated gene product 15 (ISG15). Ub is known to be highly conserved among eukaryotes, but surprisingly, ISG15 is highly divergent among animals. The ramifications of this sequence divergence to the recognition of ISG15 by coronavirus PLPs at a structural and biochemical level are poorly understood. Therefore, the activity of PLPs from SARS-CoV, MERS-CoV, and mouse hepatitis virus was evaluated against seven ISG15s originating from an assortment of animal species susceptible, and not, to certain coronavirus infections. Excitingly, our kinetic, thermodynamic, and structural analysis revealed an array of different preferences among PLPs. Included in these studies is the first insight into a coronavirus PLPs interface with ISG15 via SARS-CoV PLpro in complex with the principle binding domain of human ISG15 (hISG15) and mouse ISG15s (mISG15s). The first X-ray structure of the full-length mISG15 protein is also reported and highlights a unique, twisted hinge region of ISG15 that is not conserved in hISG15, suggesting a potential role in differential recognition. Taken together, this new information provides a structural and biochemical understanding of the distinct specificities among coronavirus PLPs observed and addresses a critical gap of how PLPs can interact with ISG15s from a wide variety of species.
                  Cover page: Structural Insights into the Interaction of Coronavirus Papain-Like Proteases and Interferon-Stimulated Gene Product 15 from Different Species.
                  • Article
                  • Peer Reviewed
                  UC Santa Cruz Previously Published Works (2021)
                  Porcine epidemic diarrhea is a devastating porcine disease that is caused by the alphacoronavirus porcine epidemic diarrhea virus (PEDV). Like other members of the Coronaviridae family, PEDV encodes a multifunctional papain-like protease 2 (PLP2) that has the ability to process the coronavirus viral polyprotein to aid in RNA replication and antagonize the host innate immune response through cleavage of the regulatory proteins ubiquitin (Ub) and/or interferon-stimulated gene product 15 (ISG15) (deubiquitination and deISGylation, respectively). Because Betacoronavirus PLPs have been well characterized, it was sought to determine how PLP2 from the alphacoronavirus PEDV differentiates itself from its related counterparts. PEDV PLP2 was first biochemically characterized, and a 3.1 Å resolution crystal structure of PEDV PLP2 bound to Ub was then solved, providing insight into how Alphacoronavirus PLPs bind to their preferred substrate, Ub. It was found that PEDV PLP2 is a deubiquitinase and readily processes a variety of di-Ub linkages, in comparison with its Betacoronavirus counterparts, which have a narrower range of di-Ub activity but process both Ub and ISG15.
                    Cover page: Structural insights into the interaction of papain-like protease 2 from the alphacoronavirus porcine epidemic diarrhea virus and ubiquitin.
                    • Article
                    • Peer Reviewed
                    UC Santa Cruz Previously Published Works (2017)
                    Antiviral responses are regulated by conjugation of ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) to proteins. Certain classes of viruses encode Ub- or ISG15-specific proteases belonging to the ovarian tumor (OTU) superfamily. Their activity is thought to suppress cellular immune responses, but studies demonstrating the function of viral OTU proteases during infection are lacking. Crimean-Congo hemorrhagic fever virus (CCHFV, family Nairoviridae) is a highly pathogenic human virus that encodes an OTU with both deubiquitinase and deISGylase activity as part of the viral RNA polymerase. We investigated CCHFV OTU function by inactivating protease catalytic activity or by selectively disrupting its deubiquitinase and deISGylase activity using reverse genetics. CCHFV OTU inactivation blocked viral replication independently of its RNA polymerase activity, while deubiquitinase activity proved critical for suppressing the interferon responses. Our findings provide insights into viral OTU functions and support the development of therapeutics and vaccines.
                      Cover page: Crimean-Congo Hemorrhagic Fever Virus Suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease.
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