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Results for “molecular dynamic simulation assay”

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PubMed2026

Integrated computational and experimental benchmarking of Bacillus phage endolysins reveals the relationship between peptidoglycan-fragment recognition descriptors and antibacterial performance.

Protein-based antibacterials such as bacteriophage endolysins offer a targeted therapeutic strategy against Gram-positive pathogens. However, prioritizing the most effective candidates from the large sequence diversity available remains a significant challenge. Here we present a standardized computational-experimental benchmarking framework that evaluates seven phage-derived endolysin variants (E1, E2, E3, E7, E10, E12, and E15) identified from Bacillus genomes. We combined molecular docking and residue-level interaction mapping against muramyl dipeptide (MDP), a minimal conserved peptidoglycan motif, with 1000-ns molecular dynamics simulations, MM/PBSA binding free-energy estimation, and matched functional inhibition assays against Staphylococcus aureus and Micrococcus luteus. Computational analyses revealed generally favorable MDP recognition across variants, albeit with notable differences in contact patterns and complex stability profiles. Experimental screening identified E2 as the most potent antibacterial agent against both species, while E7 and E1 performed strongly in selected computational metrics. Integrated analysis showed only modest correlations between computational descriptors of fragment recognition/stability and observed antibacterial performance. This study establishes a practical comparative benchmarking platform for endolysin candidate prioritization, nominates E2 and E7 as promising candidates for further development, and highlights E1 as a potential structural scaffold for rational engineering, while explicitly demonstrating both the utility and the current limitations of using minimal peptidoglycan fragments as proxies for full cell-wall recognition in lysin benchmarking.

Endopeptidases↗

PubMed2004

Potent inhibitors of the Plasmodium falciparum enzymes plasmepsin I and II devoid of cathepsin D inhibitory activity.

The hemoglobin-degrading aspartic proteases plasmepsin I (Plm I) and plasmepsin II (Plm II) of the malaria parasite Plasmodium falciparum have lately emerged as putative drug targets. A series of C(2)-symmetric compounds encompassing the 1,2-dihydroxyethylene scaffold and a variety of elongated P1/P1' side chains were synthesized via microwave-assisted palladium-catalyzed coupling reactions. Binding affinity calculations with the linear interaction energy method and molecular dynamics simulations reproduced the experimental binding data obtained in a Plm II assay with very good accuracy. Bioactive conformations of the elongated P1/P1' chains were predicted and agreed essentially with a recent X-ray structure. The compounds exhibited picomolar to nanomolar inhibition constants for the plasmepsins and no measurable affinity to the human enzyme cathepsin D. Some of the compounds also demonstrated significant inhibition of parasite growth in cell culture. To the best of our knowledge, these plasmepsin inhibitors represent the most selective reported to date and constitute promising lead compounds for further optimization.

Amides↗

PubMed2007

Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior.

The dynamic properties of helix 12 in the ligand binding domain of nuclear receptors are a major determinant of AF-2 domain activity. We investigated the molecular and structural basis of helix 12 mobility, as well as the involvement of individual residues with regard to peroxisome proliferator-activated receptor alpha (PPARalpha) constitutive and ligand-dependent transcriptional activity. Functional assays of the activity of PPARalpha helix 12 mutants were combined with free energy molecular dynamics simulations. The agreement between the results from these approaches allows us to make robust claims concerning the mechanisms that govern helix 12 functions. Our data support a model in which PPARalpha helix 12 transiently adopts a relatively stable active conformation even in the absence of a ligand. This conformation provides the interface for the recruitment of a coactivator and results in constitutive activity. The receptor agonists stabilize this conformation and increase PPARalpha transcription activation potential. Finally, we disclose important functions of residues in PPARalpha AF-2, which determine the positioning of helix 12 in the active conformation in the absence of a ligand. Substitution of these residues suppresses PPARalpha constitutive activity, without changing PPARalpha ligand-dependent activation potential.

Amino Acid Sequence↗

PubMed2006

Antileukemic activity of shepherdin and molecular diversity of hsp90 inhibitors.

