Browsing by Author "Wu, Chunxiang"

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    Allosteric differences dictate GroEL complementation of E. coli
    (Wiley, 2022) Sivinski, Jared; Ngo, Duc; Zerio, Christopher J.; Ambrose, Andrew J.; Watson, Edmond R.; Kaneko, Lynn K.; Kostelic, Marius M.; Stevens, Mckayla; Ray, Anne-Marie; Park, Yangshin; Wu, Chunxiang; Marty, Michael T.; Hoang, Quyen Q.; Zhang, Donna D.; Lander, Gabriel C.; Johnson, Steven M.; Chapman, Eli; Biochemistry and Molecular Biology, School of Medicine
    GroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies.
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    Functional Differences between E. coli and ESKAPE Pathogen GroES/GroEL
    (American Society for Microbiology, 2021-01-12) Sivinski, Jared; Ambrose, Andrew J.; Panfilenko, Iliya; Zerio, Christopher J.; Machulis, Jason M.; Mollasalehi, Niloufar; Kaneko, Lynn K.; Stevens, Mckayla; Ray, Anne-Marie; Park, Yangshin; Wu, Chunxiang; Hoang, Quyen Q.; Johnson, Steven M.; Chapmana, Eli; Biochemistry and Molecular Biology, School of Medicine
    As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli, we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli, but only in the strict absence of either E. coli GroEL (P. aeruginosa) or both E. coli GroES and GroEL (E. faecium). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coli groESL was replaced with ESKAPE groESL, demonstrated similar growth kinetics to wild-type E. coli, but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity.
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    Longitudinal IgG antibody responses to Plasmodium vivax blood-stage antigens during and after acute vivax malaria in individuals living in the Brazilian Amazo
    (PLoS, 2022-11-23) Tashi, Tenzin; Upadhye, Aditi; Kundu, Prasun; Wu, Chunxiang; Menant, Sébastien; Soares, Roberta Reis; Ferreira, Marcelo U.; Longley, Rhea J.; Mueller, Ivo; Hoang, Quyen Q.; Tham, Wai-Hong; Rayner, Julian C.; Scopel, Kézia K. G.; Lima-Junior, Josué C.; Tran, Tuan M.; Medicine, School of Medicine
    Background To make progress towards malaria elimination, a highly effective vaccine targeting Plasmodium vivax is urgently needed. Evaluating the kinetics of natural antibody responses to vaccine candidate antigens after acute vivax malaria can inform the design of serological markers of exposure and vaccines. Methodology/Principal findings The responses of IgG antibodies to 9 P. vivax vaccine candidate antigens were evaluated in longitudinal serum samples from Brazilian individuals collected at the time of acute vivax malaria and 30, 60, and 180 days afterwards. Antigen-specific IgG correlations, seroprevalence, and half-lives were determined for each antigen using the longitudinal data. Antibody reactivities against Pv41 and PVX_081550 strongly correlated with each other at each of the four time points. The analysis identified robust responses in terms of magnitude and seroprevalence against Pv41 and PvGAMA at 30 and 60 days. Among the 8 P. vivax antigens demonstrating >50% seropositivity across all individuals, antibodies specific to PVX_081550 had the longest half-life (100 days; 95% CI, 83–130 days), followed by PvRBP2b (91 days; 95% CI, 76–110 days) and Pv12 (82 days; 95% CI, 64–110 days). Conclusion/Significance This study provides an in-depth assessment of the kinetics of antibody responses to key vaccine candidate antigens in Brazilians with acute vivax malaria. Follow-up studies are needed to determine whether the longer-lived antibody responses induced by natural infection are effective in controlling blood-stage infection and mediating clinical protection.
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    Longitudinal IgG antibody responses to Plasmodium vivax blood-stage antigens during and after acute vivax malaria in individuals living in the Brazilian Amazon
    (Public Library of Science, 2022-11-23) Tashi, Tenzin; Upadhye, Aditi; Kundu, Prasun; Wu, Chunxiang; Menant, Sébastien; Reis Soares, Roberta; Ferreira, Marcelo U.; Longley, Rhea J.; Mueller, Ivo; Hoang, Quyen Q.; Tham, Wai-Hong; Rayner, Julian C.; Scopel, Kézia K. G.; Lima-Junior , Josué C.; Tran, Tuan M.; Medicine, School of Medicine
    Background: To make progress towards malaria elimination, a highly effective vaccine targeting Plasmodium vivax is urgently needed. Evaluating the kinetics of natural antibody responses to vaccine candidate antigens after acute vivax malaria can inform the design of serological markers of exposure and vaccines. Methodology/principal findings: The responses of IgG antibodies to 9 P. vivax vaccine candidate antigens were evaluated in longitudinal serum samples from Brazilian individuals collected at the time of acute vivax malaria and 30, 60, and 180 days afterwards. Antigen-specific IgG correlations, seroprevalence, and half-lives were determined for each antigen using the longitudinal data. Antibody reactivities against Pv41 and PVX_081550 strongly correlated with each other at each of the four time points. The analysis identified robust responses in terms of magnitude and seroprevalence against Pv41 and PvGAMA at 30 and 60 days. Among the 8 P. vivax antigens demonstrating >50% seropositivity across all individuals, antibodies specific to PVX_081550 had the longest half-life (100 days; 95% CI, 83-130 days), followed by PvRBP2b (91 days; 95% CI, 76-110 days) and Pv12 (82 days; 95% CI, 64-110 days). Conclusion/significance: This study provides an in-depth assessment of the kinetics of antibody responses to key vaccine candidate antigens in Brazilians with acute vivax malaria. Follow-up studies are needed to determine whether the longer-lived antibody responses induced by natural infection are effective in controlling blood-stage infection and mediating clinical protection.
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    Structure and Function of the G Domain of Parkinson's Disease-Associated Protein LRRK2
    (2019-08) Wu, Chunxiang; Hoang, Quyen Q.; Foroud, Tatiana M.; Hurley, Thomas D.; Johnson, Steven M.; Zhang, Zhong-Yin
    Mutations in the gene encoding for leucine rich repeats kinase 2 (LRRK2) are commonly found in Parkinson’s disease. Recently, we found that the disease-associated point mutations at residue R1441 in the G domain (ROC) of LRRK2 resulted in perturbation of its GTPase activity. In this study, we compare the biochemical and biophysical properties of the ROC domain of LRRK2 carrying the PD-associated mutations at residue R1441 with those of the wild-type. We found that the disease-associated mutations (R1441C/G/H) showed marked quaternary structure compared to wild-type, in that the latter existed in solution in both monomeric and dimeric conformations dynamically regulated by GDP/GTP binding state, while we detected only monomeric conformation for three disease-associated mutants. To understand the structural basis for this plasticity and the activity reduction in the mutants, we solved a 1.6 Å crystal structure of the wild type ROC that shows a stable dimeric conformation in which the switch motifs and inter-switch regions mediate extensive interactions at the dimer interface. Residue R1441, where PD-associated mutations occur, forms exquisite interactions at the interface, thus suggesting a critical role of this residue in maintaining a dynamic dimer-monomer interconversion and conformational flexibility of the switch motifs. Consistently, substituting R1441 for other arbitrary mutations (R1441K/S/T) lead to similar perturbation of GTPase activity and dimerization defects as observed in the disease-associated mutants. Locking the ROC domain in either dimeric or monomeric conformations by engineered disulfide bond alters the binding affinity to GTP (but not GDP) and significantly reduce GTPase activity, thus suggesting that the dynamic dimer-monomer interconversion and conformational plasticity are essential for ROC function as a molecular switch modulating the kinase activity of LRRK2.