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1.
J Biol Chem ; 300(9): 107649, 2024 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-39122011

RESUMEN

Amaryllidaceae alkaloids are a diverse group of alkaloids exclusively reported from the Amaryllidaceae plant family. In planta, their biosynthesis is still not fully characterized; however, a labeling study established 4'-O-methylnorbelladine as the key intermediate compound of the pathway. Previous reports have characterized O-methyltransferases from several Amaryllidaceae species. Nevertheless, the formation of the different O-methylnorbelladine derivatives (3'-O-methylnorbelladine, 4'-O-methylnorbelladine, and 3'4'-O-dimethylnorbelladine), the role, and the preferred substrates of O-methyltransferases are not clearly understood. In this study, we performed the biochemical characterization of an O-methyltransferase candidate from Narcissus papyraceus (NpOMT) in vitro and in vivo, following biotransformation of norbelladine in Nicotiana benthamiana having transient expression of NpOMT. Docking analysis was further used to investigate substrate preferences, as well as key interacting residues of NpOMT. Our study shows that NpOMT methylates norbelladine preferentially at the 4'-OH position in vitro and in planta. Interestingly, NpOMT also catalyzed the synthesis of 3',4'-O-dimethylnorbelladine from norbelladine and 4'-O-methylnorbelladine during in vitro enzymatic assay. Furthermore, we show that NpOMT methylates 3,4-dihydroxybenzylaldehyde and caffeic acid in a nonregiospecific manner to produce meta/para monomethylated products. This study reveals a novel catalytic potential of an Amaryllidaceae O-methyltransferase and its ability to regioselectively methylate norbelladine in the heterologous host N. benthamiana.

2.
Proc Natl Acad Sci U S A ; 121(28): e2402543121, 2024 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-38959031

RESUMEN

The outer membrane (OM) of gram-negative bacteria serves as a vital organelle that is densely populated with OM proteins (OMPs) and plays pivotal roles in cellular functions and virulence. The assembly and insertion of these OMPs into the OM represent a fundamental process requiring specialized molecular chaperones. One example is the translocation and assembly module (TAM), which functions as a transenvelope chaperone promoting the folding of specific autotransporters, adhesins, and secretion systems. The catalytic unit of TAM, TamA, comprises a catalytic ß-barrel domain anchored within the OM and three periplasmic polypeptide-transport-associated (POTRA) domains that recruit the TamB subunit. The latter acts as a periplasmic ladder that facilitates the transport of unfolded OMPs across the periplasm. In addition to their role in recruiting the auxiliary protein TamB, our data demonstrate that the POTRA domains mediate interactions with the inner surface of the OM, ultimately modulating the membrane properties. Through the integration of X-ray crystallography, molecular dynamic simulations, and biomolecular interaction methodologies, we located the membrane-binding site on the first and second POTRA domains. Our data highlight a binding preference for phosphatidylglycerol, a minor lipid constituent present in the OM, which has been previously reported to facilitate OMP assembly. In the context of the densely OMP-populated membrane, this association may serve as a mechanism to secure lipid accessibility for nascent OMPs through steric interactions with existing OMPs, in addition to creating favorable conditions for OMP biogenesis.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas de Escherichia coli , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Periplasma/metabolismo , Dominios Proteicos , Pliegue de Proteína
3.
Biochemistry ; 63(10): 1257-1269, 2024 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-38683758

RESUMEN

Interactions between SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four analogs thereof with model bacterial membranes were studied using Fourier-transform infrared spectroscopy (FTIR) and molecular dynamics (MD) simulations. MD trajectory analyses showed that the N-terminal segment of the peptide analogs has many contacts with the polar heads of membrane phospholipids, while the central α helix interacts strongly with the hydrophobic core of the membranes. The peptides also had a marked influence on the wave numbers associated with the phase transition of phospholipids organized as liposomes in both the interface and aliphatic chain regions of the infrared spectra, supporting the interactions observed in the MD trajectories. In addition, interesting links were found between peptide interactions with the aliphatic chains of membrane phospholipids, as determined by FTIR and from the MD trajectories, and the membrane permeabilization capacity of these peptide analogs, as previously demonstrated. To summarize, the combined experimental and computational efforts have provided insights into crucial aspects of the interactions between the investigated peptides and bacterial membranes. This work thus makes an original contribution to our understanding of the molecular interactions underlying the antimicrobial activity of these GAPDH-related antimicrobial peptides from Scombridae.


