ABSTRACT
Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world's population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.
ABSTRACT
Stimulator of interferon genes (STING) is a central component of the cytosolic nucleic acids sensing pathway and as such master regulator of the type I interferon response. Due to its critical role in physiology and its' involvement in a variety of diseases, STING has been a focus for drug discovery. Targeted protein degradation (TPD) has emerged as a promising pharmacology for targeting previously considered undruggable proteins by hijacking the cellular ubiquitin proteasome system (UPS) with small molecules. Here, we identify AK59 as a STING degrader leveraging HERC4, a HECT-domain E3 ligase. Additionally, our data reveals that AK59 is effective on the common pathological STING mutations, suggesting a potential clinical application of this mechanism. Thus, these findings introduce HERC4 to the fields of TPD and of compound-induced degradation of STING, suggesting potential therapeutic applications.
Subject(s)
Membrane Proteins , Proteolysis , Ubiquitin-Protein Ligases , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Humans , Membrane Proteins/metabolism , Membrane Proteins/genetics , Proteolysis/drug effects , HEK293 Cells , Animals , Mutation , Proteasome Endopeptidase Complex/metabolism , UbiquitinationABSTRACT
BACKGROUND & AIMS: In the classic form of α1-antitrypsin deficiency (ATD), the misfolded α1-antitrypsin Z (ATZ) variant accumulates in the endoplasmic reticulum (ER) of liver cells. A gain-of-function proteotoxic mechanism is responsible for chronic liver disease in a subgroup of homozygotes. Proteostatic response pathways, including conventional endoplasmic reticulum-associated degradation and autophagy, have been proposed as the mechanisms that allow cellular adaptation and presumably protection from the liver disease phenotype. Recent studies have concluded that a distinct lysosomal pathway called endoplasmic reticulum-to-lysosome completely supplants the role of the conventional macroautophagy pathway in degradation of ATZ. Here, we used several state-of-the-art approaches to characterize the proteostatic responses more fully in cellular systems that model ATD. METHODS: We used clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing coupled to a cell selection step by fluorescence-activated cell sorter to perform screening for proteostasis genes that regulate ATZ accumulation and combined that with selective genome editing in 2 other model systems. RESULTS: Endoplasmic reticulum-associated degradation genes are key early regulators and multiple autophagy genes, from classic as well as from ER-to-lysosome and other newly described ER-phagy pathways, participate in degradation of ATZ in a manner that is temporally regulated and evolves as ATZ accumulation persists. Time-dependent changes in gene expression are accompanied by specific ultrastructural changes including dilation of the ER, formation of globular inclusions, budding of autophagic vesicles, and alterations in the overall shape and component parts of mitochondria. CONCLUSIONS: Macroautophagy is a critical component of the proteostasis response to cellular ATZ accumulation and it becomes more important over time as ATZ synthesis continues unabated. Multiple subtypes of macroautophagy and nonautophagic lysosomal degradative pathways are needed to respond to the high concentrations of misfolded protein that characterizes ATD and these pathways are attractive candidates for genetic variants that predispose to the hepatic phenotype.