BACKGROUND: Mitochondrial energy can be stored as ATP or released as heat by uncoupling protein 1 (UCP1) during non-shivering thermogenesis in brown adipose tissue. UCP1, located in the inner mitochondrial membrane, reduces the proton gradient in the presence of long-chain fatty acids (FA). FA act as weak, protein-independent uncouplers, with the transport of the FA anion across the membrane being the rate-limiting step. According to the fatty acid cycling hypothesis, UCP1 catalyzes this step through an as-yet-undefined mechanism. METHODS: We used computational and experimental techniques, including all-atom molecular dynamics (MD) simulations, membrane conductance measurements, and site-directed mutagenesis. RESULTS: We identified two novel pathways for fatty acid anion translocation (sliding) at the UCP1 protein-lipid interface, ending at key arginine residues R84 and R183 in a nucleotide-binding region. This region forms a stable complex with fatty acid anion, which is crucial for anion transport. Mutations of these two arginines reduced membrane conductance, consistent with the MD simulation prediction that the arachidonic acid anion slides between helices H2-H3 and H4-H5, terminating at R84 and R183. Protonation of the arachidonic acid anion predicts its release from the protein-lipid interface, allowing it to move to either cytosolic or matrix leaflets of the membrane. CONCLUSION: We provide a novel, detailed mechanism by which UCP1 facilitates fatty acid anion transport, as part of the fatty acid cycling process originally proposed by Skulachev. The residues involved in this transport are conserved in other SLC25 proteins, suggesting the mechanism may extend beyond UCP1 to other members of the superfamily.

• Klíčová slova
• anion transporter, cardiolipin, fatty acid cycling, mitochondrial SLC25 family, molecular dynamics simulations, proton transport mechanism,
• MeSH
• iontové kanály * metabolismus   chemie   genetika   MeSH
• iontový transport fyziologie   MeSH
• lidé MeSH
• mastné kyseliny * metabolismus   MeSH
• mitochondriální proteiny * metabolismus   chemie   genetika   MeSH
• mutageneze cílená MeSH
• simulace molekulární dynamiky * MeSH
• uncoupling protein 1 MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• iontové kanály * MeSH
• mastné kyseliny * MeSH
• mitochondriální proteiny * MeSH
• uncoupling protein 1 MeSH

The Homo sapiens Na+/H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ in exchange for protons across cell membranes, and its dysfunction results in various pathologies. The activity of HsNHA2 is specifically inhibited by the flavonoid phloretin. Using bioinformatic modeling, we predicted two amino acids (R177 and S178) as being important for the binding of phloretin to the HsNHA2 molecule. Functional expression of HsNHA2 in Saccharomyces cerevisiae and its site-directed mutagenesis revealed that while the R177T mutation resulted in an antiporter that was less sensitive to phloretin, the S178T mutation enhanced the inhibitory effect of phloretin on HsNHA2. Our data corroborate the transport properties of HsNHA2 and its interactions with an inhibitor and can be helpful for the development of new therapeutics targeting this antiporter and its pleiotropic physiological functions.

• Klíčová slova
• Na+/H+ antiporter, human NHA2, phloretin inhibition, yeast,
• MeSH
• floretin * farmakologie   chemie   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• Na(+)-H(+) antiport * genetika   antagonisté a inhibitory   chemie   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• floretin * MeSH
• Na(+)-H(+) antiport * MeSH

This study introduces a novel cost-effective technique for cloning of linear DNA plasmid inserts, aiming to address the associated expenses linked with popular in vitro DNA assembly methods. Specifically, we introduce ECOLI (Efficient Cloning Of Linear Inserts), a method utilizing a PCR product-based site-directed mutagenesis. In comparison to other established in vitro DNA assembly methods, our approach is without the need for costly synthesis or specialized kits for recombination or restriction sites. ECOLI offers a fast, efficient, and economical alternative for cloning inserts up to several hundred nucleotides into plasmid constructs, thus enhancing cloning accessibility and efficiency. This method can enhance molecular biology research, as we briefly demonstrated on the Dishevelled gene from the WNT signaling pathway.

• Klíčová slova
• Dishevelled, DNA cloning, In vitro DNA assembly, Mutagenesis, PCR, Plasmid-based cloning, Site-directed mutagenesis,
• MeSH
• DNA genetika   MeSH
• klonování DNA * metody   MeSH
• mutageneze cílená * metody   MeSH
• plazmidy * genetika   MeSH
• polymerázová řetězová reakce metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH

