Cross-regulation of defective endolysosome trafficking and enhanced autophagy through TFEB in UNC13D deficiency

Several lines of evidence support the occurrence of cross-regulation between the endocytic pathway and autophagy, but the molecular mechanisms regulating this process are not well-understood. Here, we show that the calcium sensor UNC13D regulates the molecular mechanism of late endosomal trafficking and endosomal maturation, and defects in UNC13D lead to macroautophagy upregulation. <i>unc13d</i>-null cells showed impaired endosomal trafficking and defective endocytic flux. The defective phenotypes were rescued by the expression of UNC13D but not by its STX7-binding-deficient mutant. This defective endosomal function in UNC13D-deficient cells resulted in increased autophagic flux, increased long-lived protein degradation, decreased SQSTM1/p62 protein levels and increased autolysosome formation as determined by biochemical, microscopy and structural methods. The autophagic phenotype was not associated with increased recruitment of the UNC13D-binding proteins and autophagy regulators, RAB11 or VAMP8, but was caused, at least in part, by TFEB-mediated upregulation of a subset of autophagic and lysosomal genes, including <i>Atg9b</i>. Downregulation of TFEB decreased <i>Atg9b</i> levels and decreased macroautophagy in <i>unc13d</i>-null cells. UNC13D upregulation corrected the defects in endolysosomal trafficking and decreased the number of accumulated autophagosomes in a cellular model of the lysosomal-storage disorder cystinosis, under both fed and starvation conditions, identifying UNC13D as an important new regulatory molecule of autophagy regulation in cells with lysosomal disorders. ACTB: actin, beta; CTSB: cathepsin B; EEA1: early endosome antigen 1; ESCRT: endosomal sorting complex required for transport; FHL3: familial hemophagocytic; lymphohistiocytosis type 3; HEX: hexosaminidase; HLH: hemophagocytic lymphohistiocytosis; LSD: lysosomal storage disorder; MEF: mouse embryonic fibroblast; SEM: standard errors of the mean; SNARE: soluble n-ethylmaleimide-sensitive-factor attachment receptor; STX: syntaxin; SYT7: synaptotagmin VII; TFE3: transcription factor E3; TFEB: transcription factor EB; TIRF: total internal reflection fluorescence ULK1: unc-51 like kinase 1; UNC13D: unc-13 homolog d; VAMP: vesicle-associate membrane protein; WT: wild-type

多项证据表明内吞途径与自噬之间存在交叉调控，但调控这一过程的分子机制尚未完全阐明。本研究发现，钙感受器UNC13D（UNC-13同源蛋白D）可调控晚期内体运输与内体成熟的分子机制，且UNC13D功能缺陷会导致巨自噬（macroautophagy）上调。UNC13D敲除（unc13d-null）细胞表现出内体运输受损、内吞通量异常。上述缺陷表型可通过过表达UNC13D得以挽救，但无法通过其STX7结合缺陷突变体实现挽救。生化、显微镜及结构生物学分析显示，UNC13D缺陷细胞的内体功能异常会引发自噬通量升高、长寿命蛋白质降解增强、SQSTM1/p62蛋白水平降低，以及自噬溶酶体形成增加。该自噬表型并非由UNC13D结合蛋白与自噬调控因子RAB11或VAMP8的招募增加所介导，而是至少部分通过TFEB（转录因子EB）介导的部分自噬与溶酶体基因（包括Atg9b）的上调所导致。下调TFEB的表达可降低UNC13D敲除细胞中Atg9b的水平，并减弱巨自噬水平。在营养充足与饥饿条件下，过表达UNC13D均可纠正溶酶体贮积症（lysosomal storage disorder, LSD）胱氨酸病（cystinosis）细胞模型中的内体溶酶体运输缺陷，并减少积累的自噬体数量，证实UNC13D是溶酶体疾病细胞中自噬调控的重要新型调控分子。以下为相关术语注释：ACTB：肌动蛋白β（actin, beta）；CTSB：组织蛋白酶B（cathepsin B）；EEA1：早期内体抗原1（early endosome antigen 1）；ESCRT：内体分选复合物所需转运蛋白（endosomal sorting complex required for transport）；FHL3：家族性噬血细胞性淋巴组织细胞增生症3型（familial hemophagocytic lymphohistiocytosis type 3）；HEX：己糖胺酶（hexosaminidase）；HLH：噬血细胞性淋巴组织细胞增生症（hemophagocytic lymphohistiocytosis）；LSD：溶酶体贮积症（lysosomal storage disorder）；MEF：小鼠胚胎成纤维细胞（mouse embryonic fibroblast）；SEM：均值标准误（standard errors of the mean）；SNARE：可溶性N-乙基马来酰亚胺敏感因子附着受体（soluble n-ethylmaleimide-sensitive-factor attachment receptor）；STX：突触融合蛋白（syntaxin）；SYT7：突触结合蛋白VII（synaptotagmin VII）；TFE3：转录因子E3（transcription factor E3）；TFEB：转录因子EB（transcription factor EB）；TIRF：全内反射荧光成像（total internal reflection fluorescence）；ULK1：UNC-51样激酶1（unc-51 like kinase 1）；UNC13D：UNC-13同源蛋白D（unc-13 homolog d）；VAMP：囊泡相关膜蛋白（vesicle-associated membrane protein）；WT：野生型（wild-type）