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Comparative Protein Modelling by Satisfaction of Spatial Restraints
Acta Crystallographica Section A: Foundations and Advances
... Similarly, for the signi cantly downregulated gene Histone -H2AC14, modeling of its structure was performed based on the translated amino acid sequence. This model was aligned with the sequence of PDB model-5KGF Chain C using PyMOL, with red-highlighted segments indicating mismatches, damage, or deletions [113][114][115][116]. Ramachandran plots and model-template sequence alignment data were retrieved from the SWISS-Model database server [117]. ...
Glioblastoma multiforme (GBM) is one of the most common and aggressive forms of malignant brain cancer in adults and is classified based on its isocitrate dehydrogenase (IDH) mutation. Surgery, radiotherapy, and Temozolomide (TMZ) are the standard treatment methods for GBM. Here we present a combination therapy of cold atmospheric plasma (CAP) and TMZ as a key treatment for GBM. CAP works by increasing reactive oxygen and nitrogen species (RONS) and targets the spread of the tumor. In this study, we performed the transcriptomic analysis of U-87MG cells by high throughput deep RNA-Seq analysis to quantify differential gene expression across the genome. Furthermore, we studied various signaling pathways and predicted structural changes of consequential proteins to elucidate the functional changes caused by up or down-regulation of the most altered genes. Our results demonstrate that combination treatment downregulated key genes like p53, histones, DNA damage markers, cyclins, in the following pathways: MAPK, P53, DNA damage and cell cycle. Moreover, in silico studies were conducted for further investigation to verify these results, and the combination of CAP & TMZ showed a significant antitumor effect in the GBM cells leading to apoptosis and damaged key proteins. Further studies of the impact of TMZ on gene expression, biochemical pathways, and protein structure will lead to improved treatment approaches for GBM.
... In this study, we utilized the hSERT structure (PDB ID: 5I71; (Coleman et al., 2016)). To generate homology models of hDAT based on the hSERT structure, we employed MODELER (Šali & Blundell, 1993). ...
3,4‐Methylenedioxymethamphetamine (MDMA, ‘ecstasy’) is re‐emerging in clinical settings as a candidate for the treatment of specific neuropsychiatric disorders (e.g. post‐traumatic stress disorder) in combination with psychotherapy. MDMA is a psychoactive drug, typically regarded as an empathogen or entactogen, which leads to transporter‐mediated monoamine release. Despite its therapeutic potential, MDMA can induce dose‐, individual‐, and context‐dependent untoward effects outside safe settings. In this study, we investigated whether three new methylenedioxy bioisosteres of MDMA improve its off‐target profile. In vitro methods included radiotracer assays, transporter electrophysiology, bioluminescence resonance energy transfer and fluorescence‐based assays, pooled human liver microsome/S9 fraction incubations, metabolic stability studies, isozyme mapping, and liquid chromatography coupled to high‐resolution mass spectrometry. In silico methods included molecular docking. Compared with MDMA, all three MDMA bioisosteres (ODMA, TDMA, and SeDMA) showed similar pharmacological activity at human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) but decreased agonist activity at 5‐HT2A/2B/2C receptors. Regarding their hepatic metabolism, they differed from MDMA, with N‐demethylation being the only metabolic route shared, and without forming phase II metabolites. In addition, TDMA showed an enhanced intrinsic clearance in comparison to its congeners. Additional screening for their interaction with human organic cation transporters (hOCTs) and plasma membrane monoamine transporter (hPMAT) revealed a weaker interaction of the MDMA analogs with hOCT1, hOCT2, and hPMAT. Our findings suggest that these new MDMA bioisosteres might constitute appealing therapeutic alternatives to MDMA, sparing the primary pharmacological activity at hSERT, hDAT, and hNET, but displaying a reduced activity at 5‐HT2A/2B/2C receptors and alternative hepatic metabolism. Whether these MDMA bioisosteres may pose lower risk alternatives to the clinically re‐emerging MDMA warrants further studies. image
... Nucleotide ligands at the M-and I-sites were removed as their physiological roles are to prevent filament formation (which is irrelevant in this simulation), while Mg 2+ -TTP was kept at the S-site because of its essential role in dimer formation. Modeller (49) was then used to build unmodeled regions. Both α C-terminal tails were manually moved away from the TrxA binding interface in COOT as transient interactions between the C-termini and the TrxA binding interface were observed in many of our MD runs (results not shown). ...