Quantum and molecular mechanics (QM/MM) and QM-only (cluster model) modeling techniques represent the two workhorses in mechanistic understanding of enzyme catalysis. One of the stringent tests for QM/MM and/or QM approaches is to provide quantitative answers to real-world biochemical questions, such as the effect of single-point mutations on enzyme kinetics. This translates into predicting the relative activation energies to 1-2 kcal·mol-1 accuracy; such predictions can be used for the rational design of novel enzyme variants with desired/improved characteristics. Herein, we employ glutamate carboxypeptidase II (GCPII), a dizinc metallopeptidase, also known as the prostate specific membrane antigen, as a model system. The structure and activity of this major cancer antigen have been thoroughly studied, both experimentally and computationally, which makes it an ideal model system for method development. Its reaction mechanism is quite well understood: the reaction coordinate comprises a "tetrahedral intermediate" and two transition states and experimental activation Gibbs free energy of ∼17.5 kcal·mol-1 can be inferred for the known kcat ≈ 1 s-1. We correlate experimental kinetic data (including the E424H variant, newly characterized in this work) for various GCPII mutants (kcat = 8.6 × 10-5 s-1 to 2.7 s-1) with the energy profiles calculated by QM/MM and QM-only (cluster model) approaches. We show that the near-quantitative agreement between the experimental values and the calculated activation energies (ΔH⧧) can be obtained and recommend the combination of the two protocols: QM/MM optimized structures and cluster model (QM) energetics. The trend in relative activation energies is mostly independent of the QM method (DFT functional) used. Last but not least, a satisfactory correlation between experimental and theoretical data allows us to provide qualitative and fairly simple explanations of the observed kinetic effects which are thus based on a rigorous footing.

• MeSH
• glutamátkarboxypeptidasa II * genetika   metabolismus   MeSH
• kinetika MeSH
• kvantová teorie MeSH
• lidé MeSH
• mutageneze cílená MeSH
• simulace molekulární dynamiky * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• glutamátkarboxypeptidasa II * MeSH

Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

• MeSH
• AICDA (aktivací indukovaná cytidindeamináza) MeSH
• aktivace lymfocytů MeSH
• Burkittův lymfom genetika   imunologie   MeSH
• cytidindeaminasa genetika   MeSH
• genetická transkripce MeSH
• imunoglobuliny genetika   metabolismus   MeSH
• kur domácí MeSH
• lidé MeSH
• mutace genetika   MeSH
• mutageneze cílená MeSH
• podskupiny B-lymfocytů imunologie   MeSH
• ptačí proteiny genetika   metabolismus   MeSH
• RNA-polymerasa II genetika   metabolismus   MeSH
• rozmanitost protilátek MeSH
• somatická hypermutace imunoglobulinových genů MeSH
• zesilovače transkripce genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• AICDA (aktivací indukovaná cytidindeamináza) MeSH
• cytidindeaminasa MeSH
• imunoglobuliny MeSH
• ptačí proteiny MeSH
• RNA-polymerasa II MeSH

The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.

• MeSH
• buněčné linie MeSH
• cirkulární dichroismus MeSH
• cystein genetika   MeSH
• dánio pruhované MeSH
• down regulace MeSH
• isoindoly farmakologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• mozek metabolismus   MeSH
• mutageneze cílená MeSH
• organoselenové sloučeniny farmakologie   MeSH
• proteiny 14-3-3 chemie   genetika   metabolismus   MeSH
• proteiny dánia pruhovaného chemie   metabolismus   MeSH
• stabilita proteinů účinky léků   MeSH
• tyrosin-3-monooxygenasa metabolismus   MeSH
• vazba proteinů účinky léků   MeSH
• vazebná místa účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• ebselen MeSH Prohlížeč
• isoindoly MeSH
• organoselenové sloučeniny MeSH
• proteiny 14-3-3 MeSH
• proteiny dánia pruhovaného MeSH
• tyrosin-3-monooxygenasa MeSH
• YWHAZ protein, human MeSH Prohlížeč

Larvae of many lepidopteran species produce a mixture of secretory proteins, known as silk, for building protective shelters and cocoons. Silk consists of a water-insoluble silk filament core produced in the posterior silk gland (PSG) and a sticky hydrophilic coating produced by the middle silk gland (MSG). In Bombyx mori, the fiber core comprises three proteins: heavy chain fibroin (Fib-H), light chain fibroin (Fib-L) and fibrohexamerin (Fhx, previously referred to as P25). To learn more about the role of Fhx, we used transcription activator-like effector nuclease (TALEN) mutagenesis and prepared a homozygous line with a null mutation in the Fhx gene. Our characterization of cocoon morphology and silk quality showed that the mutation had very little effect. However, a detailed inspection of the secretory cells in the posterior silk gland (PSG) of mid-last-instar mutant larvae revealed temporary changes in the morphology of the endoplasmic reticulum. We also observed a morphological difference in fibroin secretory globules stored in the PSG lumen of Fhx mutants, which suggests that their fibroin complexes have a slightly lower solubility. Finally, we performed an LC-MS-based quantitative proteomic analysis comparing mutant and wild-type (wt) cocoon proteins and found a high abundance of a 16 kDa secretory protein likely involved in fibroin solubility. Overall, our study shows that whilst Fhx is dispensable for silk formation, it contributes to the stability of fibroin complexes during intracellular transport and affects the morphology of fibroin secretory globules in the PSG lumen.

