Multiple versions of a parent gene can function within molecular systems as gene duplicates (paralogs) and alternatively spliced isoforms. Proteins related in this manner often serve redundant roles, though they can be selectively or randomly prescribed unique functions. The present collection of three manuscripts details the evolution of members of the deubiquitinating enzyme superfamily. The first manuscript delineates the chronology of USP4, USP15 and USP11 emergence and concludes that the presumed ancestor, USP11, is in fact a recent duplicate and that, at minimum, one copy of USP4 or USP15 is required for organismal viability. The second determines that the long and short isoforms of mammalian USP4 are maintained by natural selection to occupy discrete spatial roles. The final manuscript broadens the scope and objectively draws the genealogy of all deubiquitinating enzymes, with emphasis on significant points of functional divergence of paralogs within innate immunity and DNA repair pathways.

A large fraction of the proteome displays cell cycle-dependent expression, which is important for cells to accurately grow and divide. Cyclical protein expression requires protein degradation via the ubiquitin proteasome system (UPS), and several ubiquitin ligases (E3) have established roles in this regulation. Less is understood about the roles of deubiquitinating enzymes (DUB), which antagonize E3 activity. A few DUBs have been shown to interact with and deubiquitinate cell cycle-regulatory E3s and their protein substrates, suggesting DUBs play key roles in cell cycle control. However, in vitro studies and characterization of individual DUB deletion strains in yeast suggest that these enzymes are highly redundant, making it difficult to identify their in vivo substrates and therefore fully understand their functions in the cell. To determine if DUBs play a role in the cell cycle, I performed a screen to identify specific DUB targets in vivo and then explored how these interactions contribute to cell cycle control. I conducted an in vivo overexpression screen to identify specific substrates of DUBs from a sample of UPS-regulated proteins and I determined that DUBs regulate different subsets of targets, confirming they display specificity in vivo. Five DUBs regulated the largest number of substrates, with Ubp10 stabilizing 40% of the proteins tested. Deletion of Ubp10 delayed the G1-S transition and reduced expression of Dbf4, a regulatory subunit of Cdc7 kinase, demonstrating Ubp10 is important for progression into S-phase. We hypothesized that compound deletion strains of these five DUBs would be deficient in key cellular processes because they regulated the largest number of cell cycle proteins from our screen. I performed genetic analysis to determine if redundancies exist between these DUBs. Our results indicate that most individual and combination deletion strains do not have impaired proliferation, with the exception of cells lacking UBP10. However, I observed negative interactions in some combinations when cells were challenged by different stressors. This implies the DUB network may activate redundant pathways only upon certain environmental conditions. While deletion of UBP10 impaired proliferation under standard growth conditions, I discovered that deletion of the proteasome-regulatory DUBs Ubp6 or Ubp14 rescues the cell cycle defect inubp10∆ cells. This suggests in the absence of Ubp10 substrates such as Dbf4 are rapidly degraded by the proteasome, but deletion of proteasome-associated DUBs restores cell cycle progression. Our work demonstrates that in unperturbed cells DUBs display specificity for their substrates in vivo and that a coordination of DUB activities promotes cell cycle progression.

Yes / Mislocalised membrane proteins (MLPs) present a risk to the cell due to exposed hydrophobic amino acids which cause MLPs to aggregate. Previous studies identified SGTA as a key component of the machinery that regulates the quality control of MLPs. Overexpression of SGTA promotes deubiqutination of MLPs resulting in their accumulation in cytosolic inclusions, suggesting SGTA acts in collaboration with deubiquitinating enzymes (DUBs) to exert these effects. However, the DUBs that play a role in this process have not been identified. In this study we have identified the ubiquitin specific peptidase 5 (USP5) as a DUB important in regulating the quality control of MLPs. We show that USP5 is in complex with SGTA, and this association is increased in the presence of an MLP. Overexpression of SGTA results in an increase in steady-state levels of MLPs suggesting a delay in proteasomal degradation of substrates. However, our results show that this effect is strongly dependent on the presence of USP5. We find that in the absence of USP5, the ability of SGTA to increase the steady state levels of MLPs is compromised. Moreover, knockdown of USP5 results in a reduction in the steady state levels of MLPs, while overexpression of USP5 increases the steady state levels. Our findings suggest that the interaction of SGTA with USP5 enables specific MLPs to escape proteasomal degradation allowing selective modulation of MLP quality control. These findings progress our understanding of aggregate formation, a hallmark in a range of neurodegenerative diseases and type II diabetes, as well as physiological processes of aggregate clearance.