BACKGROUND: Heat shock protein 90 (Hsp90) is a molecular chaperone that is involved in signaling pathways for cell proliferation, survival, and cellular adaptation. Inhibitors of Hsp90 are being examined as cancer therapeutic agents, but the molecular mechanism of their anticancer activity is still unclear. We investigated Hsp90 as a therapeutic target for acute myeloid leukemia (AML) by use of the Hsp90 inhibitor shepherdin (a novel peptidyl antagonist of the interaction between Hsp90 and survivin, which is a regulator of cell proliferation and cell viability in cancer). METHODS: We studied protein interactions by molecular dynamics simulations and conducted competition experiments by use of enzyme-linked immunosorbent assay (ELISA). Shepherdin[79-83], a novel variant carrying the survivin sequence from Lys-79 through Gly-83, or its scrambled peptide was made permeable to cells by adding the antennapedia helix III carrier sequence. Apoptosis, Hsp90 client protein expression, and mitochondrial dysfunction were evaluated in AML types (myeloblastic, monocytic, and chronic myelogenous leukemia in blast crisis), patient-derived blasts, and normal mononuclear cells. Effects of shepherdin on tumor growth were evaluated in AML xenograft tumors in mice (n = 6). Organ tissues were examined histologically. RESULTS: Shepherdin[79-83] bound to Hsp90, inhibited formation of the survivin-Hsp90 complex, and competed with ATP binding to Hsp90. Cell-permeable shepherdin[79-83] induced rapid (within 30 minutes) and complete (with concentrations inducing 50% cell death of 24-35 microM) killing of AML types and blasts, but it did not affect normal mononuclear cells. Shepherdin[79-83] made contact with unique residues in the ATP pocket of Hsp90 (Ile-96, Asp-102, and Phe-138), did not increase Hsp70 levels in AML cells, disrupted mitochondrial function within 2 minutes of treatment, and eliminated the expression of Hsp90 client proteins. Shepherdin[79-83] abolished growth of AML xenograft tumors (mean of control group = 1698 mm3 and mean of treated group = 232 mm3; difference = 1466 mm3, 95% confidence interval = 505.8 to 2426; P = .008) without systemic or organ toxicity and inhibited Hsp90 function in vivo. CONCLUSIONS: Shepherdin is a novel Hsp90 inhibitor with a unique mechanism of anticancer activity.

Acute Disease↗

PubMed2026

Novel environmental contaminant 6PPD-quinone promotes malignant phenotypes in colorectal cancer cells and identifies candidate response-associated genes.

6PPD-quinone (6PPDQ), an oxidative transformation product of the widely used tire antioxidant 6PPD, is a ubiquitous environmental contaminant with bioaccumulation potential and widespread human exposure. Recent epidemiological evidence indicates a positive association between urinary 6PPDQ levels and colorectal cancer (CRC) risk; however, its biological effects on CRC-related phenotypes and associated molecular responses remain unclear. We integrated bioinformatics analysis, prognostic modeling, molecular docking and dynamics simulations, and in vitro experiments to investigate cellular and molecular responses to 6PPDQ in CRC models. Predicted 6PPDQ targets were intersected with CRC prognosis-related genes from The Cancer Genome Atlas, followed by functional enrichment and LASSO regression to construct a prognostic risk model, with 1-, 3-, and 5-year AUC values of 0.727, 0.754, and 0.778, respectively. Molecular docking and 100-ns molecular dynamics simulations suggested interactions between 6PPDQ and candidate proteins, including CPT2, SHC2, SRMS, and STK35. Functional assays showed that 6PPDQ exposure altered proliferation, wound-closure capacity, and invasion in Caco-2 and HCT116 cells across the nanomolar concentration range, with non-monotonic and cell-line-dependent responses. In contrast, NCM460 cells showed no increase in EdU incorporation at 10 or 100 nM, whereas reduced proliferation at higher concentrations was accompanied by increased LDH release. 6PPDQ also altered the expression of several prognosis-associated candidate genes. These findings identify cellular phenotypes and candidate molecular responses associated with 6PPDQ exposure under the tested in vitro conditions, but do not establish their causal roles or in vivo relevance. Further mechanistic and in vivo studies are required.

Humans↗

PubMed2004

Specificity of Amaranthus leucocarpus syn. hypocondriacus lectin for O-glycopeptides.