Asunto(s)
Péptidos Antimicrobianos , Membrana Celular , Proteínas de Peces , Animales , Secuencia de Aminoácidos , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Proteínas de Peces/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Simulación de Dinámica Molecular , Espectroscopía Infrarroja por Transformada de Fourier
4.
PLoS Genet ; 20(4): e1011252, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38683847

RESUMEN

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.


Asunto(s)
Farmacorresistencia Fúngica , Antagonistas del Ácido Fólico , Metotrexato , Mutación , Pneumocystis carinii , Tetrahidrofolato Deshidrogenasa , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Pneumocystis carinii/genética , Pneumocystis carinii/enzimología , Pneumocystis carinii/efectos de los fármacos , Antagonistas del Ácido Fólico/farmacología , Farmacorresistencia Fúngica/genética , Metotrexato/farmacología , Regulación Alostérica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Humanos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Dominio Catalítico/genética
5.
Antibiotics (Basel) ; 12(9)2023 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-37760707

RESUMEN

The antimicrobial activity of SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four chemical analogs thereof was determined under different physicochemical conditions, including different pH values, the presence of monovalent and divalent cations, and after a heating treatment. The toxicity of these five peptides was also studied with hemolytic activity assays, while their stability under human gastrointestinal conditions was evaluated using a dynamic in vitro digestion model and chromatographic and mass spectrometric analyses. The antibacterial activity of all analogs was found to be inhibited by the presence of divalent cations, while monovalent cations had a much less pronounced impact, even promoting the activity of the native SJGAP. The peptides were also more active at acidic pH values, but they did not all show the same stability following a heat treatment. SJGAP and its analogs did not show significant hemolytic activity (except for one of the analogs at a concentration equivalent to 64 times that of its minimum inhibitory concentration), and the two analogs whose digestibility was studied degraded very rapidly once they entered the stomach compartment of the digestion model. This study highlights for the first time the characteristics of antimicrobial peptides from Scombridae or homologous to GAPDH that are directly related to their potential clinical or food applications.

6.
ACS Appl Mater Interfaces ; 15(14): 17922-17937, 2023 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-37010879

RESUMEN

Mining practices, chiefly froth flotation, are being critically reassessed to replace their use of biohazardous chemical reagents in favor of biofriendly alternatives as a path toward green processes. In this regard, this study aimed at evaluating the interactions of peptides, as potential floatation collectors, with quartz using phage display and molecular dynamics (MD) simulations. Quartz-selective peptide sequences were initially identified by phage display at pH = 9 and further modeled by a robust simulation scheme combining classical MD, replica exchange MD, and steered MD calculations. Our residue-specific analyses of the peptides revealed that positively charged arginine and lysine residues were favorably attracted by the quartz surface at basic pH. The negatively charged residues at pH 9 (i.e., aspartic acid and glutamic acid) further showed affinity toward the quartz surface through electrostatic interactions with the positively charged surface-bound Na+ ions. The best-binding heptapeptide combinations, however, contained both positively and negatively charged residues in their composition. The flexibility of peptide chains was also shown to directly affect the adsorption behavior of the peptide. While attractive intrapeptide interactions were dominated by a weak peptide-quartz binding, the repulsive self-interactions in the peptides improved the binding propensity to the quartz surface. Our results showed that MD simulations are fully capable of revealing mechanistic details of peptide adsorption to inorganic surfaces and are an invaluable tool to accelerate the rational design of peptide sequences for mineral processing applications.