Deubiquitinating enzymes (DUBs) constitute a diverse protein family. The specificities and functions of the majority of DUBs are unknown, although their impact on many biological and pathological processes is widely appreciated. This dissertation entails a detailed characterization of otubain 1 (OTUB1), an ovarian tumor domain-containing DUB. The presented work describes OTUB1’s specificity, localization, protein interactions, importance in infection with Yersinia, and proposes a novel model of regulation of its enzymatic activity. I first discuss the structural and biochemical properties of OTUB1, demonstrating its selectivity towards ubiquitin and NEDD8. Moreover, I show that OTUB1 cleaves lys48- but not lys63-linked polyubiquitin, emphasizing its role in ubiquitin-mediated proteasomal degradation. Mass spectrometric identification of interaction partners and localization studies suggest possible involvement of OTUB1 in RNA processing and cell morphology. Furthermore, I demonstrate that invasion of the host cells by the enterobacteria Yersinia can be altered by changing OTUB1 expression. This effect is dependent on the catalytic activity of OTUB1 and its ability to stabilize RhoA-GTP prior to infection. YpkA and OTUB1 modulate RhoA-GTP stability in opposing ways, leading to cytoskeletal rearrangements that may be involved in bacterial invasion. Moreover, OTUB1 is post-translationally modified by phosphorylation that modulates its ability to stabilize RhoA-GTP and counteracts its effect on bacterial invasion. These findings provide a novel entry point for the manipulation of the host—pathogen interactions. Lastly, a kinase screen revealed that FER, an oncogenic kinase with a role in cell morphology, phosphorylates OTUB1, as demonstrated by overexpression, siRNA and in vitro studies. The phosphorylated site was mapped to tyr26 and the activity-based labeling revealed that this modification interferes with the deubiquitinating activity of OTUB1. In summary, the results presented in this thesis confirm that OTUB1 exerts properties of a “classical DUB” and uncover some of its physiological functions.

O proteassoma 26S é uma estrutura em forma de barril com um núcleo catalítico 20S que é flanqueado por tampas 19S em ambos os lados. Nosso grupo tem demonstrado que o proteassoma 26S é crítico para o desenvolvimento e sobrevivência do Schistosoma mansoni, sendo que mais de 95% dos casais de vermes tratados com o inibidor de proteassoma MG132 apresentaram alteração na postura dos ovos e viabilidade. O tampão 19S é o complexo regulador e funciona no desdobramento e na desubiquitinação das proteínas, antes de sua entrada no complexo 20S, usando enzimas desubiquitinadoras (DUBS). Recentemente, foi demonstrado que a inibição das DUBS, UCHL5 e USP14, que estão reversivelmente ligadas à partícula 19S do complexo 26S, chamada b-AP15, resulta em autofagia, seguida de morte celular, pois leva uma variedade de células de mamíferos ao estresse oxidativo. O objetivo geral deste trabalho foi avaliar o efeito da droga sobre o desenvolvimento reprodutivo de S. mansoni e como possível alvo terapêutico, tendo como objetivos específicos determinar os seus efeitos: na inibição da oviposição in vitro de casais de parasitas Schistosoma mansoni através da contagem dos ovos e expressão do gene p14; avaliar se os órgãos reprodutores dos parasitas sofreram alguma modificação estrutural; se a substância utilizada leva a autofagia; se o tratamento dos casais de parasita com a droga leva a apoptose, ocasionada pela ativação de caspase-3. Este estudo é o primeiro a documentar o papel da droga b-AP15 como um agente esquistossomicida, pois desencadeia alterações ultraestruturais em casais de vermes de S. mansoni. Os seguintes métodos foram utilizados para analisar as alterações: