Aha1 stimulates tau aggregation
Lindsey B. Shelton, Jeremy D. Baker, Dali Zheng, Brian S.J. Blagg, and Laura J. Blair
Department of Molecular Medicine and USF Health Byrd Institute, University of South Florida, Tampa, Florida
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana
The microtubule associated protein tau pathologically accumulates inside neurons in Alzheimer’s disease (AD) and other tauopathies. Chaperones, such as heat shock protein 90kDa (Hsp90), have been shown to preserve pathological tau. Inhibition of the ATPase activity of the abundantly expressed 90kDa heat shock protein (Hsp90) reduces tau levels in vivo. This ATPase activity is also regulated by a diverse set of Hsp90 co-chaperones. In particular, the activator of Hsp90 ATPase homolog 1 (Aha1) is the only known stimulator of this ATPase activity. However, the effects of Aha1 on Hsp90-mediated tau fibrilization have not been well-characterized. Since it is known that Hsp90 promotes tau fibrilization, we hypothesized that Aha1 would further stimulate tau aggregation through stimulation of Hsp90 ATP hydrolysis. We have now found Aha1 is co-localized and co-immunoprecipitates with pathogenic tau in AD brain tissue. In recombinant and cell models, Aha1 coordinated with Hsp90 to accelerate insoluble tau accumulation, which was prevented by an Aha1 mutation that reduces Hsp90 interaction. In vivo, Over-expression of Aha1 in the hippocampus of rTg4510 tau transgenic mice increased oligomeric and insoluble tau, concomitant with reduced neuronal viability. Recently, we have determined that ablation of Aha1 levels or activity slowed the accumulation of pathogenic tau. Overall, these data suggest that Hsp90 ATP hydrolysis promotes tau oligomer and fibril formation, and small molecule inhibitors of Aha1 may be beneficial for the prevention or treatment of tau-related diseases.
Membrane-Active Hydantoin Derivatives as Antibiotic Agents
Ma Su, Donglin Xia, Peng Teng,Alekhya Nimmagadda, Chao Zhang,Timothy Odom,
Annie Cao,Yong Hu, and Jianfeng Cai
University of South Florida
Hydantoin (imidazolidinedione) derivatives such as nitrofurantoin are small molecules that have aroused considerable interest recently due to
their low rate of bacterial resistance. However, their moderate antimicrobial activity may hamper their application combating antibiotic resistance in the long run. Herein, we report the design of bacterial membrane-active hydantoin derivatives, from which we identified compounds that show much more potent antimicrobial activity than nitrofurantoin against a panel of clinically relevant Gram-positive and Gram-negative bacterial strains. These compounds are able to act on bacterial membranes, analogous to natural host-defense peptides. Additionally, these hydantoin compounds not only kill bacterial pathogens rapidly but also prevent the development of methicillin resistant Staphylococcus aureus (MRSA) bacterial resistance under the tested conditions. More intriguingly, the lead compound exhibited in vivo efficacy that is much superior to vancomycin by eradicating bacteria and suppressing inflammation caused by MRSA-induced pneumonia in a rat model, demonstrating its promising therapeutic potential.
One-Bead-Two-Compound Macrocyclic γ-AApeptide Screening Library against EphA2
Yan Shi, Jianfeng Cai*
University of South Florida
Identification of molecular ligands that recognize peptides or proteins is significant, but poses a fundamental challenge in chemical biology and biomedical sciences. Development of cyclic peptidomimetic library is scarce and thus discovery of cyclic peptidomimetic ligands for protein targets is rare. Herein we report the unprecedented One-Bead-Two-Compound (OBTC) combinatorial library based on a novel class of the macrocyclic peptidomimetics γ-AApeptides. In order to develop the library, we utilized the coding peptide tags synthesized with Dde-protected α-amino acids, which were proved to be orthogonal to solid phase synthesis of γ-AApeptides. Employing the thioether linkage, the desired macrocyclic γ-AApeptides were found to be effective for ligand identification. Screening the library against the receptor tyrosine kinase EphA2 led to the discovery of one lead compound which tightly bound to EphA2 (Kd: 81 nM) and potently antagonized EphA2-mediated signaling. This new approach of macrocyclic peptidomimetic library may lead to a novel platform which provides unique source of ligands for biomacromolecular surface recognition and function modulation.
