Chu, L. M. | bioRxiv Science

bioRxiv2024

Direct expression of CPT1a enables a high throughput platform for the discovery of CPT1a modulators

Carnitine palmitoyltransferase 1 (CPT1), which catalyzes the rate-limiting step of fatty acid oxidation, has been implicated in therapeutic approaches to several human diseases characterized by aberrant lipid metabolism. Isoform-specific quantification of CPT1 activity is essential in the characterization of small molecule inhibitors of CPT1, but several existing means to quantify enzymatic activity, including the use of radioisotope labeled carnitine, are not amenable to scalable, high throughput screening. Here, we demonstrate that mitochondrial extracts from Expi293 cells transfected with a CPT1a plasmid are a reliable and robust source of catalytically active human CPT1. Moreover, with a source of catalytically active enzyme in hand, we modified a previously reported colorimetric method of coenzyme A (CoA) easily scalable to a 96-well format for the screening of CPT1a inhibitors. This assay platform was validated by two previously reported inhibitors of CPT1a: R-etomoxir and perhexiline. To further demonstrate the applicability of this method in small molecule screening, we prepared and screened a library of 87 known small molecule APIs, validating the inhibitory effect of chlorpromazine on CPT1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/628575v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f69f0corg.highwire.dtl.DTLVardef@43eb95org.highwire.dtl.DTLVardef@11fa21corg.highwire.dtl.DTLVardef@1cbe405_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Chromosomal-level reference genome of the incense tree Aquilaria sinensis

Trees in the genus Aquilaria (Thymelaeaceae) are known as lign aloes, and are native to the forests of southeast Asia. Lign aloes produce agarwood as an antimicrobial defence. Agarwood has a long history of cultural and medicinal use, and is of considerable commercial value. However, due to habitat destruction and over collection, lign aloes are threatened in the wild. We present a chromosomal-level assembly for Aquilaria sinensis, a lign aloe endemic to China known as the incense tree, based on Illumina short-read, 10X Genomics linked-read, and Hi-C sequencing data. Our 783.8Mbp A. sinensis genome assembly is of high physical contiguity, with a scaffold N50 of 87.6Mbp, and high completeness, with a 95.8% BUSCO score for eudicotyledon genes. We include 17 transcriptomes from various plant tissues, providing a total of 35,965 gene models. We reveal the first complete set of genes involved in sesquiterpenoid production, plant defence, and agarwood production for the genus Aquilaria, including genes involved in the biosynthesis of sesquiterpenoids via the mevalonic acid (MVA), 1-deoxy-D-xylulose-5-phosphate (DXP), and methylerythritol phosphate (MEP) pathways. We perform a detailed repeat content analysis, revealing that transposable elements account for [~]61% of the genome, with major contributions from gypsy-like and copia-like LTR retroelements. We also provide a comparative analysis of repeat content across sequenced species in the order Malvales. Our study reveals the first chromosomal-level genome assembly for a tree in the genus Aquilaria and provides an unprecedented opportunity to address a variety of applied, genomic and evolutionary questions in the Thymelaeaceae more widely.

genomics↗