Microscopia Eletrônica de Transmissão (MET), Varredura (MEV) e Confocal; ensaio quantitativo colorimétrico baseado no brometo de 3- (4,5-dimetiltiazol-2-il) -2,5-difenil tetrazólio (MTT), atividade do proteassoma através do substrato Suc-Leu-Le-Al-Tyr-AMC específico para atividade de quimotripsina-like; western blotting; Reação de Polimerase em Cadeia em tempo real e TUNEL. Foram testadas várias concentrações do fármaco (0,2; 0,4; 0,8; 1,6; 3,2 até 50 µM), sendo que a partir de 1,6 µM de b-AP15 ocorreu a inibição da produção de ovos dos casais de parasitas tratados in vitro por 24 h, não havendo alteração da viabilidade, mas mostrou alterações a partir da dosagem de 3,2 µM. A partir de 1,6 µM ocorreram alterações celulares e tegumentares e vermes adultos tratados com 50 µM estavam mortos. O Western blotting mostrou acúmulo de proteínas poliubiquitinadas de alto peso molecular na presença de 1,6µM, havendo mudanças na atividade quimotripsina-like do proteassoma 20S. Os resultados mostraram que o b-AP15 altera a oviposição, a viabilidade e leva à morte de casais de parasitas, reforçando a hipótese de que o sistema ubiquitina/ proteassoma e que as enzimas deubiquitinadoras ligadas a partícula 19S são essenciais para a biologia de S. mansoni. / The 26S proteasome is a barrel structure with a catalytic core 20S that is flanked by 19S caps on both sides. Our group has been showed that 26S proteasomes are critical for Schistosoma mansoni development and survival, being that more than 95% of worms pairs treated with the proteasome inhibitor MG132 showed alteration on egg laying and viability. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14, which are reversibly bound to the 19S particle of the 26S complex, called b-AP15 , results in cell death because it leads to autophagy, followed by cell death in a variety of mammalian cells. The general objective of this work was to evaluate the effect of the drug on the reproductive development of S. mansoni and as a possible therapeutic target. The specific objectives to determine its effects: in inhibiting the in vitro oviposition of couples of Schistosoma mansoni parasites by counting eggs and p14 gene expression; evaluate whether the reproductive organs of the parasites have undergone some structural modification; if the substance used leads to autophagy; if the treatment of the parasite couples with the drug leads to apoptosis, caused by the activation of caspase-3. This study is the first to document the role of the b-AP15 drug as a schistosomicidal agent, as it triggers ultrastructural changes in couples of S. mansoni worms. The following methods were used to analyze the changes: Transmission Electron Microscopy (MET), Scanning (SEM) and Confocal; Colorimetric assay based on 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), proteasome activity through Suc-Leu-Le-Al-Tyr-AMC substrate specific for chymotrypsin-like activity; western blotting; Polymerase chain reaction in real time and TUNEL. Various concentrations of the drug (0.2, 0.4, 0.8, 1.6, 3.2 to 50 ?M) were tested. 1.6 ?M b-AP15 occurred the inhibition of egg production of couples of parasites treated in vitro for 24 h, with no change in viability, but showed changes from the dosage of 3.2 ?M. Cell and tegumentary changes occurred in 1.6 ?M and adult worms treated with 50 ?M were dead. Western blotting showed accumulation of high molecular weight polyubiquitinated proteins in the presence of 1.6?M, with changes in the chymotrypsin-like activity of the 20S proteasome. The results showed that b-AP15 alters the oviposition, viability and leads to the death of couples of parasites, reinforcing the hypothesis that the ubiquitin / proteasome system and the deubiquitinating enzymes bound to the 19S particle are essential for the biology of S. mansoni.