Amaranthus leucocarpus syn. hypochondriacus lectin (ALL) has been shown to be specific for N-acetyl-D-galactosamine (GalNAc). In this work, we determined a value of 1.0 x 10(-2) M for the association constant of ALL for GalNAc, calculated using fluorescence spectroscopy assays. Using neoglycopeptides obtained by in vitro O-glycosylation, we determined the main features of O-glycopeptides recognized by ALL using molecular dynamics simulations, capillary electrophoresis, and ELISA. Neo-glycopeptides were obtained by in vitro O-glycosylation reaction using microsomal preparations of murine thymocytes, human gastric fundus and colonic mucosa. ELISA assays were performed with peroxidase-labeled murine monoclonal IgG2, kappa light chain (5D4) antibodies against ALL. Among the in vitro neoglycopeptides, only those of TTSAPTTS containing GalNAc at Thr in #2 and #6 reacted with ALL. Neither the TTSAPTTS glycopeptide, containing a unique GalNAc residue at Thr in #2, nor others (with more than two GalNAc residues) interacted with the lectin. Computational docking assays of the lower energy conformers for interactions between glycopeptides and lectins confirmed that ALL recognized GalNAc residues when they are spaced out in glycan structures, whereas GalNAc residues arranged in clusters prevented interaction with the lectin, indicating that ALL is specific for a special GalNAc-containing motif found in different O-glycoproteins.

Acetylgalactosamine↗

PubMed2006

Construction of a small peptide library related to inhibitor OM99-2 and its structure-activity relationship to beta-secretase.

AIM: To develop probes for detecting the binding specificity between beta-secretase and substrate, and provide reliable biological activity data for further researching encircling substrate-based inhibitors. METHODS: To prepare the inhibitors, the hydroxyethylene (HE) segment including P1 and P1'was synthesized after multi-step reactions; the combination of all segments was then completed through solid phase synthesis. Recombinant human beta-secretase ectodomain (amino acid residues 1-460) was expressed as a secreted protein with a C-terminal His tag in insect cells using baculovirus infection, and all compounds were evaluated in this beta-secretase enzyme assay. In order to understand the interaction in detail, the theoretical methods, namely molecular dynamics (MD) simulation and molecular mechanics-generalized-born surface area (MM-GBSA) analysis, were performed on the complex of beta-secretase and OM99-2 to obtain the geometrical and energetical information. RESULTS: We designed and constructed a positional scanning combinatorial library including 16 compounds; all members of the library were synthesized based on HE dipeptide isostere. Structure-activity relationship studies at the P4-P1 and P1' -P4'positions led to the discoveries of P and P'sides binding specificity and potent inhibitors 14, 18, and 22. The binding free energy on the whole system and every residue were compared to the biological assay result. CONCLUSION: The removal of P4' yielded inhibitor 22 (A3 *B2) with high potency; further truncation of P3'gave inhibitor 18 (A3 *B1) with equal activity, implying that the right side of the inhibitors play a less important role and could be easily simplified, while change on the P side may cause substantial results.

Amyloid Precursor Protein Secretases↗

PubMed2006

Determination of binding site residues responsible for the subunit selectivity of novel marine-derived compounds on kainate receptors.

Dysiherbaine (DH) and related molecules are high-affinity, subunit-selective kainate receptor (KAR) ligands originally isolated from a marine sponge. To elucidate why DH, an agonist, and MSVIII-19, a competitive antagonist, bind selectively to glutamate receptor (GluR) 5 but not to the KA2 KAR subunit, we used molecular dynamics simulations to generate binding models that were tested experimentally in radioligand binding and electrophysiological assays. Three candidate sites, Val685, Leu735, and Ser741 in GluR5, corresponding to Ile669, Phe719, and Met725 in KA2, were predicted to underlie the distinct binding profiles of the marine toxins. Single or multiple reciprocal mutations introduced into the receptor subunits produced a variety of effects on binding affinity. Most notably, mutation of Met725 to serine in KA2 increased the affinity of DH by 350-fold; in contrast, mutation of one or more of the residues in GluR5 did not markedly alter DH binding. MSVIII-19 affinity for the KA2 subunit was significantly increased in multiple site mutants, and reciprocal mutations in the GluR5 subunit produced substantial (700-fold) reductions in MSVIII-19 affinity. Physiological characterization of the double- and triple-mutant subunits demonstrated altered functional behavior consistent with the changes in binding affinity. The results provide experimental support for the importance of these three ligand binding domain (LBD) residues and suggest steric hindrance in the KA2 subunit LBD is largely responsible for the very low affinity for the two compounds. In this study, we identified the molecular basis for subunit selectivity of these marine-derived molecules on KARs, which could facilitate the rational design of selective ligands with distinct pharmacological profiles.