Understanding the structural dynamics associated with enzymatic catalysis has been a long-standing goal of structural biology. A wide range of motions, from small side-chain fluctuations to large domain rearrangements, have been implicated in enzyme function by experimental and computational studies. However, because structural techniques generally depend on averaging, direct visualization of conformational landscapes during turnover has been challenging. Here, we report the conformational landscapes of a class I ribonucleotide reductase (RNR) in various stages of turnover using single-particle cryo-electron microscopy (cryo-EM) and a combination of classification and deep-learning-based analyses. RNRs are responsible for the conversion of ribonucleotides to deoxyribonucleotides, a reaction that is essential for all DNA-based life. Class I RNRs, used by humans and other aerobic organisms, perform a complex series of chemical steps that are coupled with the dynamics of two highly mobile subunits, which can be resolved by EM. We demonstrate that despite the dimeric nature of the enzyme and its intrinsic dynamics, remarkable asymmetry is maintained across the class I RNR complex that physically segregates the two halves of its turnover cycle.
... A model of the actomyosin complex An actin-myosin 1:1 complex was modelled based on chains A and F of a cryo-EM structure (PDB:5JLH (von der Ecken et al., 2016)). The missing loops of non-muscle myosin IIC were modelled with MODELLER (Šali and Blundell, 1993) via UCSF Chimera (Pettersen et al., 2004). Molecular dynamic simulation was performed with GROMACS (https://www.gromacs.org/ ...
Actin is an evolutionarily conserved cytoskeletal protein with crucial roles in cell polarity, division, migration, and muscle contraction. Actin function is regulated in part by posttranslational modifications. One such modification in non-muscle cells is arginylation, in which an arginine residue is added to the N-terminus of β-actin. What is the structure of arginylated β-actin (R-β-actin), are its interactions with other proteins altered and what phenotypes result when R-β-actin is the sole actin isoform present in the cell? Here we report the 4.2 Å structure of ADP-bound human R-β-actin filaments, the overall structure of which is nearly identical to the filaments made of non-arginylated actin. In vitro functional assays using isoform-pure actins with defined post-translational modifications reveal that the interaction between myosin-II and actin is altered upon actin arginylation, due to frequent detachment of myosin-II from R-actin filaments. In vivo, we find that replacement of the only actin gene in Schizosaccharomyces pombe with a synthetic gene encoding R-Sp-actin reduces Arp2/3-based actin patches while thickening the formin-induced actin. Furthermore, consistent with altered interactions between myosin-II and R-actin filaments, the assembly and constriction of cytokinetic actomyosin ring are perturbed in the R-Sp-actin cells. Thus, despite the overall structural similarity of arginylated and non-arginylated actin filaments, actin arginylation affects actin filament assortment into distinct subcellular structures and its interaction with myosin II.
... Finally, the geometry of the resulting model is regularized by using a force field in the case of loop modelling with Promod-II [23]. If it does not give satisfactory results, an alternative model is built with MODELLER [24]. ...
Human gut Bacteroides species encode numerous (eight or more) tightly regulated capsular polysaccharides (CPS). Specialized paralogs of the universal transcription elongation factor NusG, called UpxY (Y), and an anti-Y UpxZ (Z) are encoded by the first two genes of each CPS operon. The Y-Z regulators combine with promoter inversions to limit CPS transcription to a single operon in most cells. Y enhances transcript elongation whereas Z inhibits noncognate Ys. How Y distinguishes among cognate CPS operons and how Z inhibits only noncognate Ys are unknown. Using in-vivo nascent-RNA sequencing and p romoter-less in v itr o transcription (PIVoT), we establish that Y recognizes a paused RNA polymerase via sequences in both the exposed non-template DNA and the upstream duplex DNA. Y association is aided by novel ‘pause-then-escape’ nascent RNA hairpins. Z binds non-cognate Ys to directly inhibit Y association. This Y-Z hierarchical regulatory program allows Bacteroides to create CPS subpopulations for optimal fitness.