O proteassoma 26S é uma estrutura em forma de barril com um núcleo catalítico 20S que é flanqueado por tampas 19S em ambos os lados. Nosso grupo tem demonstrado que o proteassoma 26S é crítico para o desenvolvimento e sobrevivência do Schistosoma mansoni, sendo que mais de 95% dos casais de vermes tratados com o inibidor de proteassoma MG132 apresentaram alteração na postura dos ovos e viabilidade. O tampão 19S é o complexo regulador e funciona no desdobramento e na desubiquitinação das proteínas, antes de sua entrada no complexo 20S, usando enzimas desubiquitinadoras (DUBS). Recentemente, foi demonstrado que a inibição das DUBS, UCHL5 e USP14, que estão reversivelmente ligadas à partícula 19S do complexo 26S, chamada b-AP15, resulta em autofagia, seguida de morte celular, pois leva uma variedade de células de mamíferos ao estresse oxidativo. O objetivo geral deste trabalho foi avaliar o efeito da droga sobre o desenvolvimento reprodutivo de S. mansoni e como possível alvo terapêutico, tendo como objetivos específicos determinar os seus efeitos: na inibição da oviposição in vitro de casais de parasitas Schistosoma mansoni através da contagem dos ovos e expressão do gene p14; avaliar se os órgãos reprodutores dos parasitas sofreram alguma modificação estrutural; se a substância utilizada leva a autofagia; se o tratamento dos casais de parasita com a droga leva a apoptose, ocasionada pela ativação de caspase-3. Este estudo é o primeiro a documentar o papel da droga b-AP15 como um agente esquistossomicida, pois desencadeia alterações ultraestruturais em casais de vermes de S. mansoni. Os seguintes métodos foram utilizados para analisar as alterações: Microscopia Eletrônica de Transmissão (MET), Varredura (MEV) e Confocal; ensaio quantitativo colorimétrico baseado no brometo de 3- (4,5-dimetiltiazol-2-il) -2,5-difenil tetrazólio (MTT), atividade do proteassoma através do substrato Suc-Leu-Le-Al-Tyr-AMC específico para atividade de quimotripsina-like; western blotting; Reação de Polimerase em Cadeia em tempo real e TUNEL. Foram testadas várias concentrações do fármaco (0,2; 0,4; 0,8; 1,6; 3,2 até 50 µM), sendo que a partir de 1,6 µM de b-AP15 ocorreu a inibição da produção de ovos dos casais de parasitas tratados in vitro por 24 h, não havendo alteração da viabilidade, mas mostrou alterações a partir da dosagem de 3,2 µM. A partir de 1,6 µM ocorreram alterações celulares e tegumentares e vermes adultos tratados com 50 µM estavam mortos. O Western blotting mostrou acúmulo de proteínas poliubiquitinadas de alto peso molecular na presença de 1,6µM, havendo mudanças na atividade quimotripsina-like do proteassoma 20S. Os resultados mostraram que o b-AP15 altera a oviposição, a viabilidade e leva à morte de casais de parasitas, reforçando a hipótese de que o sistema ubiquitina/ proteassoma e que as enzimas deubiquitinadoras ligadas a partícula 19S são essenciais para a biologia de S. mansoni. / The 26S proteasome is a barrel structure with a catalytic core 20S that is flanked by 19S caps on both sides. Our group has been showed that 26S proteasomes are critical for Schistosoma mansoni development and survival, being that more than 95% of worms pairs treated with the proteasome inhibitor MG132 showed alteration on egg laying and viability. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14, which are reversibly bound to the 19S particle of the 26S complex, called b-AP15 , results in cell death because it leads to autophagy, followed by cell death in a variety of mammalian cells. The general objective of this work was to evaluate the effect of the drug on the reproductive development of S. mansoni and as a possible therapeutic target. The specific objectives to determine its effects: in inhibiting the in vitro oviposition of couples of Schistosoma mansoni parasites by counting eggs and p14 gene expression; evaluate whether the reproductive organs of the parasites have undergone some structural modification; if the substance used leads to autophagy; if the treatment of the parasite couples with the drug leads to apoptosis, caused by the activation of caspase-3. This study is the first to document the role of the b-AP15 drug as a schistosomicidal agent, as it triggers ultrastructural changes in couples of S. mansoni worms. The following methods were used to analyze the changes: Transmission Electron Microscopy (MET), Scanning (SEM) and Confocal; Colorimetric assay based on 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), proteasome activity through Suc-Leu-Le-Al-Tyr-AMC substrate specific for chymotrypsin-like activity; western blotting; Polymerase chain reaction in real time and TUNEL. Various concentrations of the drug (0.2, 0.4, 0.8, 1.6, 3.2 to 50 ?M) were tested. 1.6 ?M b-AP15 occurred the inhibition of egg production of couples of parasites treated in vitro for 24 h, with no change in viability, but showed changes from the dosage of 3.2 ?M. Cell and tegumentary changes occurred in 1.6 ?M and adult worms treated with 50 ?M were dead. Western blotting showed accumulation of high molecular weight polyubiquitinated proteins in the presence of 1.6?M, with changes in the chymotrypsin-like activity of the 20S proteasome. The results showed that b-AP15 alters the oviposition, viability and leads to the death of couples of parasites, reinforcing the hypothesis that the ubiquitin / proteasome system and the deubiquitinating enzymes bound to the 19S particle are essential for the biology of S. mansoni.

