Valadez Jessica A, Cuajungco Math P
Department of Biological Science, and Center for Applied Biotechnology Studies, California State University Fullerton, CA 92831, USA.
Department of Biological Science, and Center for Applied Biotechnology Studies, California State University Fullerton, CA 92831, USA; Mental Health Research Institute, Melbourne Brain Centre, Parkville, Victoria 3052, Australia.
Gene. 2015 Jan 25;555(2):194-202. doi: 10.1016/j.gene.2014.11.003. Epub 2014 Nov 6.
Transient receptor potential mucolipin (TRPML) proteins belong to the TRP superfamily of non-selective cation channels. The TRPML1, -2, and -3 proteins are encoded by Mucolipin (MCOLN)-1, -2 and -3 genes, respectively. TRPML1 has been associated with mucolipidosis type IV (MLIV), while no disease phenotype has been linked with TRPML2 or -3 protein. The TRPML proteins share high sequence similarities, form hetero-tetramers, and serve in membrane trafficking, autophagy, and metal homeostasis. Previous studies suggest that TRPML2 serves a role in the immune system; however, the evidence is mostly indirect. We hypothesize that if TRPML2 is involved in immune function its expression would be likely regulated by an immune-associated transcription factor protein. Thus, we set out to identify the core promoter region and the transcription factor responsible for MCOLN2 gene expression. Using dual-luciferase assay and over-expression analyses, we reveal for the first time that B-cell lineage specific activator protein (BSAP), also known as paired box 5 (PAX5), controls MCOLN2 expression. Specifically, heterologous expression of PAX5 in HEK-293 cells significantly increased endogenous MCOLN2 transcript and TRPML2 protein levels, while RNA interference targeting endogenous PAX5 reduced its effect. Site-directed mutagenesis studies showed that the core promoter and PAX5 binding region to be between -79 and -60 base pairs upstream of the transcriptional start site. Thus, our findings add to a growing list of evidence for TRPML2's possible involvement in the immune system. The knowledge gained from this study could be used to further characterize the role of TRPML2 in B-cell development and function.
瞬时受体电位黏脂素(TRPML)蛋白属于非选择性阳离子通道的TRP超家族。TRPML1、-2和-3蛋白分别由黏脂素(MCOLN)-1、-2和-3基因编码。TRPML1与IV型黏脂贮积症(MLIV)相关,而尚未发现TRPML2或-3蛋白与疾病表型有关。TRPML蛋白具有高度的序列相似性,形成异源四聚体,并在膜运输、自噬和金属稳态中发挥作用。先前的研究表明TRPML2在免疫系统中起作用;然而,证据大多是间接的。我们假设,如果TRPML2参与免疫功能,其表达可能受免疫相关转录因子蛋白的调控。因此,我们着手确定负责MCOLN2基因表达的核心启动子区域和转录因子。通过双荧光素酶测定和过表达分析,我们首次揭示B细胞谱系特异性激活蛋白(BSAP),也称为配对盒5(PAX5),控制MCOLN2的表达。具体而言,PAX5在HEK-293细胞中的异源表达显著增加了内源性MCOLN2转录本和TRPML2蛋白水平,而靶向内源性PAX5的RNA干扰降低了其作用。定点诱变研究表明,核心启动子和PAX5结合区域位于转录起始位点上游-79至-60碱基对之间。因此,我们的发现为TRPML2可能参与免疫系统增加了越来越多的证据。从这项研究中获得的知识可用于进一步阐明TRPML2在B细胞发育和功能中的作用。