Atomic model for core modifying region of human fatty acid synthase in complex with Denifanstat.

Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a 16-carbon chain fatty acid that is the primary precursor of lipid metabolism and an important intracellular signaling molecule. FASN is an attractive drug target in diabetes, cancer, fatty liver diseases, and viral infections. Here, we develop an engineered full-length human FASN (hFASN) that enables isolation of the condensing and modifying regions of the protein post-translation. The engineered protein enables electron cryo-microscopy (cryoEM) structure determination of the core modifying region of hFASN to 2.7 Å resolution. Examination of the dehydratase dimer within this region reveals that unlike its close homolog, porcine FASN, the catalytic cavity is close-ended and is accessible only through one opening in the vicinity of the active site. The core modifying region exhibits two major global conformational variabilities that describe long-range bending and twisting motions of the complex in solution. Finally, we solved the structure of this region bound to an anti-cancer drug, Denifanstat (i.e., TVB-2640), demonstrating the utility of our approach as a platform for structure guided design of future hFASN small molecule inhibitors.

Impact of demographic, environmental, socioeconomic, and government intervention on the spreading of COVID-19.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is a worldwide epidemiological emergency, and the risk factors for the multiple waves with new COVID-19 strains are concerning. This study aims to identify the most significant risk factors for spreading COVID-19 to help policymakers take early measures for the next waves. METHODS: We conducted the study on randomly selected 29 countries where the pandemic had a downward trend in the daily active cases curve as of June 10, 2020. We investigated the association with the standardized spreading index and demographical, environmental, socioeconomic, and government intervention. To standardize the spreading index, we accounted for the number of tests and the timeline bias. Furthermore, we performed multiple linear regression to identify the relative importance of the variables. RESULTS: In the correlation analysis, air pollution, PM2.5 (r = 0.37, p = 0.0466), number of days to impose lockdown from first case (r = 0.38, p = 0.0424) and total confirmed cases on the first lockdown (r = 0.61, p = 0.0004) were associated with outcome measures. In the adjusted model, air pollution ( ß 1  = 4.5, p = 0.0127, |t| = 3.1) and overweight prevalence ( ß 1  = 4.7, p = 0.0187, |t| = 2.9) were the most significant exposure variable for spreading of COVID-19. CONCLUSION: Our findings showed that countries with larger PM2.5 values and comparatively more overweight populations are at higher risk of spreading COVID-19. Proper preventive measures may reduce the spreading.

Structural Characterization of Endogenous Tuberous Sclerosis Protein Complex Revealed Potential Polymeric Assembly.

Tuberous sclerosis protein complex (pTSC) nucleates a proteinaceous signaling hub that integrates information about the internal and external energy status of the cell in the regulation of growth and energy consumption. Biochemical and cryo-electron microscopy studies of recombinant pTSC have revealed its structure and stoichiometry and hinted at the possibility that the complex may form large oligomers. Here, we have partially purified endogenous pTSC from fasted mammalian brains of rat and pig by leveraging a recombinant antigen binding fragment (Fab) specific for the TSC2 subunit of pTSC. We demonstrate Fab-dependent purification of pTSC from membrane-solubilized fractions of the brain homogenates. Negative stain electron microscopy of the samples purified from pig brain demonstrates rod-shaped protein particles with a width of 10 nm, a variable length as small as 40 nm, and a high degree of conformational flexibility. Larger filaments are evident with a similar 10 nm width and a ≤1 µm length in linear and weblike organizations prepared from pig brain. Immunogold labeling experiments demonstrate linear aggregates of pTSC purified from mammalian brains. These observations suggest polymerization of endogenous pTSC into filamentous superstructures.

Structure of the murine lysosomal multienzyme complex core.

The enzymes ß-galactosidase (GLB1) and neuraminidase 1 (NEU1; sialidase 1) participate in the degradation of glycoproteins and glycolipids in the lysosome. To remain active and stable, they associate with PPCA [protective protein cathepsin A (CTSA)] into a high-molecular weight lysosomal multienzyme complex (LMC), of which several forms exist. Genetic defects in these three proteins cause the lysosomal storage diseases GM1-gangliosidosis/mucopolysaccharidosis IV type B, sialidosis, and galactosialidosis, respectively. To better understand the interactions between these enzymes, we determined the three-dimensional structure of the murine LMC core. This 0.8-MDa complex is composed of three GLB1 dimers and three CTSA dimers, adopting a triangular architecture maintained through six copies of a unique GLB1-CTSA polar interface. Mutations in this contact surface that occur in GM1-gangliosidosis prevent formation of the LMC in vitro. These findings may facilitate development of therapies for lysosomal storage disorders.

Electron cryomicroscopy observation of acyl carrier protein translocation in type I fungal fatty acid synthase.

During fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.