Alanine↗

PubMed2026

Proteomic regulation of anti-proliferative and anti-migratory activity by potent phytochemicals from Pistacia integerrima J.L. Steward Ex Brandis via PI3K, AKT1, and KRAS for Lung Cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of mortality worldwide and remains a major therapeutic challenge due to high metastasis, drug resistance and limited treatments. Pistacia integerrima J.L. Steward Ex Brandis (PI) consists of flavonoids, steroids, terpenoids and phenolic compounds reported for pharmacological activities. The efficacy of potent bioactives from P. integerrima may be ascertained employing cytotoxic, antiproliferative, anti-migratory, and anti-metastatic evaluations in A549 NSCLC cells with proteomic profiling, molecular docking, and dynamics simulation study. METHODS AND RESULTS: PI EtAc produced significant dose-dependent cytotoxicity in A549 cells (100 µg/mL, p < 0.0001 in the MTT assay. There was a pronounced decrease in colony formation after treatment with EtAc, with 18.41% (p < 0.002), and markedly. Furthermore. PI EtAC markedly inhibited cell migration emphasized by wound healing and Transwell migration (p < 0.01) assays, indicating reduced metastatic migratory potential. Proteomic analysis demonstrated significant downregulation of Endoglin (CD105), KLK5 and MMP-2, indicating suppression of angiogenic and metastatic signalling pathways in the Human XL Oncology protein array. The interaction of major PI phytochemicals with key NSCLC-associated targets was recorded in Molecular docking, revealing favourable binding affinities of kaempferol, β- sitosterol, luteolin, and quercetin towards several oncogenic targets, including AKT1(- 7.6 kcal/mol), PI3K(- 9.4 kcal/mol), KRAS (- 8.5 kcal/mol) and MMP9 (- 8.1 kcal/mol). Molecular dynamics simulation confirmed the structural stability of the kaempferol -AKT1 complex throughout the 100 ns simulation. CONCLUSION: Pistacia integerrima bioactives exhibited significant anti-proliferative, anti-migratory, and anti-metastatic activities in vitro, which may provide scientific rationale identifying newer promising candidates for NSCLC.

Humans↗

PubMed2026

Visual Detection and Stratification of Pathogenic mtDNA SNV Heteroplasmy by Balancing FnCas12a Signal Output and Allelic Discrimination.

Assessment of pathogenic mitochondrial DNA (mtDNA) single-nucleotide variant (SNV) heteroplasmy is important for molecular diagnostics, yet rapid visual profiling remains analytically challenging because an assay must combine single-nucleotide allelic discrimination, mutant-fraction-associated readout, and suitable target access. Herein, we report VISTA (visual identification and stratification of targeted mtDNA alleles), a broad-PAM FnCas12a assay that rebalances trans-cleavage signal output and mutant-wild-type discrimination for visual mtDNA SNV heteroplasmy analysis. VISTA uses unmodified FnCas12a with relaxed TTN PAM recognition and integrates crRNA spacer-length engineering with PEG8000/acBSA reaction tuning to improve the practical signal-discrimination balance without nuclease engineering. At the m.3243A>G model locus, spacer truncation enhanced mutant-wild-type discrimination, while molecular-dynamics simulations identified spacer-dependent differences between matched and mismatched complexes at the crRNA-DNA interface. The optimized assay resolved defined synthetic m.3243A>G heteroplasmy gradients by fluorescence imaging and was further adapted to lateral-flow detection. In locus-specific analyses of a deidentified collection of 74 peripheral-blood samples, fluorescence and lateral-flow readouts achieved ROC AUC values above 0.9 for mutant-allele classification after target-region amplification. Fluorescence supported heteroplasmy-associated profiling, whereas lateral flow provided a visual, semiquantitative readout for relative ranking based on the T/C ratio rather than absolute heteroplasmy measurement. VISTA therefore provides an accessible dual-readout analytical strategy for visual detection and heteroplasmy-associated profiling by tuning the FnCas12a signal output and allelic discrimination.

