410 research outputs found
The Crucial Step in Ether Phospholipid Biosynthesis: Structural Basis of a Noncanonical Reaction Associated with a Peroxisomal Disorder
No full textSummaryEther phospholipids are essential constituents of eukaryotic cell membranes. Rhizomelic chondrodysplasia punctata type 3 is a severe peroxisomal disorder caused by inborn deficiency of alkyldihydroxyacetonephosphate synthase (ADPS). The enzyme carries out the most characteristic step in ether phospholipid biosynthesis: formation of the ether bond. The crystal structure of ADPS from Dictyostelium discoideum shows a fatty-alcohol molecule bound in a narrow hydrophobic tunnel, specific for aliphatic chains of 16 carbons. Access to the tunnel is controlled by a flexible loop and a gating helix at the protein-membrane interface. Structural and mutagenesis investigations identify a cluster of hydrophilic catalytic residues, including an essential tyrosine, possibly involved in substrate proton abstraction, and the arginine that is mutated in ADPS-deficient patients. We propose that ether bond formation might be orchestrated through a covalent imine intermediate with the flavin, accounting for the noncanonical employment of a flavin cofactor in a nonredox reaction
Binding Affinity Ranking at the Molecular Initiating Event (BARMIE): an open- source computational pipeline for ecological hazard ranking of endocrine disrupting chemicals
No full textOne of the key challenges in ecological risk assessment lies in identifying the chemicals that pose the greatest threat and determining the species that are most vulnerable to their effects. Computational prediction of protein binding affinity can help in assessing the risk of chemicals to species. In this study we developed and validated an open-source tool called BARMIE (Binding Affinity Ranking at the Molecular Initiating Event) to rank chemical hazards and identify species that are most susceptible based on the binding affinity of the chemical to steroid receptor proteins. As an exemplar of BARMIE’s output we focus on 163 teleost fish glucocorticoid receptors (GRs) and the natural ligand cortisol and 10 synthetic glucocorticoid (GCs) drugs and five other potential chemical GR agonists. The hazard ranking is based on the likelihood that the chemicals with the highest binding affinity are likely to outcompete cortisol at the receptor binding site. In this analysis, halcinonide, a GC, was predicted to be the most hazardous based on its binding affinity and the superorder Protacanthopterygii species, including the Esociformes and Salmoniformes, were identified as the most vulnerable. This computational pipeline can be expanded to evaluate more chemicals, species, and proteins as part of an in silico chemical hazard assessment tool
Machine Learning Prediction of Allosteric Drug Activity from Molecular Dynamics
No full textAllosteric drugs have been attracting increasing interest over the past few years. In this context, it is common practice to use high-throughput screening for the discovery of non-natural allosteric drugs. While the discovery stage is supported by a growing amount of biological information and increasing computing power, major challenges still remain in selecting allosteric ligands and predicting their effect on the target protein's function. Indeed, allosteric compounds can act both as inhibitors and activators of biological responses. Computational approaches to the problem have focused on variations on the theme of molecular docking coupled to molecular dynamics with the aim of recovering information on the (long-range) modulation typical of allosteric proteins
The MYC-induced Long Non-Coding RNA MINCR as a potential novel player in cell cycle regulation in prostate cancer
No full textAmplifications of chromosome 8q24 are found in various diseases, including prostate cancer, especially in cases of severe disease. Although this region, named "gene desert," contains few protein-coding genes, with c-MYC being one of the notable oncogenes within it, it is rich in long non-coding RNAs, some of which are linked to c-MYC expression.
Among them, MINCR (MYC-induced Long Non-Coding RNA) has emerged as a significant player in different cancers and also in non-tumor-related diseases such as osteoarthritis and amyotrophic lateral sclerosis.
Given the lack of information regarding the relevance of MINCR and 8q24 amplification in prostate cancer, this study aims to investigate the specific role of MINCR in the disease. By identifying patients where only MINCR chromosomal subregion is amplified without concurrent amplification of c-MYC, or patients with increased MINCR expression and no overexpression of c-MYC, we confirmed MINCR upregulation in prostate cancer cell lines and tissues compared to normal counterparts, particularly in metastatic or severe disease cases.
Bona fide MINCR isoforms were reannotated by analyzing long-read sequencing data. Overall, we found that MINCR has a preferentially cytosolic localization in prostate cell lines. Analysis of publicly available datasets demonstrated a positive correlation between MINCR expression and cell cycle-related genes, including c-MYC targets. ASO-mediated loss of function studies revealed that silencing of MINCR by independent gapmer ASOs targeting all the isoforms led to decreased cell proliferation. RNA-sequencing on MINCR-knocked down cells identified pathways related to cell cycle regulation and mitotic spindle formation among down-regulated genes and inflammatory response and cholesterol homeostasis in up-regulated ones.
