Spivak Igor, Guldiken Nurdan, Usachov Valentyn, Schaap Frank, Damink Steven W M Olde, Bouchecareilh Marion, Lehmann Alexandra, Fu Lei, Mo Fa-Rong, Ensari Gökce Kobazi, Hufnagel Franziska, Fromme Malin, Preisinger Christian, Strnad Pavel
Medical Department III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Aachen, Germany.
Department of Surgery, Maastricht University Medical Center and NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, Netherlands.
Liver Int. 2025 Jan;45(1):e16207. doi: 10.1111/liv.16207.
The homozygous PiZ mutation (PIZZ genotype) constitutes the predominant cause of severe alpha-1 antitrypsin (AAT) deficiency and leads to liver disease via hepatocellular AAT aggregation. We systematically analysed the composition of AAT aggregates and studied the impact of bile acids.
AAT inclusions were isolated from livers of PiZ overexpressing mice and PIZZ humans via fluorescence-activated and immunomagnetic sorting (FACS/MACS), while insoluble proteins were obtained via Triton-X extraction. Inclusion composition was evaluated through mass-spectrometry (MS), immunoblotting and immunostaining. Hepatocytes with versus without AAT aggregates were obtained via microdissection. Serum bile acids were assessed in 57 PIZZ subjects and 19 controls. Mice were administered 2% cholic acid (CA)-supplemented chow for 7 days.
MS identified the key endoplasmic reticulum chaperone 78 kDa glucose-regulated protein (GRP78) in FACS/MACS pulldowns. GRP78 was also enriched in insoluble fractions from PiZ mice versus wild types and detected in insoluble fractions/MACS isolates from PIZZ liver explants. In cultured cells/primary hepatocytes, PiZ overexpression was associated with increased GRP78 mRNA/protein levels. In human livers, hepatocytes with AAT aggregates had higher GRP78 levels than hepatocytes without. PIZZ subjects displayed higher serum bile acid levels than controls and the highest levels were seen in individuals with liver injury/fibrosis. In PiZ mice, CA-mediated bile acid challenge resulted in increased liver injury and translocation of GRP78 into the aggregates.
Our results demonstrate that GRP78 is sequestered within AAT inclusions. Bile acid accumulation, as seen in PIZZ subjects with liver disease, may promote GRP78 segregation and thereby augment liver damage.
NCT02929940.
纯合子PiZ突变（PIZZ基因型）是严重α1抗胰蛋白酶（AAT）缺乏的主要原因，通过肝细胞AAT聚集导致肝脏疾病。我们系统分析了AAT聚集体的组成，并研究了胆汁酸的影响。
通过荧光激活和免疫磁珠分选（FACS/MACS）从过表达PiZ的小鼠肝脏和PIZZ人类肝脏中分离AAT包涵体，同时通过Triton-X提取获得不溶性蛋白质。通过质谱（MS）、免疫印迹和免疫染色评估包涵体组成。通过显微切割获得有和没有AAT聚集体的肝细胞。在57名PIZZ受试者和19名对照中评估血清胆汁酸。给小鼠喂食添加2%胆酸（CA）的饲料7天。
MS在FACS/MACS下拉实验中鉴定出关键的内质网伴侣78 kDa葡萄糖调节蛋白（GRP78）。与野生型相比，GRP78在PiZ小鼠的不溶性组分中也富集，并在PIZZ肝外植体的不溶性组分/MACS分离物中检测到。在培养细胞/原代肝细胞中，PiZ过表达与GRP78 mRNA/蛋白水平升高相关。在人类肝脏中，有AAT聚集体的肝细胞比没有的肝细胞具有更高的GRP78水平。PIZZ受试者的血清胆汁酸水平高于对照组，在有肝损伤/纤维化的个体中水平最高。在PiZ小鼠中，CA介导的胆汁酸刺激导致肝损伤增加和GRP78向聚集体的转位。
我们的结果表明GRP78被隔离在AAT包涵体内。在患有肝脏疾病的PIZZ受试者中所见的胆汁酸积累可能促进GRP78分离，从而加剧肝损伤。
NCT02929940。
