Rodrigues da Silva Eduardo Vagner, Torres Caroline, Nemamiah Escolarique Ribeiro Hariel, Santana Travaglini Berti de Correia Camila Rolemberg, de Oliveira de Castro Taissa, da Costa Mancin Giovanna, Zanchetta Venancio Mayla Gabriela, Abdel Baqui Munira Muhammad, Teixeira Felipe Roberti, Gomes Marcelo Damário
Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil.
Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, Brazil; Current address: Department of BioMolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil.
Arch Biochem Biophys. 2025 Feb;764:110272. doi: 10.1016/j.abb.2024.110272. Epub 2024 Dec 15.
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 Å, 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.
UBC13是在墨西哥利什曼原虫中描述的人类泛素结合E2酶的直系同源物，无法产生缺乏该基因的无效突变体，这表明其在这种寄生虫中具有重要功能。婴儿利什曼原虫是内脏利什曼病的病原体，这种疾病是最严重的类型，如果不治疗可能会致命。泛素化过程已成为杀利什曼原虫化合物的作用靶点，表明其在寄生虫体内平衡中具有重要功能。因此，对泛素化过程的分子特征进行研究可能有助于更好地理解利什曼病的分子和细胞基础。在此，我们对婴儿利什曼原虫中的LINF_350017900基因进行了表征，该基因被命名为LinfUBC13，它是墨西哥利什曼原虫中UBC13以及人类UBE2D家族的E2直系同源物，与UBE2D1、UBE2D2和UBE2D3具有72 - 74%的同一性。LinfUbc13包含保守的催化残基，包括Cys86和HPN基序，这些对于泛素结合活性至关重要。结构分析显示LinfUbc13与人类UBE2D蛋白高度相似，均方根偏差（RMSD）为0.4 Å，表明功能保守。重组LinfUbc13得以表达，并显示能从E1接受泛素，形成硫酯中间体。功能测定表明LinfUbc13通过人类HDM2 E3连接酶将泛素转移至p53，证实了其在泛素化中的作用。亚细胞定位显示LinfUbc13分布于整个寄生虫细胞质中。这些发现突出了婴儿利什曼原虫与人类之间泛素 - 蛋白酶体系统的保守性质，表明LinfUbc13是一种在寄生虫发育中起关键作用的E2酶。