Briefly, MINCR is overexpressed in prostate cancer and correlates with disease severity. It exhibits pro-proliferative and oncogenic activities, and its silencing results in downregulation of cell cycle-promoting genes, highlighting its potential as a therapeutic target, especially for cases resistant to traditional treatments. Further investigations into the activities of the single isoforms, the relationship between MINCR and the c-MYC pathway, and its additional cellular functions, are warranted
A single tyrosine hydroxyl group almost entirely controls the NADPH specificity of Plasmodium falciparum ferredoxin-NADP(+) reductase
No full textPlasmodium falciparum ferredoxin-NADP(+) reductase (FNR) is a FAD-containing enzyme that, in addition to be a promising target of novel antimalarial drugs, represents an excellent model of plant-type FNRs. The cofactor specificity of FNRs depends on differences in both k(cat) and K(m) values for NADPH and NADH. Here, we report that deletion of the hydroxyl group of the conserved Y258 of P. falciparum FNR, which interacts with the 2'-phosphate group of NADPH, selectively decreased the k(cat) of the NADPH-dependent reaction by a factor of 2 to match that of the NADH-dependent one. Rapid-reaction kinetics, active-site titrations with NADP(+), and anaerobic photoreduction experiments indicated that this effect may be the consequence of destabilization of the catalytically competent conformation of bound NADPH. Moreover, because the Y258F replacement increased the K(m) for NADPH 4-fold and decreased that for NADH 3-fold, it led to a drop in the ability of the enzyme to discriminate between the coenzymes from 70- to just 1.5-fold. The impact of the Y258F change was not affected by the presence of the H286Q mutation, which is known to enhance the catalytic activity of the enzyme. Our data highlight the major role played by the Y258 hydroxyl group in determining the coenzyme specificity of P. falciparum FNR. From the general standpoint of engineering the kinetic properties of plant-type FNRs, although P. falciparum FNR is less strictly NADPH-dependent than its homologues, the almost complete abolishment of coenzyme selectivity reported here has never been accomplished before through a single mutation
Functionally important residues from graph analysis of coevolved dynamic couplings
No full textData availability:
All files required to run the simulations (topology, coordinates, input), processed trajectories (xtc), corresponding coordinates (pdb), can be downloaded from https://doi.org/10.57760/sciencedb.15876 (PDC-3) and https://doi.org/10.5281/zenodo.13693144 (SHV-1). DyNoPy is available at https://github.com/alepandini/DyNoPy, (copy archived at Pandini, 2024).
The following previously published data sets were used
Haider S (2024) Science Data Bank Ω-Loop mutations control the dynamics of the active site by modulating a network of hydrogen bonds in PDC-3 β-lactamase.
https://doi.org/10.57760/sciencedb.15876
Haider S (2024) Zenodo Functionally Important Residues from Graph Analysis of Co-evolved Dynamical Couplings.
https://doi.org/10.5281/zenodo.13693144This article reports the analysis of coevolutionary patterns and dynamical information for identifying functionally relevant sites. These findings are considered important due to the broad utility of the unified framework and network analysis capable of revealing communities of key residues that go beyond the residue-pair concept. The data are solid and the results are clearly presented.Leverhulme Trust (RPG-2017-222)
James A Garnett
Alessandro Pandin
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No full textErscheinungsvermerk in Vorlageform: IN VENETIA, Per Alessandro de'Vecchi. M DC XXII
Role of the His57-Glu214 ionic couple located in the active site of Mycobacterium tuberculosis FprA
No full textMycobacterium tuberculosis FprA is a NADPH-ferredoxin reductase, functionally and structurally similar to the mammalian adrenodoxin reductase. It is presumably involved in supplying electrons to one or more of the pathogen's cytochrome P450s through reduced ferredoxins. It has been proposed on the basis of crystallographic data (Bossi, R. T., et al. (2002) Biochemistry 41, 8807-8818) that the highly conserved His57 and Glu214 whose side chains are H-bonded are involved in catalysis. Both residues were individually changed to nonionizable amino acyl residues through site-directed mutagenesis. Steady-state kinetics showed that the role of Glu214 in catalysis is negligible. On the contrary, the substitutions of His57 markedly impaired the catalytic efficiency of FprA for ferredoxin in the physiological reaction. Furthemore, they decreased the k(cat)/K(m) value for NADPH in the ferricyanide reduction. Rapid-reaction (stopped-flow) kinetic analysis of the isolated reductive half-reaction of wild-type and His57Gln forms of FprA with NADPH and NADH allowed a detailed description of the mechanism of enzyme-bound FAD reduction, with the identification of the intermediates involved. The His57Gln mutation caused a 6-fold decrease in the rate of hydride transfer from either NADPH or NADH to the enzyme-bound FAD cofactor. The 3D structure of FprA-H57Q, obtained at 1.8 A resolution, explains the inefficient hydride transfer of the mutant in terms of a suboptimal geometry of the nicotinamide-isoalloxazine interaction in the active site. These data demonstrate the role of His57 in the correct binding of NADPH to FprA for the subsequent steps of the catalytic cycle to proceed at a high rate
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No full textErscheinungsvermerk in Vorlageform: IN VENETIA, Per Alessandro de'Vecchi. M DC XXII
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