Chi-Yuan Chou

Structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease

Papain‐like protease (PLpro) is one of two cysteine proteases involved in the proteolytic processing of the polyproteins of Severe acute respiratory syndrome coronavirus (SARS‐CoV). PLpro also shows significant in vitro deubiquitinating and de‐ISGylating activities, although the detailed mechanism is still unclear. Here, the crystal structure of SARS‐CoV PLpro C112S mutant in complex with ubiquitin (Ub) is reported at 1.4 Å resolution. The Ub core makes mostly hydrophilic interactions with PLpro, while the Leu‐Arg‐Gly‐Gly C‐terminus of Ub is located in the catalytic cleft of PLpro, mimicking the P4–P1 residues and providing the first atomic insights into its catalysis. One of the O atoms of the C‐terminal Gly residue of Ub is located in the oxyanion hole consisting of the main‐chain amides of residues 112 and 113. Mutations of residues in the PLpro–Ub interface lead to reduced catalytic activity, confirming their importance for Ub binding and/or catalysis. The structure also revealed an N‐cyclohexyl‐2‐aminethanesulfonic acid molecule near the catalytic triad, and kinetic studies suggest that this binding site is also used by other PLpro inhibitors. Overall, the structure provides a foundation for understanding the molecular basis of coronaviral PLpro catalysis.

Thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of Middle East respiratory syndrome coronavirus

Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) is a new highly pathogenic human coronaviruses that emerged in Jeddah and Saudi Arabia and has quickly spread to other countries in Middle East, Europe and North Africa since 2012. Up to 17 December 2014, it has infected at least 938 people with a fatality rate of about 36% globally. This has resulted in an urgent need to identify antiviral drugs that are active against MERS-CoV. The papain-like protease (PLpro) of MERS-CoV represents an important antiviral target as it is not only essential for viral maturation, but also antagonizes interferon stimulation of the host via its deubiquitination activity. Here, we report the discovery that two SARS-CoV PLpro inhibitors, 6-mercaptopurine (6MP) and 6-thioguanine (6TG), as well as the immunosuppressive drug mycophenolic acid, are able to inhibit MERS-CoV PLpro. Their inhibition mechanisms and mutually binding synergistic effect were also investigated. Our results identify for the first time three inhibitors targeting MERS-CoV PLpro and these can now be used as lead compounds for further antiviral drug development.

Structural and functional characterization of MERS coronavirus papain-like protease

BackgroundsA new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PLpro) is expressed, and its structural and functional consequences are elucidated.ResultsCircular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PLpro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PLpro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PLpro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PLpro. A natural mutation, Leu105, is the major reason for this difference.ConclusionsOverall, MERS-CoV PLpro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PLpro family, which is applicable to develop strategies inhibiting PLpro against highly pathogenic coronaviruses.

Mechanism for controlling the monomer-dimer conversion of SARS coronavirus main protease.

The Severe acute respiratory syndrome coronavirus (SARS‐CoV) main protease (Mpro) cleaves two virion polyproteins (pp1a and pp1ab); this essential process represents an attractive target for the development of anti‐SARS drugs. The functional unit of Mpro is a homodimer and each subunit contains a His41/Cys145 catalytic dyad. Large amounts of biochemical and structural information are available on Mpro; nevertheless, the mechanism by which monomeric Mpro is converted into a dimer during maturation still remains poorly understood. Previous studies have suggested that a C‐terminal residue, Arg298, interacts with Ser123 of the other monomer in the dimer, and mutation of Arg298 results in a monomeric structure with a collapsed substrate‐binding pocket. Interestingly, the R298A mutant of Mpro shows a reversible substrate‐induced dimerization that is essential for catalysis. Here, the conformational change that occurs during substrate‐induced dimerization is delineated by X‐ray crystallography. A dimer with a mutual orientation of the monomers that differs from that of the wild‐type protease is present in the asymmetric unit. The presence of a complete substrate‐binding pocket and oxyanion hole in both protomers suggests that they are both catalytically active, while the two domain IIIs show minor reorganization. This structural information offers valuable insights into the molecular mechanism associated with substrate‐induced dimerization and has important implications with respect to the maturation of the enzyme.

UV-induced fin damage in zebrafish as a system for evaluating the chemopreventive potential of broccoli and cauliflower extracts

This study applied broccoli and cauliflower extracts (whole, floret, and stem) to zebrafish larvae in parallel to receive 100 mJ/cm2 of UVB six times, and recorded their fin malformation phenotypes. Chemopreventive effects of each group, including UVB, whole-, floret-, and stem-extracts of broccoli and cauliflower on fin development were evaluated using Kaplan-Meier analysis, log-rank test, and Cox proportional hazards regression. Results showed that (1) zebrafish fins in the UVB + whole broccoli extract group are 6.20~9.32-times more likely to return to normal fins than ones in the UVB only group, but fins in the UVB + whole cauliflower extract group are only 5.13~11.10-times more likely to recover, indicated that whole broccoli and cauliflower extract had similar chemopreventive ability on fin development; and (2) the broccoli stem has the highest antioxidant capacity among other groups. In conclusion, zebrafish can be used as a system for evaluating the efficacy of other UVB protective compounds.

