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.