Molecular characterization of the E2 conjugating enzyme LinfUbc13 in Leishmania infantum

Author(s)

da Silva, Eduardo Vagner Rodrigues ; Torres, Caroline ; Ribeiro, Hariel Nemamiah Escolarique ; de Correia, Camila Rolemberg Santana Travaglini Berti ; de Castro, Taissa de Oliveira ; Mancin, Giovanna da Costa ; Venancio, Mayla Gabriela Zanchetta ; Baqui, Munira Muhammad Abdel ; Teixeira, Felipe Roberti ; Gomes, Marcelo Damario

• Document type: Journal article
• Source: Archives of Biochemistry and Biophysics; v. 764, p. 9-pg., 2024-12-19.

Abstract

UBC13 is an orthologue of Homo sapiens ubiquitin-conjugation E2 enzymes described in Leishmania mexicana, a null mutant lacking this gene cannot be produced, suggesting essential functions in this parasite. Leishmania infantum is an etiological agent of visceral leishmaniasis, the most severe type of disease that is potentially fatal if untreated. The ubiquitination process has been targeted for leishmanicidal compounds, indicating its essential function in parasite homeostasis. Therefore, the molecular characterization of the ubiquitination process may provide a better understanding of the molecular and cellular basis of leishmaniasis. Here, we characterized the gene LINF_350017900 in Leishmania infantum, which was named LinfUBC13, an E2 orthologue of UBC13 in Leishmania mexicana and the UBE2D family in Homo sapiens, sharing 72-74 % identity with UBE2D1, UBE2D2, and UBE2D3. LinfUbc13 contains conserved catalytic residues, including Cys86 and the HPN motif, which are essential for ubiquitin-conjugating activity. Structural analysis revealed a high similarity between LinfUbc13 and human UBE2D proteins, with a root-mean-square deviation (RMSD) of 0.4 & Aring;, suggesting conserved functions. Recombinant LinfUbc13 was expressed and shown to accept ubiquitin from E1, forming a thioester intermediate. Functional assays demonstrated that LinfUbc13 transfers ubiquitin to p53 through human HDM2 E3 ligase, confirming its role in ubiquitination. Subcellular localization showed that LinfUbc13 was distributed throughout the parasite cytoplasm. These findings highlight the conserved nature of the ubiquitin-proteasome system between Leishmania infantum and Homo sapiens, showing that LinfUbc13 is an E2 enzyme that plays a crucial role in parasitic development. (AU)

• FAPESP's process: 21/10971-0 - Biochemical characterization of CRLs (Cullin RING ligases) E3 ubiquitin-ligases in Leishmania infantum
• Grantee: Camila Rolemberg Santana Travaglini Berti de Correia
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 22/02933-3 - Role of the ubiquitin proteasome system in proliferation, differentiation and infectivity of Leishmania infantum
• Grantee: Felipe Roberti Teixeira
• Support Opportunities: Research Grants - Initial Project
• FAPESP's process: 22/16270-6 - In silico characterization of the E3 ubiquitin CRL1 ligase complex (Cullin RING-ligases) in Leishmania infantum
• Grantee: Caroline Torres
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 23/17920-7 - Characterization of the interaction of Trypanosoma cruzi with the host cell nuclear machinery investigating repair pathways and measurement of DNA damage
• Grantee: Munira Muhammad Abdel Baqui
• Support Opportunities: Regular Research Grants
• FAPESP's process: 24/01732-0 - Functional characterization of proteins from the Ubiquitin Proteasome System of L. infantum
• Grantee: Taissa de Oliveira de Castro
• Support Opportunities: Scholarships in Brazil - Scientific Initiation