Screening of CPT1A-Targeting Lipid Metabolism Modulators Using Mitochondrial Membrane Chromatography

Abstract

Carnitine palmitoyltransferase 1A (CPT1A), which resides on the mitochondrial outer membrane, serves as the rate-limiting enzyme of fatty acid β-oxidation. Identifying the compounds targeting CPT1A warrants a promising candidate for modulating lipid metabolism. In this study, we developed a CPT1A-overexpressed mitochondrial membrane chromatography (MMC) to screen the compounds with affinity for CPT1A. Cells overexpressing CPT1A were cultured, and subsequently, their mitochondrial membrane was isolated and immobilized on amino-silica gel cross-linked by glutaraldehyde. After packing the mitochondrial membrane column, retention components of MMC were performed with LC/MS, whose analytic peaks provided structural information on compounds that might interact with mitochondrial membrane proteins. With the newly developed MMC−LC/MS approach, several Chinese traditional medicine extracts, such as Scutellariae Radix and Polygoni Cuspidati Rhizoma et Radix (PCRR), were analyzed. Five noteworthy compounds, baicalin, baicalein, wogonoside, wogonin, and resveratrol, were identified as enhancers of CPT1A enzyme activity, with resveratrol being a new agonist for CPT1A. The study suggests that MMC serves as a reliable screening system for efficiently identifying modulators targeting CPT1A from complex extracts.

Twin Strep-Tag Modified CPT1A Mitochondrial Membrane Chromatography in Screening Lipid Metabolism Regulators

“…Based on our previously reported method, the Twin Strep-Tag CPT1A/MMSP was characterized using immunofluorescence (IF). The surface morphology of the stationary phase was characterized using the scanning electron microscopy (SEM) instrument.…”

“…The specificity of the Twin Strep-Tag CPT1A/MMC column was investigated by examining the retention behaviors of baicalin on the Twin Strep-Tag CPT1A/MMC column, blank SiO 2 –Strep Tactin column, and NC-HEK293/MMC column, respectively. Concurrently, based on our previously reported method, we prepared an MMC column targeting CPT1A by immobilizing the CPT1A membrane protein onto glutaraldehyde-modified silica (GD-CPT1A/MMC). Intrareproducibility was assessed using the relative standard deviation (RSD%) of retention times obtained from six consecutive injections of baicalin in the same column.…”

“…Dysfunction of mitochondria leads to severe diseases, and treatments with drugs and nutrients targeting mitochondrial membrane proteins have been proven to be an effective solution for disease control and therapy. , Therefore, screening mitochondrial targeting drugs or effectors is of great significance. Mitochondrial membrane chromatography (MMC) , stands out as a bioaffinity chromatography technique for studying receptor–ligand interactions and screening effector molecules targeting mitochondria. MMC excels in preserving the integrity of the mitochondrial membrane, the surrounding environment, and the three-dimensional structure of membrane receptors to the fullest extent.…”

Screening and analysis of the targeted compounds in Choerospondias axillaris extract by receptor chromatographic column with immobilized angiotensin II type 1 receptor

“…In recent years, high-performance affinity chromatography has been confirmed as an efficient method and has gained popularity in the field of bioactive compound screening. The stationary phase used in these applications is composed of immobilized receptors, serum albumin, enzymes, cell membranes, and nucleic acids (Li and Hage, 2017;Sun et al, 2019;Li et al, 2011;Ji et al, 2023;Su et al, 2024). Benefiting from high specificity and stability, immobilized receptor chromatography is considered the most robust and powerful method for investigating binding interactions between drugs and receptors.…”