DNA, Mitochondrial↗

PubMed2000

Molecular dynamics study on lipid A from Escherichia coli: insights into its mechanism of biological action.

Structural properties of the Escherichia coli lipid A moiety were analysed by means of molecular mechanics and molecular dynamics simulations and compared to synthetic monophospho and dephospho analogues with different biological activities in the Limulus assay. The conformation of glucosamine disaccharide headgroup, order and packing of fatty acid chains, solvation of phosphate groups, coordination by water molecules, sodium counterions and models of cationic amino acid side chains were described in terms of mean values, mean residence times, radial distribution functions, coordination numbers, solvation and interaction energies. Solvation and polar interactions of the phosphate groups were correlated to known biological activities the lipid A variants. The observed relationship between the biological effect and the number and position of the phosphate groups were explained with the help of simple mechanistic models of lipid A action. The possible mechanism of action involving specific binding of lipid A disaccharide headgroup to cationic residues of a receptor model was compared with an alternative mechanism, which assumes a relationship between the ability to adopt non-lamellar supramolecular structures and the biological activity. Conclusions are drawn about the probable mode of lipid A action. Implications for rational drug design of endotoxin-neutralising agents are discussed.

Carbohydrate Conformation↗

PubMed2026

Resveratrol Attenuates Gemcitabine Resistance in Hepatocellular Carcinoma Cells by Inhibiting Thymidylate Synthase.

BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Gemcitabine (Gem) is a commonly used drug against HCC, but its efficacy is limited by the development of resistance. Resveratrol (Res), a natural polyphenol with antitumor activity, may reverse Gem resistance in HCC, although the mechanism remains unclear. METHODS: The effects of Res on the proliferation, apoptosis, cell cycle, and invasion of Hep3B and HuH-7 cells were assessed via cell counting kit-8 (CCK-8), clonogenic, flow cytometry, and Transwell assays, respectively. Potential Res targets were predicted by network pharmacology, and markers of HCC prognosis were identified from the cancer genome atlas (TCGA) data. The interaction between Res and thymidylate synthase (TYMS) was validated by molecular docking and dynamics simulation. A Gem-resistant HuH-7 cell line (HuH-7/GR) was established, and when these cells were treated with Res combined with Gem, the effect on Gem sensitivity was detected by CCK-8 assay, clonogenic assay, and flow cytometry. Finally, a subcutaneous nude mouse model of HCC was used to evaluate the in vivo effects of Res combined with Gem. RESULTS: Res inhibited HCC cell proliferation, induced apoptosis and G2/M arrest, and suppressed invasion in a concentration-dependent manner. Network pharmacology and TCGA analysis identified TYMS as an important target gene for Res. TYMS was highly expressed in HCC tissues and correlated with poor prognosis. Res treatment reduced TYMS expression, while molecular docking and simulation showed stable binding of Res to TYMS. TYMS levels were elevated in HuH-7/GR resistant cells. Res combined with Gem was found to reverse drug resistance, inhibit proliferation and colony formation, and induce apoptosis. The Res + Gem combination group showed the smallest tumor volume in the in vivo model. CONCLUSION: By attenuating Gem resistance through TYMS inhibition, Res holds promise as a clinically viable adjunct to Gem-based chemotherapy, offering a potential strategy to improve outcomes in HCC patients.

Resveratrol↗

PubMed1998

Synthesis of cyclic dipeptide templates, their incorporation into peptides and studies on their conformational and biological properties.