• Klíčová slova
• BMSK0001030, BMSK0001060, ER stress, ER whorls, Gene editing, Targeted mutagenesis,
• MeSH
• bourec * genetika   ultrastruktura   MeSH
• endoplazmatické retikulum metabolismus   ultrastruktura   MeSH
• fibroiny genetika   metabolismus   ultrastruktura   MeSH
• hedvábí * chemie   genetika   MeSH
• mutace MeSH
• mutageneze cílená metody   MeSH
• slinné žlázy * cytologie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fhx protein, Bombyx mori MeSH Prohlížeč
• fibroiny MeSH
• hedvábí * MeSH

The wide variety of protein structures and functions results from the diverse properties of the 20 canonical amino acids. The generally accepted hypothesis is that early protein evolution was associated with enrichment of a primordial alphabet, thereby enabling increased protein catalytic efficiencies and functional diversification. Aromatic amino acids were likely among the last additions to genetic code. The main objective of this study was to test whether enzyme catalysis can occur without the aromatic residues (aromatics) by studying the structure and function of dephospho-CoA kinase (DPCK) following aromatic residue depletion. We designed two variants of a putative DPCK from Aquifex aeolicus by substituting (a) Tyr, Phe and Trp or (b) all aromatics (including His). Their structural characterization indicates that substituting the aromatics does not markedly alter their secondary structures but does significantly loosen their side chain packing and increase their sizes. Both variants still possess ATPase activity, although with 150-300 times lower efficiency in comparison with the wild-type phosphotransferase activity. The transfer of the phosphate group to the dephospho-CoA substrate becomes heavily uncoupled and only the His-containing variant is still able to perform the phosphotransferase reaction. These data support the hypothesis that proteins in the early stages of life could support catalytic activities, albeit with low efficiencies. An observed significant contraction upon ligand binding is likely important for appropriate organization of the active site. Formation of firm hydrophobic cores, which enable the assembly of stably structured active sites, is suggested to provide a selective advantage for adding the aromatic residues.

• Klíčová slova
• aromatic amino acids, catalysis evolution, genetic code evolution, protein disorder, protein structure evolution,
• MeSH
• Aquifex enzymologie   genetika   MeSH
• bakteriální proteiny chemie   genetika   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem chemie   genetika   MeSH
• katalytická doména MeSH
• katalýza MeSH
• mutageneze cílená MeSH
• sekundární struktura proteinů MeSH
• substituce aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• dephospho-CoA kinase MeSH Prohlížeč
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH

Protein engineering is the discipline of developing useful proteins for applications in research, therapeutic, and industrial processes by modification of naturally occurring proteins or by invention of de novo proteins. Modern protein engineering relies on the ability to rapidly generate and screen diverse libraries of mutant proteins. However, design of mutant libraries is typically hampered by scale and complexity, necessitating development of advanced automation and optimization tools that can improve efficiency and accuracy. At present, automated library design tools are functionally limited or not freely available. To address these issues, we developed Mutation Maker, an open source mutagenic oligo design software for large-scale protein engineering experiments. Mutation Maker is not only specifically tailored to multisite random and directed mutagenesis protocols, but also pioneers bespoke mutagenic oligo design for de novo gene synthesis workflows. Enabled by a novel bundle of orchestrated heuristics, optimization, constraint-satisfaction and backtracking algorithms, Mutation Maker offers a versatile toolbox for gene diversification design at industrial scale. Supported by in silico simulations and compelling experimental validation data, Mutation Maker oligos produce diverse gene libraries at high success rates irrespective of genes or vectors used. Finally, Mutation Maker was created as an extensible platform on the notion that directed evolution techniques will continue to evolve and revolutionize current and future-oriented applications.

• Klíčová slova
• PCR-based accurate synthesis, directed evolution, gene synthesis, multi site-directed mutagenesis, protein design, protein engineering, site-scanning saturation mutagenesis, synthetic biology,
• MeSH
• algoritmy MeSH
• Escherichia coli genetika   MeSH
• genová knihovna MeSH
• kodon genetika   MeSH
• mutace * MeSH
• mutageneze cílená metody   MeSH
• mutageneze * MeSH
• mutantní proteiny MeSH
• oligonukleotidy genetika   MeSH
• počítačová simulace MeSH
• proteiny genetika   MeSH
• řízená evoluce molekul metody   MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kodon MeSH
• mutantní proteiny MeSH
• oligonukleotidy MeSH
• proteiny MeSH

Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.

• Klíčová slova
• HEK293T cells, P2X7 receptor, deactivation, dye uptake, extracellular vestibule, gating, mutagenesis,
• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• gating iontového kanálu MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• kinetika MeSH
• konzervovaná sekvence MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• luminescentní proteiny chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• mutantní proteiny chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• purinergní receptory P2X7 chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• statická elektřina MeSH
• substituce aminokyselin MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• luminescentní proteiny MeSH
• mutantní proteiny MeSH
• P2rx7 protein, rat MeSH Prohlížeč
• purinergní receptory P2X7 MeSH
• rekombinantní fúzní proteiny MeSH
• yellow fluorescent protein, Bacteria MeSH Prohlížeč