N-Acyltransferases and Fatty Acid Amides
Brian G. O'Flynn, Ryan L. Anderson, Gabriela Suarez, Dylan Wallis, and David J. Merkler
Department of Chemistry
University of South Florida
Fatty acid amides are a family of cell signaling lipids with the general structure of R-CO-NH-Y. This structural simplicity belies a wealth of diversity amongst this lipid family as the R-group is derived from fatty acids (R-COOH) and the Y-group is derived from biogenic amines (H2N-Y). The fatty acid amide family is divided into classes, defined by parent amines. Examples include the N-acylethanolamines (NAEs, R-CO-NH-CH2-CH2OH) and the N-acylglycines (NAGs,
R-CO-NH-CH2-COOH). Other classes of fatty acid amides are known. The best known fatty acid amide is N-arachidonoylethanolamine (anandamide), a fatty acid amide found in the human brain that binds to the cannabinoid receptors.
We have a long interest in the enzymes of fatty acid amide biosynthesis. We identified an enzyme that oxidizes the NAGs to the to the primary fatty acid amides and showed that inhibiting this enzyme led to the cellular accumulation of the NAGs. We have characterized a number of insect N-acyltransferases (from D. melanogaster, B. mori, and T. castaneum) that catalyze the acyl-CoA-dependent formation of fatty acid amides from an amine acyl-acceptor substrate. Knock-out experiments in D. melanogaster validate our in vitro substrate specificity studies demonstrating that one novel N-acyltransferases, arylalkyl N-acyltransferase-like 2 (AANATL2), does catalyze the formation of N-acyldopamines in vivo. We developed a straightforward platform technology to rapidly identify substrates for our panel of uncharacterized insect N-acyltransferases. Our application of this technology lead to identification of an enzyme in D. melanogaster, agmatine N-acetyltransferase (AgmNAT), which catalyzes the formation of N-acetylagmatine, a virtually unknown metabolite. We have determined the X-ray structure of AgmNAT. Our work on AgmNAT hints at an unknown reaction in arginine metabolism and points to a novel class on fatty acid amides, the N-acylagmatines. The presentation will also include our results on the kinetic and chemical mechanisms of the novel N-acyltransferases.
Biological N–N bond formation: From the nitrogen cycle to microbial natural products
Jonathan D. Caranto
University of Central Florida
Nitrous oxide (N2O) participates in ozone layer depletion and is a potent greenhouse gas, with a global warming potential 300 times greater than that of carbon dioxide. The atmospheric N2O concentration has surged 120% since the pre-industrial era largely due to increased fertilizer use. An estimated 60–75% of this gas is emitted as a by-product of microbial biochemical pathways that mediate the nitrogen cycle. Designing inhibitors for N2O-producing enzymes in these pathways could decrease N2O emissions, but this approach requires knowledge of the enzymatic mechanisms.
Agricultural N2O is largely a by-product of nitrification and denitrification that are mediated by nitrifying and denitrifying microorganisms, respectively. Nitrification oxidizes ammonia to nitrite or nitrate while denitrification reduces nitrate to dinitrogen or N2O. In denitrification, N2O is an obligate intermediate or the final product of the pathway that is generated from the two-electron reduction of two molecules of nitric oxide (NO). Occasionally, this N2O escapes the microorganism and is released into the environment. By contrast, N2O is not an obligate intermediate of nitrification. However, recent work provided evidence for NO as an obligate intermediate of nitrification. Nitrifying microorganisms frequently possess enzymatic machinery for nitrifier denitrification, which includes a respiratory NO reductase that can reduce NO to N2O. In addition to this NO reduction pathway, an alternate oxidative N2O-generating pathway was recently characterized, for which hydroxylamine (NH2OH) is oxidized to N2O.