Increased post-translation modification of eIF5A contributes to TDP43 proteinopathy
Shayna Smeltzer, Zain Quadri, Frank Zamudio, Jordan Hunter, Daniel C Lee, Maj-Linda B Selenica
College of Pharmacy, University of South Florida
The hallmark of TDP-43 proteinopathy is loss of nuclear function and accumulation as cytoplasmic inclusions. Recent evidence suggests for unique accumulation of TDP-43 in stress granules (SG) as disease progresses. In patients, TDP-43 pathology results in impairment of motor neuron function (ALS) and cognitive dysfunction (FTD). Hypusination of eIF5A (eIF5AhypK50) denotes its activation and cytoplasmic localization where it also binds to various RNA binding proteins. Its overall cellular function is translational elongation but translation inhibition in stress granules also occurs, as shown in stress-induced cellular models. While this is a common feature of SG biology, the activity of eIF5AhypK50 in SG can be problematic for TDP-43 proteinopathies. It can lead to further seeding of TDP-43 and translation of aberrant truncated TDP-43 forms adding to cytoplasmic aggregation once SGs dissolve. We show increased levels of enzymes responsible for hypusination in brain tissue from AD patient as well as in TDP-43 animal models. The animal model also displayed significant increase in hypusination levels, suggesting that its augmentation underlies the progression of the disease. Further, we know that protein–protein binding occurs between eIF5AhypK50 and TDP-43, and just by inhibiting hypusination, phosphorylated and total TDP-43 levels are reduced in the cytoplasm. It is however unknown the mechanism by which this occur. We predict that inhibition of hypusination will reduce TDP-43 burden and provide a strategy for therapies in TDP-43 proteinopathy. We hypothesize that eIF5AhypK50 regulates TDP-43 trafficking via several potential mechanisms, including protein-protein interactions, promoting cytoplasmic accumulation, translational regulation and the government of SG biology. We predict that it is through these mechanisms that eIF5AhypK50 orchestrates TDP-43 trafficking to cytoplasm and determines the biological signature of SGs. Our team has developed several unique tools to evaluate the efficacy of such approaches and dissect the mechanism of action through which eIF5AhypK50 affects TDP-43 pathology. We already know that pharmacological inhibition effectively reduces TDP-43 accumulation in SG in cells. However, we do not know if modulations of eIF5AhypK50 have neuronal efficacy or if it yields beneficial outcomes in the brain. Therefore, we have employed several innovative techniques, siRNA screening, antisense oligonucleotides and viral constructs to examine the neuronal role of eIF5AhypK50 in primary neurons and in TDP43 transgenic model.
Identification of suramin as a potent and specific inhibitor of the mammalian high mobility group protein AT-hook 2 (HMGA2)-DNA interactions
Linjia Su1,2, Prem P. Chapagain1,3, Steve Vasile4, Layton Smith4, and Fenfei Leng1,2,*
1 Biomolecular Sciences Institute and 2 Department of Chemistry and Biochemistry, 3 Department of Physics, Florida International University, Miami, FL 33199
4 Sanford-Burnham Center for Chemical Genomics at Sanford-Burnham Medical Research Institute, Orlando, Florida 32827, USA
The mammalian high mobility group protein AT-hook 2 (HMGA2) is a nuclear protein associated with epithelial-mensenchymal transition (EMT) during cell development and differentiation. This protein plays an important role in the formation of a variety of tumors including malignant tumors, such as melanoma, lung cancer and hepatocellular carcinoma. These results suggest that HMGA2 is a potential therapeutic target of anticancer drugs. HMGA2 is a DNA minor-groove binding protein specifically recognizing the minor groove of AT-rich DNA sequences. Previous results showed that HMGA2-DNA interactions are a potential target for chemical intervention. In this study, we developed an AlphaScreen HTS Assay to screen inhibitors targeting HMGA2-DNA interactions. We are particularly interested in identifying non DNA-binding compounds that inhibit HMGA2-DNA interactions due to the fact that DNA-binding compounds are highly cytotoxic. After the HTS campaign, several non DNA-binding compounds have been identified to potently inhibit HMGA2-DNA interactions. Among them is suramin, a negatively charged antiparasitic drug. Suramin potently inhibits HMGA2-DNA interaction with an IC50 of 2.78±0.10 micro molar. We also found that suramin and analogues strongly bind to HMGA2, suggesting that the inhibition of HMGA2-DNA interactions is through suramin binding to HMGA2 and therefore blocking HMGA2’s DNA binding capacity. Suramin is an anticancer agent that inhibits tumor growth and metastasis for certain cancers including pancreatic cancer, prostate cancer, melanoma, and etc. The anti-cancer mechanism of suramin is still illusive. The discovery of suramin as a potent inhibitor of HMGA2-DNA interactions suggests that the anti-cancer activities of suramin may stem from its inhibition of HMGA2-DNA interactions in vivo and opens a door for future research in suramin as an anticancer agent.
Bacterial large subunit ribosome assembly
Riley Gentry, Jared Childs, Eda Koculi
University of Central Florida
The ribosome is the protein-RNA complex responsible for protein production in every known organism. While the structure of bacterial ribosome is known from crystallographic studies, the knowledge of the intermediates’ structures and compositions remains largely unknown. Because ribosome assembly in bacteria is a very fast and efficient process, the intermediates in ribosome assembly do not accumulate in cell in quantities sufficient to perform biophysical investigation. To increase the amount of intermediates accumulated during large subunit ribosome assembly, we expressed in Escherichia coli the helicase inactive R331A DbpA protein. Expression of R331A DbpA produces the accumulation of three large subunit intermediates. Our experiments demonstrate that the intermediates belong to three different stages of the large subunit ribosome assembly and that they rearrange to form the 50S large subunit via three parallel pathways. This is the first time that the existence of multiple pathways of large subunit ribosome assembly was observed experimentally. The ribosomal RNA is extensively modified in bacteria, and the modifications are shown to influence both the structure of the ribosome and its function. Using Next Generation Sequencing we determined that most of the modifications occur in the early stages of large subunit ribosome assembly. Lastly, our chemical modification experiments combined with Next Generation Sequencing demonstrate that the maturation of the peptidyltransferase center is the last step of large subunit ribosome assembly, preventing in this way the formation of malfunctioning ribosomes. This conclusion agrees with the data previously gathered on the bacterial and mitochondrial ribosomes.
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)