CREG1 promotes lysosomal biogenesis and function

CREG1 is a small glycoprotein which has been proposed as a transcription repressor, a secretory ligand, a lysosomal, or a mitochondrial protein. This is largely because of lack of antibodies for immunolocalization validated through gain- and loss-of-function studies. In the present study, we demonstrate, using antibodies validated for immunofluorescence microscopy, that CREG1 is mainly localized to the endosomal-lysosomal compartment. Gain- and loss-of-function analyses reveal an important role for CREG1 in both macropinocytosis and clathrin-dependent endocytosis. CREG1 also promotes acidification of the endosomal-lysosomal compartment and increases lysosomal biogenesis. Functionally, overexpression of CREG1 enhances macroautophagy/autophagy and lysosome-mediated degradation, whereas knockdown or knockout of CREG1 has opposite effects. The function of CREG1 in lysosomal biogenesis is likely attributable to enhanced endocytic trafficking. Our results demonstrate that CREG1 is an endosomal-lysosomal protein implicated in endocytic trafficking and lysosomal biogenesis. <b>Abbreviations:</b> AIFM1/AIF: apoptosis inducing factor mitochondria associated 1; AO: acridine orange; ATP6V1H: ATPase H+ transporting V1 subunit H; CALR: calreticulin; CREG: cellular repressor of E1A stimulated genes; CTSC: cathepsin C; CTSD: cathepsin D; EBAG9/RCAS1: estrogen receptor binding site associated antigen 9; EIPA: 5-(N-ethyl-N-isopropyl)amiloride; ER: endoplasmic reticulum; GFP: green fluorescent protein; HEXA: hexosaminidase subunit alpha; IGF2R: insulin like growth factor 2 receptor; LAMP1: lysosomal associated membrane protein 1; M6PR: mannose-6-phosphate receptor, cation dependent; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; PDIA2: protein disulfide isomerase family A member 2; SQSTM1/p62: sequestosome 1; TF: transferrin; TFEB: transcription factor EB

CREG1是一种小型糖蛋白（glycoprotein），曾被提出可作为转录抑制子、分泌配体、溶酶体或线粒体蛋白。这一观点的提出主要源于缺乏通过功能获得与功能丧失实验验证的免疫定位抗体。在本研究中，我们使用经免疫荧光显微镜验证的抗体，证实CREG1主要定位于内体-溶酶体区室。功能获得与功能丧失分析显示，CREG1在巨胞饮作用和网格蛋白依赖的内吞作用中均发挥重要调控作用。CREG1还可促进内体-溶酶体区室的酸化，并增加溶酶体的生物发生。在功能层面，过表达CREG1可增强巨自噬/自噬及溶酶体介导的降解过程，而敲低或敲除CREG1则会产生相反的效应。CREG1在溶酶体生物发生中的功能，可能源于其增强的内吞运输能力。本研究结果证实，CREG1是一种参与内吞运输与溶酶体生物发生的内体-溶酶体蛋白。**缩写：** AIFM1/AIF：凋亡诱导因子线粒体相关1（apoptosis inducing factor mitochondria associated 1）；AO：吖啶橙（acridine orange）；ATP6V1H：ATP酶H+转运V1亚基H（ATPase H+ transporting V1 subunit H）；CALR：钙网蛋白（calreticulin）；CREG：E1A刺激基因细胞抑制因子（cellular repressor of E1A stimulated genes）；CTSC：组织蛋白酶C（cathepsin C）；CTSD：组织蛋白酶D（cathepsin D）；EBAG9/RCAS1：雌激素受体结合位点相关抗原9（estrogen receptor binding site associated antigen 9）；EIPA：5-(N-乙基-N-异丙基)阿米洛利（5-(N-ethyl-N-isopropyl)amiloride）；ER：内质网（endoplasmic reticulum）；GFP：绿色荧光蛋白（green fluorescent protein）；HEXA：己糖胺酶亚基α（hexosaminidase subunit alpha）；IGF2R：胰岛素样生长因子2受体（insulin like growth factor 2 receptor）；LAMP1：溶酶体相关膜蛋白1（lysosomal associated membrane protein 1）；M6PR：甘露糖-6-磷酸受体（阳离子依赖性）（mannose-6-phosphate receptor, cation dependent）；MAPK1/ERK2：丝裂原活化蛋白激酶1（mitogen-activated protein kinase 1）；MTORC1：雷帕霉素激酶复合物1（mechanistic target of rapamycin kinase complex 1）；PDIA2：蛋白质二硫键异构酶家族A成员2（protein disulfide isomerase family A member 2）；SQSTM1/p62：隔离体1（sequestosome 1）；TF：转铁蛋白（transferrin）；TFEB：转录因子EB（transcription factor EB）