Universidad Escuela de Administración de Negocios, Departamento de Ciencias Básicas, Bogotá, Colombia.
Universidad El Bosque, Laboratorio de Virología, Bogotá, Colombia.
Mem Inst Oswaldo Cruz. 2020 Feb 21;115:e190242. doi: 10.1590/0074-02760190242. eCollection 2020.
Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia.
To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs).
We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels.
We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation.
Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.
泛素（Ub）和泛素样蛋白（Ub-L）是细胞周期、DNA 修复、转录、染色质重塑、信号转导和蛋白质降解等复杂细胞过程的关键调节因子。肠道贾第虫具有经过实验验证的 Ub 缀合系统；然而，使用基本局部比对搜索工具（BLAST）仅鉴定出参与该过程的少数几种酶。这是因为 BLAST 识别序列相似度 < 30%时的同源功能区域的能力有限。此外，在贾第虫中，Ub-Ls 及其缀合酶尚未被充分阐明。
使用基于隐马尔可夫模型（HMM）的智能系统鉴定参与 Ub 和 Ub-Ls 缀合过程的酶。
我们在 Giardia 蛋白质组中这些途径的关键酶的功能 Pfam 结构域中进行了 HMM 搜索。鉴定的每个开放阅读框均通过序列同源性、结构域架构和转录水平进行分析。
我们鉴定了 118 个基因，其中 106 个对应于泛素化过程（Ub、E1、E2、E3 和 DUB 酶）。E3 连接酶组是最大的组，有 82 个成员；其中 71 个成员具有特征性 RING 结构域。鉴定了 4 种 Ub-Ls，并首次描述了 NEDD8 和 URM1 的缀合酶。Ub-Ls 的 3D 模型显示了典型的 β-抓握折叠。此外，我们对相应激活酶的序列分析检测到了缀合所需的必需基序。
我们的研究结果突出了贾第虫 Ub 缀合系统的复杂性，与以前报道的系统有很大不同，并为 NEDDylation 和 URMylation 酶在肠道贾第虫基因组和转录组中的存在提供了证据。
