Département de Chimie, Université du Québec à Montréal, Montréal, QC H2X 2J6, Canada.
Centre d'Excellence en Recherche sur les Maladies Orphelines-Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montréal, QC H2X 3Y7, Canada.
Cells. 2021 Nov 6;10(11):3063. doi: 10.3390/cells10113063.
Developmental and epileptic encephalopathies (DEE) are rare and serious neurological disorders characterized by severe epilepsy with refractory seizures and a significant developmental delay. Recently, DEE73 was linked to genetic alterations of the RNF13 gene, which convert positions 311 or 312 in the RNF13 protein from leucine to serine or proline, respectively (L311S and L312P). Using a fluorescence microscopy approach to investigate the molecular and cellular mechanisms affected by RNF13 protein variants, the current study shows that wild-type RNF13 localizes extensively with endosomes and lysosomes, while L311S and L312P do not extensively colocalize with the lysosomal marker Lamp1. Our results show that RNF13 L311S and L312P proteins affect the size of endosomal vesicles along with the temporal and spatial progression of fluorescently labeled epidermal growth factor, but not transferrin, in the endolysosomal system. Furthermore, GST-pulldown and co-immunoprecipitation show that RNF13 variants disrupt association with AP-3 complex. Knockdown of AP-3 complex subunit AP3D1 alters the lysosomal localization of wild-type RNF13 and similarly affects the size of endosomal vesicles. Importantly, our study provides a first step toward understanding the cellular and molecular mechanism altered by DEE73-associated genetic variations of RNF13.
发育性和癫痫性脑病（DEE）是一种罕见且严重的神经发育障碍，其特征为严重癫痫、难治性癫痫发作和明显的发育迟缓。最近，DEE73 与 RNF13 基因的遗传改变有关，该改变分别将 RNF13 蛋白中的第 311 或 312 位的亮氨酸转换为丝氨酸或脯氨酸（L311S 和 L312P）。本研究采用荧光显微镜方法研究 RNF13 蛋白变异体影响的分子和细胞机制，结果表明野生型 RNF13 广泛定位于内体和溶酶体，而 L311S 和 L312P 则不能与溶酶体标记物 Lamp1 广泛共定位。我们的结果表明，RNF13 L311S 和 L312P 蛋白会影响内体囊泡的大小，以及表皮生长因子在内涵体系统中的荧光标记的时空进展，但对转铁蛋白没有影响。此外，GST 下拉和免疫共沉淀表明，RNF13 变体破坏了与 AP-3 复合物的结合。AP-3 复合物亚基 AP3D1 的敲低改变了野生型 RNF13 的溶酶体定位，并同样影响了内体囊泡的大小。重要的是，我们的研究为理解由 RNF13 相关基因突变引起的 DEE73 改变的细胞和分子机制提供了第一步。
