CTSD/CathD/CATD（组织蛋白酶 D）是一种溶酶体天冬氨酸蛋白酶。 CTSD的一个显着特征是其双重功能，即通过在细胞外分泌酶原作为配体促进细胞增殖，并通过细胞内该酶的成熟形式促进细胞凋亡；然而，其分泌、表达和成熟的调节尚未确定。以严重农业害虫鳞翅目昆虫棉铃虫为模型，揭示了CTSD分泌、表达和成熟的双重功能和调控机制。天冬酰胺 233 (N233) 的糖基化决定了前 CTSD 的分泌。类固醇激素 20-羟基蜕皮酮 (20E) 促进 CTSD 表达。巨自噬/自噬触发 CTSD 成熟并在中肠细胞内定位，从而激活 CASP3 (caspase 3) 并促进细胞凋亡。 Pro-CTSD在蛹表皮中表达，并分泌到血淋巴中，促进成体脂肪体内复制/内复制、细胞增殖和缔合。我们的研究表明，组织中 CTSD 的差异表达和自噬介导的成熟决定了其在细胞凋亡和细胞增殖中的作用，从而决定了鳞翅目变态过程中组织的细胞命运。缩写：20E：20-羟基蜕皮酮； 3-MA：3-甲基腺嘌呤； ACTB/β-肌动蛋白：肌动蛋白β； AKT：蛋白激酶B； ATG1：自噬相关1； ATG4：自噬相关4； ATG5：自噬相关5； ATG7：自噬相关7； ATG14：自噬相关14； BSA：牛血清白蛋白； CASP3: 半胱天冬酶 3; CQ：氯喹； CTSD：组织蛋白酶D； DAPI：4',6-二脒基-2-苯基吲哚； DMSO：二甲亚砜； DPBS：杜尔贝科磷酸盐缓冲盐水； DsRNA：双链RNA； EcR：蜕皮激素受体； EcRE：蜕皮激素反应元件； EdU：5-乙炔基-2'-脱氧尿苷； Gm-CTSD：糖基化-mautre-CTSD； G-pro-CTSD: 糖基化-pro-CTSD； HaEpi：棉铃虫表皮细胞系； HE染色：苏木精伊红染色； IgG：免疫球蛋白G； IM：想象中肠； JH：保幼激素； Kr-h1：克鲁佩尔同源蛋白1； LM：幼虫中肠； M6P：6-磷酸甘露糖； PBS：磷酸盐缓冲盐水； PCD：程序性细胞死亡； PNGase：肽-N-糖苷酶F； RFP：红色荧光蛋白； RNAi：RNA干扰； SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳； SYX17: 语法蛋白 17; USP1：超气门亚型 1。
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Autophagy triggers CTSD (cathepsin D) maturation and localization inside cells to promote apoptosis.
CTSD/CathD/CATD (cathepsin D) is a lysosomal aspartic protease. A distinguishing characteristic of CTSD is its dual functions of promoting cell proliferation via secreting a pro-enzyme outside the cells as a ligand, and promoting apoptosis via the mature form of this enzyme inside cells; however, the regulation of its secretion, expression, and maturation is undetermined. Using the lepidopteran insect Helicoverpa armigera, a serious agricultural pest, as a model, we revealed the dual functions and regulatory mechanisms of CTSD secretion, expression, and maturation. Glycosylation of asparagine 233 (N233) determined pro-CTSD secretion. The steroid hormone 20-hydroxyecdysone (20E) promoted CTSD expression. Macroautophagy/autophagy triggered CTSD maturation and localization inside midgut cells to activate CASP3 (caspase 3) and promote apoptosis. Pro-CTSD was expressed in the pupal epidermis and was secreted into the hemolymph to promote adult fat body endoreplication/endoreduplication, cell proliferation, and association. Our study revealed that the differential expression and autophagy-mediated maturation of CTSD in tissues determine its roles in apoptosis and cell proliferation, thereby determining the cell fates of tissues during lepidopteran metamorphosis.Abbreviations: 20E: 20-hydroxyecdysone; 3-MA: 3-methyladenine; ACTB/β-actin: actin beta; AKT: protein kinase B; ATG1: autophagy-related 1; ATG4: autophagy-related 4; ATG5: autophagy-related 5; ATG7: autophagy-related 7; ATG14: autophagy-related 14; BSA: bovine serum albumin; CASP3: caspase 3; CQ: choroquine; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; DPBS: dulbecco's phosphate-buffered saline; DsRNA: double-stranded RNA; EcR: ecdysone receptor; EcRE: ecdysone response element; EdU: 5-ethynyl-2´-deoxyuridine; G-m-CTSD: glycosylated-mautre-CTSD; G-pro-CTSD: glycosylated-pro-CTSD; HaEpi: Helicoverpa armigera epidermal cell line; HE staining: hematoxylin and eosin staining; IgG: immunoglobin G; IM: imaginal midgut; JH: juvenile hormone; Kr-h1: krueppel homologous protein 1; LM: larval midgut; M6P: mannose-6-phosphate; PBS: phosphate-buffered saline; PCD: programmed cell death; PNGase: peptide-N-glycosidase F; RFP: red fluorescent protein; RNAi: RNA interference; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SYX17: syntaxin 17; USP1: ultraspiracle isoform 1.
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