Endogenous DNA damage arises frequently, particularly apurinic (AP) sites. These must be dealt with by cells in order to avoid genotoxic effects. DNA polymerase theta; is a newly identified enzyme encoded by the human POLQ gene. We find that POLQ has an exceptional ability to bypass an AP site, inserting A with 22% of the efficiency of a normal template, and continuing extension as avidly as with a normally paired base. POLQ preferentially incorporates A opposite an AP site and strongly disfavors C. On nondamaged templates, POLQ makes frequent errors, incorporating G or T opposite T about 1% of the time. This very low fidelity distinguishes POLQ from other A-family polymerases. POLQ has three sequence insertions between conserved motifs in its catalytic site. One insert of approximately 22 residues into the tip of the polymerase thumb subdomain is predicted to confer considerable flexibility and additional DNA contacts to affect enzyme fidelity. POLQ is the only known enzyme that efficiently carries out both the insertion and extension steps for bypass of AP sites, commonly formed as endogenous genomic lesions.
Background: Tumor cells require proficient autophagy to meet high metabolic demands and resist chemotherapy, which suggests that reducing autophagic flux might be an attractive route for cancer therapy. However, this theory in clinical cancer research remains controversial due to the limited number of drugs that specifically inhibit autophagy-related (ATG) proteins.Methods: We screened FDA-approved drugs using a novel platform that integrates computational docking and simulations as well as biochemical and cellular reporter assays to identify potential drugs that inhibit autophagy-required cysteine proteases of the ATG4 family. The effects of ATG4 inhibitors on autophagy and tumor suppression were examined using cell culture and a tumor xenograft mouse model.Results: Tioconazole was found to inhibit activities of ATG4A and ATG4B with an IC50 of 1.3 µM and 1.8 µM, respectively. Further studies based on docking and molecular dynamics (MD) simulations supported that tioconazole can stably occupy the active site of ATG4 in its open form and transiently interact with the allosteric regulation site in LC3, which explained the experimentally observed obstruction of substrate binding and reduced autophagic flux in cells in the presence of tioconazole. Moreover, tioconazole diminished tumor cell viability and sensitized cancer cells to autophagy-inducing conditions, including starvation and treatment with chemotherapeutic agents.Conclusion: Tioconazole inhibited ATG4 and autophagy to enhance chemotherapeutic drug-induced cytotoxicity in cancer cell culture and tumor xenografts. These results suggest that the antifungal drug tioconazole might be repositioned as an anticancer drug or chemosensitizer.
In this study, I present a new elastic network model, to our knowledge, that addresses insufficiencies of two conventional models-the Gaussian network model (GNM) and the anisotropic network model (ANM). It has been shown previously that the GNM is not rotation-invariant due to its energy, which penalizes rigid-body rotation (external rotation). As a result, GNM models are found contaminated with rigid-body rotation, especially in the most collective ones. A new model (EPIRM) is proposed to remove such external component in modes. The extracted internal motions result from a potential that penalizes interresidue stretching and rotation in a protein. The new model is shown to pertinently describe crystallographic temperature factors (B-factors) and protein open↔closed transitions. Also, the capability of separating internal and external motions in GNM slow modes permits reexamining important mechanochemical properties in enzyme active sites. The results suggest that catalytic residues stay closer to rigid-body rotation axes than their immediate backbone neighbors. I show that the cumulative density of states for EPIRM and ANM follow different power laws as functions of low-mode frequencies. When using a cutoff distance of 7.5 Å, The cumulative density of states of EPIRM scales faster than that of all-atom normal mode analysis and slower than that of simple lattices.
Objective: The present study was designed to determine the neuroprotective effect of Abelmoschus esculentus L. on alloxan-induced diabetic neuropathy in rats.Methods: Diabetes was induced in rats with a single intraperitoneal injection of alloxan monohydrate (130 mg/kg b.w). The ethanol extract of A. esculentus L. at a dose of 100 and 200 mg/kg of body weight was administered at single dose per day to alloxan-induced diabetic rats for 21 days. The fasting blood glucose was screened in the intermittent on day 0, day 14, and day 21. Behavioral tests such as thermal hyperalgesia test and rotarod performance test were performed to assess the thermal sensitivity and muscle grip strength. At the end of the study period, experimental animals were sacrificed and sciatic nerve tissues were obtained for histopathological investigation.Results: Animals treated with A. esculentus L. extarct at a dose of 200 mg/kg of body weight significantly reduced (p<0.05) in hyperglycemia and thermal hyperalgesia and significantly increased (p<0.05) in rotarod performance. The sciatic nerve fiber of diabetic rats receiving 200 mg/kg of body weight of A. esculentus L. extract also shows no swelling of nerve fibers, and lesser demyelination was observed.Conclusion: These findings demonstrate that A. esculentus L. exhibits significant antidiabetic and neuroprotective effect against alloxan-induced diabetic neuropathy in rats.
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