Asunto(s)
Péptidos , Cuarzo , Cuarzo/química , Péptidos/química , Secuencia de Aminoácidos , Simulación de Dinámica Molecular , Minerales , Adsorción
7.
Am J Med Genet A ; 191(6): 1508-1517, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36864778

RESUMEN

Variants of filamin C (FLNC) have been identified as rare genetic substrate for hypertrophic cardiomyopathy (HCM). Data on the clinical course of FLNC-related HCM are conflicting with some studies suggesting mild phenotypes whereas other studies have reported more severe outcomes. In this study, we present a novel FLNC variant (Ile1937Asn) that was identified in a large family of French-Canadian descent with excellent segregation data. FLNC-Ile1937Asn is a novel missense variant characterized by full penetrance and poor clinical outcomes. End stage heart failure requiring transplantation occurred in 43% and sudden cardiac death in 29% of affected family members. Other particular features of FLNC-Ile1937Asn include an early disease onset (mean age of 19 years) and the development of a marked atrial myopathy (severe biatrial dilatation with remodeling and multiple complex atrial arrhythmias) that was present in all gene carriers. The FLNC-Ile1937Asn variant is a novel, pathogenic mutation resulting in a severe form of HCM with full disease penetrance. The variant is associated with a high proportion of end-stage heart failure, heart transplantation, and disease-related mortality. Close follow-up and appropriate risk stratification of affected individuals at specialized heart centers is recommended.


Asunto(s)
Fibrilación Atrial , Cardiomiopatía Hipertrófica , Cardiomiopatía Restrictiva , Insuficiencia Cardíaca , Humanos , Cardiomiopatía Restrictiva/genética , Mutación , Filaminas/genética , Canadá , Cardiomiopatía Hipertrófica/diagnóstico , Cardiomiopatía Hipertrófica/genética , Insuficiencia Cardíaca/genética
8.
Antibiotics (Basel) ; 11(3)2022 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-35326761

RESUMEN

The structure-activity relationships and mode of action of synthesized glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-related antimicrobial peptides were investigated. Including the native skipjack tuna GAPDH-related peptide (SJGAP) of 32 amino acid residues (model for the study), 8 different peptide analogs were designed and synthesized to study the impact of net charge, hydrophobicity, amphipathicity, and secondary structure on both antibacterial and antifungal activities. A net positive charge increase, by the substitution of anionic residues or C-terminal amidation, improved the antimicrobial activity of the SJGAP analogs (minimal inhibitory concentrations of 16-64 µg/mL), whereas the alpha helix content, as determined by circular dichroism, did not have a very definite impact. The hydrophobicity of the peptides was also found to be important, especially for the improvement of antifungal activity. Membrane permeabilization assays showed that the active peptides induced significant cytoplasmic membrane permeabilization in the bacteria and yeast tested, but that this permeabilization did not cause leakage of 260 nm-absorbing intracellular material. This points to a mixed mode of action involving both membrane pore formation and targeting of intracellular components. This study is the first to highlight the links between the physicochemical properties, secondary structure, antimicrobial activity, and mechanism of action of antimicrobial peptides from scombrids or homologous to GAPDH.

9.
J Biol Chem ; 297(5): 101308, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34673030

RESUMEN

The design of allosteric modulators to control protein function is a key objective in drug discovery programs. Altering functionally essential allosteric residue networks provides unique protein family subtype specificity, minimizes unwanted off-target effects, and helps avert resistance acquisition typically plaguing drugs that target orthosteric sites. In this work, we used protein engineering and dimer interface mutations to positively and negatively modulate the immunosuppressive activity of the proapoptotic human galectin-7 (GAL-7). Using the PoPMuSiC and BeAtMuSiC algorithms, mutational sites and residue identity were computationally probed and predicted to either alter or stabilize the GAL-7 dimer interface. By designing a covalent disulfide bridge between protomers to control homodimer strength and stability, we demonstrate the importance of dimer interface perturbations on the allosteric network bridging the two opposite glycan-binding sites on GAL-7, resulting in control of induced apoptosis in Jurkat T cells. Molecular investigation of G16X GAL-7 variants using X-ray crystallography, biophysical, and computational characterization illuminates residues involved in dimer stability and allosteric communication, along with discrete long-range dynamic behaviors involving loops 1, 3, and 5. We show that perturbing the protein-protein interface between GAL-7 protomers can modulate its biological function, even when the overall structure and ligand-binding affinity remains unaltered. This study highlights new avenues for the design of galectin-specific modulators influencing both glycan-dependent and glycan-independent interactions.