No description available.

<div>The papain-like protease (PLP) domain of nonstructural protein 3 (nsp3) of the coronavirus (CoV) genome promotes viral replication by processing the CoV polyprotein (protease) and also antagonize innate immune responses by deubiquitinating (DUB) and deISGylating (deISG) host substrates. Selectively removing the DUB/deISG activities of PLP while keeping the protease activity intact is a potential strategy for designing a live attenuated virus. However, it is unclear in the literature the precise mechanism by which PLPs support CoV evasion of the innate immune system. Deciphering the substrate specificity of PLPs for host ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) can therefore help in the design of PLP mutants that selectively lack one activity for evaluating the DUB and deISG mechanism in CoV pathogenesis and replication. </div><div> In this dissertation, we investigate the structure and function of the single PLP (PLpro) from beta-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), which are dangerous viral pathogens that emerged from a zoonotic source to cause infectious disease in the human population. Additionally, we translate the knowledge gained to the equivalent PLP2 from alpha-CoV porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV), which cause fatal disease in suckling piglets on industrial pork farms and household cats, respectively. The primary objective of this work is to rationally design PLP mutants across beta- and alpha-CoVs to help attenuate CoV infection, as no antiviral or vaccine exist for human CoVs and the efficacy of PEDV vaccines are an ongoing research topic. </div><div><br></div><div>In Chapter 1, different human, animal, and the bat origin CoV strains are introduced. The CoV life-cycle and virion structure are outlined, along with the replicase complex for viral replication. The multidomain nsp3 from alpha- and beta-CoV genomes are also described with a focus on the PLP domain and its proposed cleavage sites of the viral polyprotein. The discovery of the first viral protease DUB and the multiple activities of PLPs are defined, which includes a proposed model of how DUB versus deISG activities may act in the innate immune response. This leads into the therapeutic potential of PLP for an antiviral or live attenuated vaccine, which is followed by the introduction of live attenuated vaccines and the reverse genetics system. Next, proof of concept studies on PLP2 mutants are described and the introduction is concluded by stating the ultimate goal for the design of PLP mutants.</div><div><br></div><div>In Chapter 2, we hypothesize that the flanking ubiquitin-like (Ubl2) domain of MERS-CoV PLpro is not required for its enzymatic function. We characterize the specific activity, kinetics, substrate specificity, and inhibition of the PLpro enzyme with and without the Ubl2 domain and reveal that the Ubl2 domain does not significantly alter PLpro function. We determine the structure of the core PLpro, smallest catalytic unit to 1.9 Å resolution and observed no structural changes compared to the wild-type. Additionally, we demonstrate that a purported MERS-CoV PLpro inhibitor is nonselective in non-reducing conditions and should not be pursed for therapeutic use. We show that the core PLpro enzyme i.e. without the Ubl2 domain is a stable and robust construct for crystallization and is also thermally stable based on thermal melting studies with utility for structure-based drug design. </div><div><br></div><div>In Chapter 3, we shed light on the specificity of SARS-CoV PLpro towards Ub versus ISG15 by characterizing the specific activity and kinetic parameters of SARS-CoV PLpro mutants. In addition, the structure of SARS-CoV PLpro in complex with the C-terminal domain of ISG15 is determined and compared with the Ub-bound structure. Based on the structure and kinetic