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Clickable Probes for Pathogen Proteasomes: Synthesis and Applications.
Liu, Lawrence J; Lucero, Bobby; Manriquez-Rodriguez, Cindy; Francisco, Karol R; Teixeira, Thaiz R; Yohannan, Darius J; Ballatore, Carlo; Myers, Samuel A; O'Donoghue, Anthony J; Caffrey, Conor R.
Afiliación
  • Liu LJ; Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, United States.
  • Lucero B; Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, United States.
  • Manriquez-Rodriguez C; Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, United States.
  • Francisco KR; Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, United States.
  • Teixeira TR; Center for Autoimmunity and Inflammation, La Jolla Institute for Immunology, La Jolla, California 92037, United States.
  • Yohannan DJ; Laboratory for Immunochemical Circuits, La Jolla Institute for Immunology, La Jolla, California 92037, United States.
  • Ballatore C; Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, United States.
  • Myers SA; Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, United States.
  • O'Donoghue AJ; Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, United States.
  • Caffrey CR; Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, United States.
ACS Omega ; 9(32): 34829-34840, 2024 Aug 13.
Article en En | MEDLINE | ID: mdl-39157084
ABSTRACT
The 20S proteasome is a multimeric protease complex that is essential for proteostasis in the cell. Small molecule proteasome inhibitors are approved drugs for various cancers and are advancing clinically as antiparasitics. Although tools and technologies to study the 20S proteasome have advanced, only one probe is commercially available to image proteasome activity. This probe consists of a fluorescently labeled, peptidyl vinyl sulfone that binds to one or more of the catalytic proteasome subunits. Here, we synthesized two, active site-directed epoxyketone probes, LJL-1 and LJL-2, that were based on the peptidyl backbones of the anticancer drugs, carfilzomib and bortezomib, respectively. Each probe was conjugated, via click chemistry, to a bifunctional group comprising 5-carboxytetramethylrhodamine (TAMRA) and biotin to, respectively, visualize and enrich the 20S proteasome from protein extracts of two eukaryotic pathogens, Leishmania donovani and Trichomonas vaginalis. Depending on species, each probe generated a different subunit-binding profile by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the biotin tag enabled the enrichment of the bound subunits which were then formally identified by proteomics. Species differences in the order of electrophoretic migration by the ß subunits were also noted. Finally, both probes reacted specifically with the 20S subunits in contrast to the commercial vinyl sulfone probe that cross reacted with cysteine proteases. LJL-1 and LJL-2 should find general utility in the identification and characterization of pathogen proteasomes, and serve as reagents to evaluate the specificity and mechanism of binding of new antiparasitic proteasome inhibitors.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: ACS Omega Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: ACS Omega Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos