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.

Pseudopeptide Amyloid Aggregation Inhibitors: In Silico, Single Molecule and Cell Viability Studies.

Neurodegeneration in Alzheimer's disease (AD) is defined by pathology featuring amyloid-ß (Aß) deposition in the brain. Aß monomers themselves are generally considered to be nontoxic, but misfold into ß-sheets and aggregate to form neurotoxic oligomers. One suggested strategy to treat AD is to prevent the formation of toxic oligomers. The SG inhibitors are a class of pseudopeptides designed and optimized using molecular dynamics (MD) simulations for affinity to Aß and experimentally validated for their ability to inhibit amyloid-amyloid binding using single molecule force spectroscopy (SMFS). In this work, we provide a review of our previous MD and SMFS studies of these inhibitors and present new cell viability studies that demonstrate their neuroprotective effects against Aß(1-42) oligomers using mouse hippocampal-derived HT22 cells. Two of the tested SG inhibitors, predicted to bind Aß in anti-parallel orientation, demonstrated neuroprotection against Aß(1-42). A third inhibitor, predicted to bind parallel to Aß, was not neuroprotective. Myristoylation of SG inhibitors, intended to enhance delivery across the blood-brain barrier (BBB), resulted in cytotoxicity. This is the first use of HT22 cells for the study of peptide aggregation inhibitors. Overall, this work will inform the future development of peptide aggregation inhibitors against Aß toxicity.

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.

Intrapulpal Temperature Rise During Light Activation of Restorative Composites in a Primary Molar.

PURPOSE: To investigate intrapulpal temperature rise in a primary molar during light activation of a composite restoration to determine if clinically significant pulpal temperatures (greater than 5.5 degrees Celsius) were reached. METHODS: Restorative composites (EsthetX HD, Filtek Supreme Ultra, Filtek Bulk Fill) were placed into a primary molar with occlusal preparation (1.5 mm depth; remaining pulpal floor thickness one mm). The pulp was extirpated through a root access to place a thermocouple against the pulpal roof. Temperature changes were recorded during composite restoration light polymerization with three curing lights (one quartz-tungsten-halogen, two LEDs). Sample size was 10. Samples received additional irradiation to assure complete polymerization, followed by a third irradiation for calculating the exothermic heat contribution (subtracting third irradiation temperatures from first irradiation temperatures). Cured restorations were removed after each test, and the tooth was reused. Results were analyzed with Kruskal-Wallis (α =0.05). RESULTS: Type of curing light and composite material affected the intrapulpal temperature rise, which was up to five degrees Celsius for one combination of LED-composite. CONCLUSIONS: Clinicians should be aware of the potential for clinically significant intrapulpal temperature rises when light-activating composite restorations in a primary molar with a moderately deep cavity.

A poster and a mobile healthcare application as information tools for dental trauma management.

BACKGROUND/AIMS: Prompt management of dental trauma in children affects outcomes, and multiple educational resources are available. The aim of this study was to compare subjects' accuracy in answering a survey about dental trauma management utilizing a poster and a mobile healthcare application and to determine user preference for mode of delivery of information. MATERIALS AND METHODS: A survey was administered to parents of patients in two pediatric dental practices. Questions collected demographic information, frequency of internet use, and responses to questions regarding dental trauma management for two separate scenarios. Participants used both a poster and a mobile application, but were randomly assigned as to which tool was utilized first. RESULTS: Eighty-nine surveys were usable. The majority of respondents were aged 36-45 years (50.6%), had education beyond high school (64%), and had private insurance (52.8%). Less-educated individuals were more likely to report searching the Internet (74%) compared to individuals with a graduate degree (57%) (P = 0.017). The majority of subjects answered trauma management questions correctly with both tools. However, for an avulsed permanent tooth, individuals receiving the mobile application were more likely to select: 'put the tooth back in place' (71.1%) compared to those utilizing the poster, who chose 'put the tooth in milk' (56.8%) (P = 0.004). Less-educated individuals were willing to pay more for the application (P = 0.015) and were more likely to report being interested in receiving dental information through mobile technology in the future (P = 0.006). CONCLUSIONS: Both a poster and a mobile healthcare application are effective in delivering dental trauma information.

Comparison of Amount of Primary Tooth Reduction Required for Anterior and Posterior Zirconia and Stainless Steel Crowns.

PURPOSE: To determine if aggressiveness of primary tooth preparation varied among different brands of zirconia and stainless steel (SSC) crowns. METHODS: One hundred primary typodont teeth were divided into five groups (10 posterior and 10 anterior) and assigned to: Cheng Crowns (CC); EZ Pedo (EZP); Kinder Krowns (KKZ); NuSmile (NSZ); and SSC. Teeth were prepared, and assigned crowns were fitted. Teeth were weighed prior to and after preparation. Weight changes served as a surrogate measure of tooth reduction. RESULTS: Analysis of variance showed a significant difference in tooth reduction among brand/type for both the anterior and posterior. Tukey's honest significant difference test (HSD), when applied to anterior data, revealed that SSCs required significantly less tooth removal compared to the composite of the four zirconia brands, which showed no significant difference among them. Tukey's HSD test, applied to posterior data, revealed that CC required significantly greater removal of crown structure, while EZP, KKZ, and NSZ were statistically equivalent, and SSCs required significantly less removal. CONCLUSIONS: Zirconia crowns required more tooth reduction than stainless steel crowns for primary anterior and posterior teeth. Tooth reduction for anterior zirconia crowns was equivalent among brands. For posterior teeth, reduction for three brands (EZ Pedo, Kinder Krowns, NuSmile) did not differ, while Cheng Crowns required more reduction.

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.