3D culture models to study pathophysiology of steatotic liver disease

Steatotic liver disease (SLD) refers to a spectrum of diseases caused by hepatic lipid accumulation. SLD has emerged as the leading cause of chronic liver disease worldwide. Despite this burden and many years, understanding the pathophysiology of this disease is challenging due to the inaccessibility to human liver specimens. Therefore, cell-based in vitro systems are widely used as models to investigate the pathophysiology of SLD. Culturing hepatic cells in monolayers causes the loss of their hepatocyte-specific phenotype and, consequently, tissue-specific function and architecture. Hence, three-dimensional (3D) culture models allow cells to mimic the in vivo microenvironment and spatial organization of the liver unit. The utilization of 3D in vitro models minimizes the drawbacks of two-dimensional (2D) cultures and aligns with the 3Rs principles to alleviate the number of in vivo experiments. This article provides an overview of liver 3D models highlighting advantages and limitations, and culminates by discussing their applications in pharmaceutical and biomedical research

Mitochondrial amidoxime-reducing component 1 p.Ala165Thr increases protein degradation mediated by the proteasome

Objective: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern with no effective and specific drug treatment available. The rs2642438 minor allele in mitochondrial amidoxime-reducing component 1 (MARC1) results in an aminoacidic substitution (p.Ala165Thr) and associates with protection against MASLD. However, the mechanisms behind this protective effect are unknown. In this study, we examined the consequences of this aminoacidic substitution on protein stability and subcellular localization. Methods: We overexpressed the human MARC1 A165 (wild-type) or 165T (mutant) in vivo in mice and in vitro in human hepatoma cells (HepG2 and HuH-7), generated several mutants at position 165 by in situ mutagenesis and then examined protein levels. We also generated HepG2 cells stably overexpressing MARC1 A165 or 165T to test the effect of this substitution on MARC1 subcellular localization. Results: MARC1 165T overexpression resulted in lower protein levels than A165 both in vivo and in vitro. Similarly, any mutant at position 165 showed lower protein levels compared to the wild-type protein. We showed that the 165T mutant protein is polyubiquitinated and its degradation is accelerated through lysine-48 ubiquitin-mediated proteasomal degradation. We also showed that the 165T substitution does not affect the MARC1 subcellular localization. Conclusions: This study shows that alanine at position 165 in MARC1 is crucial for protein stability, and the threonine substitution at this position leads to a hypomorphic protein variant due to lower protein levels. Our result supports the notion that lowering hepatic MARC1 protein level may be a successful therapeutic strategy for treating MASLD

Development of a Selective Dual Discoidin Domain Receptor DDR p38 Kinase Chemical Probe

Discoidin domain receptors 1 and 2 DDR1 2 play a central role in fibrotic disorders, such as renal and pulmonary fibrosis, atherosclerosis, and various forms of cancer. Potent and selective inhibitors, so called chemical probe compounds, have been developed to study DDR1 2 kinase signaling. However, these inhibitors showed undesired activity on other kinases such as the tyrosine protein kinase receptor TIE or tropomyosin receptor kinases, which are related to angiogenesis and neuronal toxicity. In this study, we optimized our recently published p38 mitogen activated protein kinase inhibitor 7 toward a potent and cell active dual DDR p38 chemical probe and developed a structurally related negative control. The structure guided design approach used provided insights into the P loop folding process of p38 and how targeting of non conserved amino acids modulates inhibitor selectivity. The developed and comprehensively characterized DDR p38 probe, 30 SR 302 , is a valuable tool for studying the role of DDR kinase in normal physiology and in disease developmen

Development of a selective dual discoidin domain receptor (DDR)/p38 kinase chemical probe

Discoidin domain receptors 1 and 2 (DDR1/2) play a central role in fibrotic disorders, such as renal and pulmonary fibrosis, atherosclerosis, and various forms of cancer. Potent and selective inhibitors, so-called chemical probe compounds, have been developed to study DDR1/2 kinase signaling. However, these inhibitors showed undesired activity on other kinases such as the tyrosine protein kinase receptor TIE or tropomyosin receptor kinases, which are related to angiogenesis and neuronal toxicity. In this study, we optimized our recently published p38 mitogen-activated protein kinase inhibitor 7 toward a potent and cell-active dual DDR/p38 chemical probe and developed a structurally related negative control. The structure-guided design approach used provided insights into the P-loop folding process of p38 and how targeting of non-conserved amino acids modulates inhibitor selectivity. The developed and comprehensively characterized DDR/p38 probe, 30 (SR-302), is a valuable tool for studying the role of DDR kinase in normal physiology and in disease development