Mitochondrial metabolism of pyruvate is essential for regulating glucose-stimulated insulin secretion.

It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in ß-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that ß-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 ß-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (KATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP(+) ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-ß-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.

Direct structural analysis of a single acyl carrier protein domain in fatty acid synthase from the fungus Saccharomyces cerevisiae.

Acyl carrier protein (ACP) is the work horse of polyketide (PKS) and fatty acid synthases (FAS) and acts as a substrate shuttling domain in these mega enzymes. In fungi, FAS forms a 2.6 MDa symmetric assembly with six identical copies of FAS1 and FAS2 polypeptides. However, ACP spatial distribution is not restricted by symmetry owing to the long and flexible loops that tether the shuttling domain to its corresponding FAS2 polypeptide. This symmetry breaking has hampered experimental investigation of substrate shuttling route in fungal FAS. Here, we develop a protein engineering and expression method to isolate asymmetric fungal FAS proteins containing odd numbers of ACP domains. Electron cryomicroscopy (cryoEM) observation of the engineered complex reveals a non-uniform distribution of the substrate shuttling domain relative to its corresponding FAS2 polypeptide at 2.9 Å resolution. This work lays the methodological foundation for experimental study of ACP shuttling route in fungi.

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.

Identification of triazenyl indoles as inhibitors of fungal fatty acid biosynthesis with broad-spectrum activity.

Rising drug resistance among pathogenic fungi, paired with a limited antifungal arsenal, poses an increasing threat to human health. To identify antifungal compounds, we screened the RIKEN natural product depository against representative isolates of four major human fungal pathogens. This screen identified NPD6433, a triazenyl indole with broad-spectrum activity against all screening strains, as well as the filamentous mold Aspergillus fumigatus. Mechanistic studies indicated that NPD6433 targets the enoyl reductase domain of fatty acid synthase 1 (Fas1), covalently inhibiting its flavin mononucleotide-dependent NADPH-oxidation activity and arresting essential fatty acid biosynthesis. Robust Fas1 inhibition kills Candida albicans, while sublethal inhibition impairs diverse virulence traits. At well-tolerated exposures, NPD6433 extended the lifespan of nematodes infected with azole-resistant C. albicans. Overall, identification of NPD6433 provides a tool with which to explore lipid homeostasis as a therapeutic target in pathogenic fungi and reveals a mechanism by which Fas1 function can be inhibited.

Steric occlusion regulates proximal interactions of acyl carrier protein domain in fungal fatty acid synthase.

The acyl carrier protein (ACP) domain shuttles substrates and reaction intermediates in type I fungal fatty acid synthases via transient protein-protein interactions. Here, using electron cryo-microscopy (cryoEM), we report the structure of a fungal FAS stalled at the dehydration reaction, which precedes the final enoyl reduction in the fatty acid biosynthesis cycle. This conformation revealed multiple contact sites between ACP and the dehydratase (DH) and enoyl reductase (ER) domains that occluded the ACP binding to the adjacent ER domain. Our data suggests a minimal path from the DH to the ER reaction site that requires minute changes in the coordinates of the structured N- and C- termini of the ACP domain.

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.