Biochemical and structural properties of zebrafish Capsulin produced by Escherichia coli

Capsulin is one of the transcription factors involved in regulating cell differentiation but its biochemical properties and structural characteristics are still unclear. In the present study, we cloned capsulin from zebrafish, which produces large numbers of transparent embryos and has well-characterized developmental stages. By alignment, the deduced amino acid sequence of zebrafish Capsulin, which contains a putative bHLH motif, shares very high homology to that of other species with an 72-82% identity. Zebrafish Capsulin was also targeted to the nucleus of mammalian cells when overexpressed by transient transfection. In order to characterize the structural and biochemical properties of zebrafish Capsulin, a recombinant zebrafish Capsulin protein was expressed and purified in Escherichia coli. By circular dichroism spectroscopy, Capsulin was shown to be 55% α-helical. The size distribution assay by analytical ultracentrifugation indicated that it existed as a monomer-dimer mixture. The results suggested that the recombinant Capsulin has a well-organized and functional structure. Finally, endogenous Capsulin was distributed mainly in the epicardial cells of zebrafish by immunohistochemistry analysis using antibodies raised against zebrafish Capsulin. The present study not only helps us to comparatively analyze capsulin genes across species, but it also provides valuable structural information for further studies of Capsulin biological function in the future.

Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein.

Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We’ve also identified four historical mutations that are able to produce a “GST-like” S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution.

Disulfiram can inhibit MERS and SARS coronavirus papain-like proteases via different modes

Abstract Severe acute respiratory syndrome coronavirus (SARS‐CoV) emerged in southern China in late 2002 and caused a global outbreak with a fatality rate around 10% in 2003. Ten years later, a second highly pathogenic human CoV, MERS‐CoV, emerged in the Middle East and has spread to other countries in Europe, North Africa, North America and Asia. As of November 2017, MERS‐CoV had infected at least 2102 people with a fatality rate of about 35% globally, and hence there is an urgent need to identify antiviral drugs that are active against MERS‐CoV. Here we show that a clinically available alcohol‐aversive drug, disulfiram, can inhibit the papain‐like proteases (PLpros) of MERS‐CoV and SARS‐CoV. Our findings suggest that disulfiram acts as an allosteric inhibitor of MERS‐CoV PLpro but as a competitive (or mixed) inhibitor of SARS‐CoV PLpro. The phenomenon of slow‐binding inhibition and the irrecoverability of enzyme activity after removing unbound disulfiram indicate covalent inactivation of SARS‐CoV PLpro by disulfiram, while synergistic inhibition of MERS‐CoV PLpro by disulfiram and 6‐thioguanine or mycophenolic acid implies the potential for combination treatments using these three clinically available drugs. HighlightsDisulfiram, a drug for use in alcohol aversion therapy, can inhibit the papain‐like proteases of MERS‐CoV and SARS‐CoV.Disulfiram is a noncompetitive inhibitor of MERS‐CoV papain‐like protease.Disulfiram, 6‐thioguanine and mycophenolic acid can synergistically inhibit MERS‐CoV papain‐like protease.Disulfiram is a competitive inhibitor of SARS‐CoV papain‐like protease.Disulfiram is a slow‐binding inhibitor that forms a covalent adduct at the active site of SARS‐CoV papain‐like protease.

Protective Role of Comfrey Leave Extracts on UV-induced Zebrafish Fin Damage

In zebrafish, UV exposure leads to fin malformation phenotypes including fin reduction or absence. The present study evaluated UV-protective activities of comfrey leaves extracts in a zebrafish model by recording fin morphological changes. Chemopreventive effects of comfrey leave extracts were evaluated using Kaplan-Meier analysis and Cox proportional hazards regression. The results showed that (1) the mean times of return to normal fin in the UV+comfrey (50 and 100 ppm) groups were 3.43 and 2.86 days and were quicker compared with that in the UV only group (4.21 days); (2) zebrafish fins in the UV+comfrey (50 and 100 ppm) groups were 2.05 and 3.25 times more likely to return to normal than those in the UV only group; and (3) comfrey leave extracts had UV-absorbance abilities and significantly reduced ROS production in UV-exposed zebrafish embryos, which may attenuate UV-mediated apoptosis. In conclusion, comfrey leaves extracts may have the potential to be developed as UV-protective agents to protect zebrafish embryos from UV-induced damage.

Porcine epidemic diarrhea virus papain-like protease 2 can be noncompetitively inhibited by 6-thioguanine

Abstract Porcine epidemic diarrhea virus (PEDV) is a coronavirus (CoV) discovered in the 1970s that infects the intestinal tract of pigs, resulting in diarrhea and vomiting. It can cause extreme dehydration and death in neonatal piglets. In Asia, modified live attenuated vaccines have been used to control PEDV infection in recent years. However, a new strain of PEDV that belongs to genogroup 2a appeared in the USA in 2013 and then quickly spread to Canada and Mexico as well as Asian and European countries. Due to the less effective protective immunity provided by the vaccines against this new strain, it has caused considerable agricultural and economic loss worldwide. The emergence of this new strain increases the importance of understanding PEDV as well as strategies for inhibiting it. Coronaviral proteases, including main proteases and papain‐like proteases, are ideal antiviral targets because of their essential roles in viral maturation. Here we provide a first description of the expression, purification and structural characteristics of recombinant PEDV papain‐like protease 2, moreover present our finding that 6‐thioguanine, a chemotherapeutic drug, in contrast to its competitive inhibition on SARS‐ and MERS‐CoV papain‐like proteases, is a noncompetitive inhibitor of PEDV papain‐like protease 2. HighlightsPEDV PL2pro exhibits much higher DUB activity than that of other PLpros in spite of their structural similarities.In contrast to its competitive inhibition on SARS‐ and MERS‐CoV PLpros, 6‐thioguanine inhibits PEDV PL2pro allosterically.Putative 6‐thioguanine binding site is proposed to render the blocking loop less flexible and therefore disfavor catalysis.6‐thioguanine can be a lead compound for anti‐coronaviral drug development.