results, the altered specificities of SARS-CoV PLpro mutants Arg167Glu, Met209Ala, and Gln233Glu are compared with the wild-type. Arg167Glu mutant exhibits DUB hyperactivity and is expected to adopt a more favorable interaction with the Arg42 of Ub. At the same time, ARG167GLU contains a shorter side-chain that hinders interaction with the unique Trp123 of ISG15 for deISG activity compared to the wild-type. These results aid in the development of SARS-CoV PLpro mutants that have directed shifts in substrate specificity for Ub versus ISG15. </div><div><br></div><div>In Chapter 4, the process and antiviral activity of ISGylation is reviewed and how viruses can modulate host-derived versus virus-derived machineries to counteract ISGylation for viral infection. MERS-CoV PLpro is cross-reactive for Ub, but less is known about its specificity towards ISG15. In this study, we determine the structure of MERS-CoV PLpro bound with ISG15 to 2.3 Å resolution and reveal a small hydrophobic pocket of ISG15 that consists of P130 and W123, which differs from Ub hydrophobic patch. We design and determine the kinetic parameters for 13 PLpro mutants and reveal that MERS-CoV PLpro only has a single ubiquitin recognition (SUb1) site. Kinetic studies show that removing the charge of the R1649 greatly enhances DUB/protease activity while mutating in an Arg near R42 of Ub or ISG15 hydrophobic region is detrimental to both DUB/deISG activities. Kinetic experiments and probe-reactivity assays showed that Val1691Arg, Val1691Lys, and His1652Arg mutants are drastically reduced DUB/deISG activities compared to the wild-type. Overall, MERS-CoV PLpro mutants with alter kinetic profiles will be useful for discovery tools and DUB/deISG deficient mutants are great candidates for removing host cell antagonism activity by PLpro for live attenuated vaccines.</div><div><br></div><div>In Chapter 5, the goal is to translate the knowledge gained in Chapters 2-4 on beta-CoVs PLpro and evaluate the substrate specificity of alpha-CoVs FIPV and PEDV PLP2 for mutagenesis experiments. First, we design and purify the core PLP2 enzymes for kinetics. PLP2s are efficient DUBs that prefer Ub to ISG15 in vitro, and this preference is conserved in beta-CoV MHV PLP2 as well as alpha-CoV NL63 PLP2. We determine the structure of alpha-CoV PEDV PLP2 to 1.95 Å resolution and reveal the unique Zn-finger coordinating Cys3-His arrangement of the alpha-CoV genus that differs from past beta-CoV PLP crystal structures. To determine residues of the SUb1 site, we generate a homology model of FIPV PLP2 and overlay our PLP2 structures with MERS-CoV PLpro bound with Ub. In addition, we create electrostatic surface maps across coronaviral PLP subfamilies to evaluate the charge distribution of the SUb1 for the rational design of several FIPV and PEDV PLP2 mutants. We evaluate the turnover of PLP mutants for FRET-based substrates and reveal that His101ArgFIPV and Asn101ArgPEDV are drastically reduced in Ub-AMC activity while their peptide activities are within 2-fold of the wild-type. These mutants show delayed reactivity for Ub probes and no longer cleave Ub-chains displaying isopeptide bonds compared to the wild-type. Results from this study reveal a hot spot in both alpha- and beta-CoVs that can be used to selectively remove DUB activity of PLPs for generating a DUB deficient PLP enzyme. </div><div><br></div><div>In this dissertation, we investigate the substrate specificity of PLPs across alpha- and beta-CoVs and develop a fingerprint for Ub and also shed light on ISG15 recognition. Specifically, hot spots were identified in the SUb1 site of different PLPs, which recognize R42 and hydrophobic Ile44 of Ub. Position 97-98 of PLPs can be used to remove DUB activity by substituting an Arg, but usually effect protease function. Substituting an Arg at position 101 and 136 of coronaviral PLPs serve as the best strategy to remove DUB function while not hindering active site functionality. The DUB/deISG deficient mutants described will be useful for inhibiting the ability of PLPs to function in the innate immune response. Ultimately, this work provides a guide for identifying attenuating mutants in existing CoVs for live attenuated vaccines and also a blueprint for engineering PLPs from new emerging CoVs. </div>