This presentation will survey metalloenzyme intermediates that have been characterized along the reaction pathways of N2O-producing enzymes. Bacterial respiratory NO reductases found in denitrification and nitrifer denitrification pathways are mixed heme/non-heme diiron enzymes. By contrast, the NO reductases found in fungal denitrifiers contain mononuclear cytochrome P450 active sites. NO reductase activity has been characterized in a third class of metalloenzyme known as flavo-diiron proteins (FDPs) that contain a non-heme diiron center. Finally, cytochrome P460 exhibits a covalently modified c-heme enzyme that generates N2O by oxidation of NH2OH instead of NO reduction. For each of these enzymes, the mechanisms and iron-nitrosyl intermediates leading to N–N bond formation and the subsequent generation of N2O will be discussed. In addition, the application of these studies to a new research direction—N–N bond formation in microbial natural product biosynthesis—will also be discussed.
Manumycin-A is a potent inhibitor of mammalian thioredoxin reductase
Anupama Tuladhar; Kathleen Rein
Department of Chemistry and Biochemistry, Florida International University, Miami, Fl, 33199.
The thioredoxin system is the major cellular reductant system present in the cell, whose role is to maintain cellular redox homeostasis. The system controls many cellular processes such as DNA synthesis and also act as an antioxidant. The thioredoxin system is comprised of thioredoxin reductase (TrxR) and thioredoxin (Trx) which reduces target protein disulfide bridges by thiol-disulfide exchange. Because it is a regulator of numerous critical cellular functions, TrxR is also a common target for many cancer drugs including cisplatin and auranofin. Recently we have shown that the Florida red tide toxin, brevetoxin can inhibit mammalian TrxR which explains the oxidative stress observed in marine organisms exposed to red tide. We have discovered several compounds which are similar to brevetoxin in size and functionality that have a similar effect on TrxR. One of these compound is manumycin A (man-A). Unlike PbTx-2, man-A a known farnesyl transferase inhibitor, inhibits both DTNB and insulin reduction which implies that the potential binding site of man-A is C-terminal selenocysteine. Inhibition of TrxR at the C-terminal tail produces a pro-oxidant known as SecTRAP (Selenium Compromised Thioredoxin Reductase-derived Apoptotic Proteins), which produces superoxide radical anion. This might explain the observed burst of ROS in cells exposed to man-A. We have also tried to characterize the molecular mechanism of action by using site-specific mutant enzymes which allow us to determine the specific site of interactions between enzyme and man-A. The result with the mutant enzymes have revealed that man-A interacts in very different ways with mitochondrial and cytoplasmic TrxRs. This study will thus identify a novel mechanism of action of man-A and thus will contribute to develop a new drug to treat cancer.
Serine and Metallo carbapenemases: deciphering broad-spectrum activity and engineering cross-class inhibitors
Orville Pemberton, Nick Torelli, Afroza Akhtar, Priyadarshini Jaishankar, Kyle DeFrees, Adam Renslo, Yu Chen
University of South Florida, University of California San Francisco
Gram-negative bacterial pathogens expressing the serine carbapenemase KPC-2 and the metallo carbapenemases NDM-1 and VIM-2 threaten the clinical utility of all β-lactam antibiotics. These enzymes have a broad-substrate profile, most likely due to the hydrophobicity and flexibility of their active sites, particularly for the metalloenzymes. Here we demonstrate that this versatility in ligand recognition may expose a potential weakness that can be exploited through rational drug design. Using a fragment-based approach, we report the identification of a series of phosphonate compounds that represent the first cross-class non-covalent inhibitors of KPC-2, NDM-1, and VIM-2. Although our lead optimization specifically targeted KPC-2, the increase in KPC-2 inhibition was mirrored by improvement in activity against NDM-1 and particularly VIM-2. The best of these compounds showed high nM affinity against both KPC-2 and VIM-2, and cell-based activity in MIC testing in combination with imipenem. These findings provide novel chemical scaffolds for antibiotic development against bacteria co-producing serine and metallo carbapenemases, and suggest that the shared ligand binding features and the increased druggability of carbapenemase active sites can be leveraged in developing high affinity cross-class inhibitors.