This study investigated the diastereoselective synthesis of three dipeptide templates 1, 2 and 3, which may be regarded as conformationally restricted analogs of H-Gly-Xaa-OH, in which Xaa constitutes an aromatic amino acid. Bond formation between alpha-C of Gly and the aromatic moiety was achieved by proton-catalyzed intramolecular electrophilic aromatic substitution. The absolute configuration of the dipeptide templates was determined by single-crystal X-ray crystallography or by nuclear Overhauser enhancement measurements. A protective group strategy was elaborated to allow their incorporation into peptide sequences by liquid phase as well as by solid-phase peptide synthesis. The templates were used to generate an enkephalin analog 15, a modified peptidic neurokinin antagonist 20 and two dermorphin derivatives (24 and 33). Molecular dynamic simulations with 15 and 20 revealed the preference for a turn-like motif for 15. The biological activity, as investigated by respective receptor binding and functional assays, was strongly diminished with all four derivatives, indicating that their receptor-relevant molecular geometries lie outside the examined conformational space.

Crystallography, X-Ray↗

PubMed2004

Altering the RNA-binding mode of the U1A RBD1 protein.

The N-terminal RNA-binding domain (RBD1) of the human U1A protein is evolutionarily designed to bind its RNA targets with great affinity and specificity. The physical mechanisms that modulate the coupling (local cooperativity) among amino acid residues on the extensive binding surface of RBD1 are investigated here, using mutants that replace a highly conserved glycine residue. This glycine residue, at the strand/loop junction of beta3/loop3, is found in U1A RBD1, and in most RBD domains, suggesting it has a specific role in modulation of RNA binding. Here, two RBD1 proteins are constructed in which that residue (Gly53) is replaced by either alanine or valine. These new proteins are shown by NMR methods and molecular dynamics simulations to be very similar to the wild-type RBD1, both in structure and in their backbone dynamics. However, RNA-binding assays show that affinity for the U1 snRNA stem-loop II RNA target is reduced by nearly 200-fold for the RBD1-G53A protein, and by 1.6 x 10(4)-fold for RBD1-G53V. The mode of RNA binding by RBD1-G53A is similar to that of RBD1-WT, displaying its characteristic non-additive free energies of base recognition and its salt-dependence. The binding mode of RBD1-G53V is altered, having lost its salt-dependence and displaying site-independence of base recognition. The molecular basis for this alteration in RNA-binding properties is proposed to result from the inability of the RNA to induce a change in the structure of the free protein to produce a high-affinity complex.

Binding Sites↗

PubMed1999

Understanding substrate specificity in human and parasite phosphoribosyltransferases through calculation and experiment.

We present molecular dynamics (MD) simulations on two enzymes: a human hypoxanthine-guanine-phosphoribosyltransferase (HGPRTase) and its analogue in the protozoan parasite Tritrichomonas foetus. The parasite enzyme has an additional ability to process xanthine as a substrate, making it a hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) [Chin, M. S., and Wang, C. C. (1994) Mol. Biochem. Parasitol. 63 (2), 221-229 (1)]. X-ray crystal structures of both enzymes complexed to guanine monoribosyl phosphate (GMP) have been solved, and show only subtle differences in the two active sites [Eads et al. (1994) Cell 78 (2), 325-334 (2); Somoza et al. (1996) Biochemistry 35 (22), 7032-7040 (3)]. Most of the direct contacts with the base region of the substrate are made by the protein backbone, complicating the identification of residues significantly associated with xanthine recognition. Our calculations suggest that the broader specificity of the parasite enzyme is due to a significantly more flexible base-binding region, and rationalize the effect of two mutations, R155E and D163N, that alter substrate specificity [Munagala, N. R., and Wang, C. C. (1998) Biochemistry 37 (47), 16612-16619 (4)]. In addition, our simulations suggested a double mutant (D106E/D163N) that might rescue the D163N mutant. This double mutant was expressed and assayed, and its catalytic activity was confirmed. Our molecular dynamics trajectories were also used with a structure-based design program, Pictorial Representation Of Free Energy Changes (PROFEC), to suggest parasite-selective derivatives of GMP. Our calculations here successfully rationalize the parasite-selectivity of two novel inhibitors derived from the computer-aided design of Somoza et al. (5) and demonstrate the utility of PROFEC in the design of species-selective inhibitors.

Animals↗

PubMed2026

Identification and validation of an alternatively spliced novel isoform of maspin that modulate genes involved in inflammatory and apoptotic pathways.