Estradiol dimers (EDs) possess significant anticancer activity by targeting tubulin dynamics. In this study, we synthesised 12 EDs variants via copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction, focusing on structural modifications within the aromatic bridge connecting two estradiol moieties. In vitro testing of these EDs revealed a marked improvement in selectivity towards cancerous cells, particularly for ED1–8. The most active compounds, ED3 (IC50 = 0.38 μM in CCRF-CEM) and ED5 (IC50 = 0.71 μM in CCRF-CEM) demonstrated cytotoxic effects superior to 2-methoxyestradiol (IC50 = 1.61 μM in CCRF-CEM) and exhibited anti-angiogenic properties in an endothelial cell tube-formation model. Cell-based experiments and in vitro assays revealed that EDs interfere with mitotic spindle assembly. Additionally, we proposed an in silico model illustrating the probable binding modes of ED3 and ED5, suggesting that dimers with a simple linker and a single substituent on the aromatic central ring possess enhanced characteristics compared to more complex dimers.
In the quest for new bioactive substances, nonribosomal peptide synthetases (NRPS) provide biodiversity by synthesizing nonproteinaceous peptides with high cellular activity. NRPS machinery consists of multiple modules, each catalyzing a unique series of chemical reactions. Incomplete understanding of the biophysical principles orchestrating these reaction arrays limits the exploitation of NRPSs in synthetic biology. Here, we use nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to solve the conundrum of how intermodular recognition is coupled with loaded carrier protein specificity in the tomaymycin NRPS. We discover an adaptor domain that directly recruits the loaded carrier protein from the initiation module to the elongation module and reveal its mechanism of action. The adaptor domain of the type found here has specificity rules that could potentially be exploited in the design of engineered NRPS machinery.
The guanine exchange factor SOS1 is a crucial node into the positive feedback regulation of the KRAS signaling pathway. Currently, the regulation of KRAS-SOS1 interactions and KRAS downstream effector proteins has become a new hotspot in the development of KRAS-driven cancer therapies. However, the detailed dynamic mechanisms of SOS1-catalyzed GDP extraction and the impact of KRAS mutations remain unknown. Herein, the main mechanisms of GDP extraction from KRAS oncogenes by means of the guanine exchange factor SOS1 are disclosed and described with full details at the atomic-level. For GDP-bound wild-type KRAS, four amino acids (Lys811, Glu812, Lys939 and Glu942) responsible for the catalytic function of SOS1 were identified. With the occurrence of KRAS-G12D mutation, the GDP extraction rate is significantly increased. The molecular interactions behind this phenomenon have been subsequently identified being mainly hydrogen bonding interactions between the mutated residue Asp12 and a positively charged pocket located at the intrinsically disordered region807−818 and composed by Ser807, Trp809, Thr810 and Lys811. Our findings provide new insights into the SOS1-KRAS interactions and facilitate the development of related anti-cancer strategies based on the blockage of the above described mechanisms.
A growing body of literature implicates host-associated microbiota in the modulation of circulating androgen levels in the host, which could have far-reaching implications for androgen-mediated diseases. However, the microbial genetic pathways involved in androgen production remain unknown. Here, we report the first host-associated microbial gene ( desF ) encoding an enzyme that catalyzes conversion of androstenedione to epitestosterone (epiT) in the gut bacterium, Clostridium scindens . Despite current dogma that epiT is a nuclear androgen-receptor (AR) antagonist, we demonstrate that epiT is a potent androgen, as assessed by its ability to promote prostate cancer cell growth and expression of prostate specific antigen (PSA). We then quantified the desF gene in fecal samples collected from individuals with advanced prostate cancer (rising blood PSA) undergoing androgen deprivation therapy combined with abiraterone acetate and prednisone (AA/P). Strikingly, fecal desF levels were elevated in a subset of individuals progressing on AA/P versus samples taken during AA/P response (stable). Importantly, we observed that AA does not inhibit the bacterial desmolase enzyme that is analogous to the human drug target of AA. We then determined that bacterial isolates from urine or prostatectomy tissue are capable of androgen production. From these isolates we detected 17β-hydroxysteroid dehydrogenase (17β-HSDH) activity, which has not been previously reported in urinary tract bacteria, and discovered the desG gene in urinary isolates encoding 17β-HSDH that catalyzed conversion of androstenedione to testosterone. Applying advanced artificial intelligence and molecular dynamics, we predict the structures and ligand binding to DesF and DesG. Using a novel bioengineered microencapsulation technique, we demonstrate that urinary androgen-producing bacterial strains can also promote prostate cancer cell growth through steroid metabolism. Taken together, our results are a significant advance for steroid microbiology in humans and suggest that these microbial biotransformations should be further studied in the context of androgen-mediated physiological processes and diseases.
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