Oncogenic protein kinase Cι drives melanoma cell epithelial-mesenchymal transition by activating vimentin through Par6/RhoA signaling
Wishrawana Sarathi Ratnayake, Christopher Apostolatos, Sloan Breedy and Mildred Acevedo-Duncan
University of South Florida
Melanoma is one of the fastest growing cancers in the United States, predicting a 14% increase in 2017 compared to 2016. The five year survival rate drops to 4% when melanoma is metastasized and responsible for ˃90% melanoma related deaths. Most available drugs target BRAF (V600E) mutation which occurs in ~60% melanoma cases, yet there is a poor prognosis and tumors acquire resistance to BRAF mutation inhibition. PKC-ι (iota) is an oncogene involved in cell cycle progression, tumorigenesis and cell survival in many cancers. We believe PKC-ι is an effective therapeutic target for invasive melanoma. We reported that PKC-ι is overexpressed in melanoma cells [Int. J. Oncol. 51(5), 1370-1382, (2017)]. In the current study, we have investigated the effects of knockdown of expression of PKC-ι and specific inhibitors on cellular properties of two malignant melanoma cell lines (SK-MEL-2 and MeWo) compared to a normal melanocyte cell line (MEL-NEO-F). Cell viability and WST-1 assay showed that both inhibitors show lesser cytotoxicity to MEL-F-NEO cell line at higher concentrations (˃7.5 μM) compare to significant toxicity on melanoma (˃1 μM). PKC-ι knockdown/ inhibition decreased the levels of total and phosphorylated levels of PKC-ι. Furthermore, levels of E-cadherin and RhoA were increased while decreasing the levels of Vimentin; a mesenchymal marker associated with EMT. Treatments with inhibitors significantly decreased the phosphorylated Vimentin (S39) while increasing phosphorylation at S33 and S56, thereby preventing the Vimentin intermediate assembly. Immunoprecipitation and reversed immunoprecipitation showed a strong interaction of PKC-ι and Vimentin and immunofluorescence staining proved the observation. mRNA expression levels of PKC-ι, Vimentin decreased upon PKC-ι knockdown. PKC-ι knockdown downregulates Par6 thereby stabilize RhoA even upon TGFβ1 stimulation. We have also showed that PKC-ι inhibition limits the translocation of activated NF-κB p65/p52 complex and β-catenin thereby regulates NF-κB and WNT/β-catenin signaling. Overall, results show that PKC-ι is essential for melanoma progression and metastasis through activation of Vimentin via TGFβ/Par6/RhoA pathway. Therefore PKC-ι could be used as effective therapeutic targets for malignant melanoma.
Phosphorylation induced cochaperone unfolding promotes kinase recruitment and client class-specific Hsp90 phosphorylation
Ashleigh Bachman, Dimitra Keramisanou1 and Ioannis Gelis
Department of Chemistry, University of South Florida, Tampa, FL, 33620, USA
Post translational modifications of the Hsp90 machinery occur as regular signaling events that mark in the timely progression through the chaperone cycle or as a response to environmental or other stimuli. During the kinase chaperone cycle both Hsp90 and the kinase-specific cochaperone, Cdc37, are subject to multiple phosphorylation events. However, the exact molecular details by which these modifications impact the kinase chaperone cycle remain unexplored. We show that Cdc37 promotes tyrosine phosphorylation of Hsp90 in a client class-specific manner, through an unprecedented mechanism. Cdc37 phosphorylation at Y298 results in partial unfolding of its C-terminal domain and the population of folding intermediates. This phosphorylation-stabilized extended conformation unmasks a high affinity SH2-binding phosphopeptide, which exhibits broad specificity over SH2 domains of non-receptor tyrosine kinase (nRTKs). Docking of these nRTKs on Cdc37 promotes efficient phosphorylation of Hsp90 at Y197, which results in Cdc37 dissociation. Thus the presence Cdc37 imprints a specific phosphorylation pattern on Hsp90 that specifically regulates the kinase chaperone cycle. Overall, we show that by providing client class specificity, Hsp90 cochaperones such as Cdc37 do not merely assist in client recruitment but also shape the post-translational modification landscape of Hsp90 in a client class-specific manner.
Bachman AB, Keramisanou D, Xu W, Beebe K, Moses MA, Vasantha Kumar MV, Gray G, Noor RE, van der Vaart A, Neckers L, Gelis I, Nature Communications, 9 (2018)