Maspin regulates cellular adhesion, migration, apoptosis, angiogenesis, and tumor suppression in a tissue and context-dependent manner. Its functional diversity is governed largely thorough extracellular matrix interactions, subcellular localization, and the reactive center loop (RCL), although the structural details are not well understood. To examine whether alternative splicing contributes to this heterogeneity, we analysed the SERPINB5 gene using a computational genomics approach and identified a novel 80 bp coding exon upstream of the first coding exon (E1). The alternatively spliced transcript was validated in human skin and esophagus by semi-nested touchdown PCR, quantitative real-time PCR, and Sanger sequencing. Recombinant B5N displayed a red-shifted fluorescence emission spectrum, indicating a more solvent-exposed conformation, which was supported by molecular dynamics simulations showing greater exposure of the nuclear localization signal (NLS) and the reactive center loop. Enzyme kinetic assays demonstrated concentration-dependent enhancement of tissue plasminogen activator (tPA) activity by both isoforms. In HaCaT cells, wildtype maspin produced stronger antiproliferative and anti-migratory effects, whereas B5N was only mildly antiproliferative. Annexin V/7-AAD staining revealed that wildtype maspin induced higher early apoptosis and cell death, while B5N produced lower overall cell death but a greater proportion of late apoptotic cells. RNA-seq of transfected HaCaT cells identified differentially expressed genes enriched in inflammatory, antiviral, and apoptotic pathways, which was validated by qPCR, and several of these were markedly upregulated in SARS-CoV-2 infected A549 cells. Thus, a novel N-terminally extended maspin isoform with differentially regulated gene profile is identified and validated in this study.

Apoptosis↗

PubMed2003

Role of base sequence context in conformational equilibria and nucleotide excision repair of benzo[a]pyrene diol epoxide-adenine adducts.

We investigate the influence of base sequence context on the conformations of the 10S (+)- and 10R (-)-trans-anti-[BP]-N(6)-dA adducts through molecular dynamics (MD) simulations with free energy calculations, and relate the structural findings to results of nucleotide excision repair (NER) assays in human cell extracts. In previous studies, these adducts were studied in the CA*A sequence context, and here we report results for the CA*C sequence. Our simulations indicate that the base sequence context affects the syn-anti conformational equilibrium in the 10S (+) adduct by modulating the barrier heights between these states on the energy surface, with a higher barrier in the CA*C case. Our nucleotide excision repair assay finds greater NER susceptibilities in the 10S (+) adduct for the CA*C sequence context. A structural rationale ties together these results. A sequence specific hydrogen bond, accompanied by a significantly increased roll and consequent bending in the 10S (+) adduct, has been found in our simulations for the CA*C sequence, which could account for the enhanced nucleotide excision repair as well as the syn-anti equilibrium difference we observe in this isomer and sequence. Such sequence specific differential repair could contribute to the existence of mutational hotspots and thereby contribute to the complexity of cancer initiation.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2025

TENT5C functions as a corepressor in the ligand-bound glucocorticoid receptor and estrogen receptor α complexes.

Terminal nucleotidyltransferase 5C (TENT5C) is a noncanonical poly(A) polymerase that promotes cancer suppression. TENT5C has been proposed to mediate the susceptibility of multiple myeloma to treatment with dexamethasone, a steroid hormone analog that binds to the glucocorticoid receptor (GR). However, the relationship between TENT5C and nuclear receptor (NR) signaling remains unclear. In this study, we investigate the regulatory role of TENT5C in the GR and estrogen receptor α (ERα) ligand complexes. We find that TENT5C acts as a corepressor of both GR and ERα. Molecular dynamics simulations indicate that the third TENT5C LXXLL motif directly interacts with ERα, but not GR. The physical interaction of TENT5C and ERα is supported by co-immunoprecipitation assays. Reporter assays show that mutations to the third TENT5C LXXLL motif disrupt TENT5C-mediated repression of ERα but do not affect the repression of the GR complex. In addition, the disruption of TENT5C poly(A) polymerase activity does not appear to affect TENT5C repression of ERα in the cell lines studied. Taken together, our findings highlight a role of TENT5C as an NR corepressor, differentially modulating GR- and ERα-induced transcriptional activity.

Receptors, Glucocorticoid↗