<p>Coronaviruses (CoVs) are single-stranded, positive-sense RNA viruses in the Coronaviridae family. Within this family are four different genera, Alpha-, Beta-, Gamma-, and Deltacoronaviruses with human-infecting CoVs spanning the Alpha- and Beta-CoV genera. Most notably, Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1) and SARS-CoV-2 are Betacoronaviruses that spread worldwide in their outbreaks from 2002-2003 (SARS-CoV-1) and 2019-2020 (SARS-CoV-2). Human-infecting Alphacoronaviruses, NL63-CoV and 229E-CoV, have caused milder infections involving respiratory disease, gastroenteritis, and in more severe cases, death. Despite milder disease, Alphacoronaviruses are the cause of 15-30% of severe upper and lower respiratory tract infections each year. There have been recent efforts in the development of potent, small-molecule inhibitors to treat SARS-CoV-2 infection but there is an ongoing need to develop new and effective anti-coronavirus therapeutics to treat other human-infecting CoVs circulating society. Coronaviruses encode two essential proteases, the papain-like protease (PLP) and the 3C-like protease. PLPs are cysteine proteases located in non-structural protein 3 (nsp3). PLPs processes the viral polyprotein, releasing the first three nonstructural proteins encoded in the virus, and also are involved in evading the innate immune response through deubiquitinating (DUB) and deISGylating activity. </p> <p><br></p> <p>This study compares the substrate specificity and catalytic function of multiple human-infecting PLPs from both Alpha- and Beta-CoVs including NL63-CoV PLP2, 229E-CoV PLP2, Canine-CoV PLP2, FIPV-CoV PLP2, PEDV-CoV PLP2, SARS-CoV-1 PLpro, and SARS-CoV-2 PLpro. Interestingly, Alphacoronavirus PLP2s have a >400-fold greater catalytic efficiency for ubiquitin compared to Betacoronaviruses PLpro. This work also identifies a non-covalent scaffold of inhibitors that has pan-CoV inhibition; however, the IC50 values are >30-fold higher for NL63-CoV PLP2 than for SARS-CoV-1 PLpro. The X-ray structures of NL63 PLP2 and 229E PLP2 were determined to 2.1 Å and 1.8 Å, respectively, and provide structural information about the substrate and inhibitor binding region that could be the result in the differences in Alpha- and Betacoronavirus PLP function. Since PLP does not function as a single-domain in vivo, it is critical to understand the function of PLP when tethered to other domains of nsp3. This study also investigates nine different constructs of SARS-CoV-2 nsp3 with increasing domains, ranging from the single PLpro domain to Ubl1-Ydomain ΔTM1-TM2. Interestingly, the longer constructs of SARS-CoV-2 nsp3 show less catalytic efficiency for Ub-AMC and greater affinity for ISG15-AMC, with 8-fold lower Km values compared to PLpro alone. Lastly, each SARS-CoV-2 nsp3 construct was inhibited by a known PLpro inhibitor, GRL-0617, with reported IC50 values ranging from 0.91 μM to 1.9 μM. These data show that GRL-0617 still remains a lead compound to be optimized for cellular potency. </p> <p><br></p> <p>Overall, this dissertation advances the understanding of the kinetic and structural differences between Alphacoronavirus PLP2 and Betacoronavirus PLpro enzymes in the efforts of developing a pan-CoV inhibitor. Additionally, these data provide initial kinetic and biophysical characterization of PLpro within the larger context of nsp3 to elucidate the function of PLpro in its most native context during coronaviral infection.</p>