Asunto(s)
Apoptosis , Galectinas , Tolerancia Inmunológica , Multimerización de Proteína , Linfocitos T/inmunología , Regulación Alostérica , Apoptosis/genética , Apoptosis/inmunología , Galectinas/química , Galectinas/genética , Galectinas/inmunología , Humanos , Células Jurkat , Multimerización de Proteína/genética , Multimerización de Proteína/inmunología
10.
J Biol Chem ; 295(26): 8708-8724, 2020 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-32371400

RESUMEN

Mammalian acetylcholinesterase (AChE) is well-studied, being important in both cholinergic brain synapses and the peripheral nervous systems and also a key drug target for many diseases. In contrast, little is known about the structures and molecular mechanism of prokaryotic acetylcholinesterases. We report here the structural and biochemical characterization of ChoE, a putative bacterial acetylcholinesterase from Pseudomonas aeruginosa Analysis of WT and mutant strains indicated that ChoE is indispensable for P. aeruginosa growth with acetylcholine as the sole carbon and nitrogen source. The crystal structure of ChoE at 1.35 Å resolution revealed that this enzyme adopts a typical fold of the SGNH hydrolase family. Although ChoE and eukaryotic AChEs catalyze the same reaction, their overall structures bear no similarities constituting an interesting example of convergent evolution. Among Ser-38, Asp-285, and His-288 of the catalytic triad residues, only Asp-285 was not essential for ChoE activity. Combined with kinetic analyses of WT and mutant proteins, multiple crystal structures of ChoE complexed with substrates, products, or reaction intermediate revealed the structural determinants for substrate recognition, snapshots of the various catalytic steps, and the molecular basis of substrate inhibition at high substrate concentrations. Our results indicate that substrate inhibition in ChoE is due to acetate release being blocked by the binding of a substrate molecule in a nonproductive mode. Because of the distinct overall folds and significant differences of the active site between ChoE and eukaryotic AChEs, these structures will serve as a prototype for other prokaryotic acetylcholinesterases.


Asunto(s)
Acetilcolinesterasa/metabolismo , Pseudomonas aeruginosa/enzimología , Acetilcolinesterasa/química , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Cinética , Modelos Moleculares , Conformación Proteica , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Especificidad por Sustrato
11.
Antiviral Res ; 170: 104561, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31323237

RESUMEN

As part of a 2015-2018 clinical trial of peramivir treatment for acute influenza infections in the elderly, an influenza B/Yamagata/16/1988-like isolate harbouring a Val430Ile neuraminidase (NA) substitution was recovered from a single patient. This substitution was detected in respiratory samples collected before and during peramivir treatment. In NA inhibition assays, oseltamivir, zanamivir and peramivir IC50s of the Val430Ile isolate were 4-, 15- and 16-fold higher compared to a wild-type (WT) strain. In reverse genetics experiments, the Ile430Val reversion restored the drug susceptible phenotype. The Val430Ile mutant and the WT strain had comparable replication kinetics in ST6GalI-MDCK cells and the NA mutation was stable after four passages in that cell line. Molecular dynamics simulations suggested that Val430Ile impacts the NA binding through a mechanism involving the catalytic Arg116 residue. The potential of some NA mutations not part of the active site to alter the susceptibility to NA inhibitors highlights the need to develop novel antiviral strategies against influenza B infections.


Asunto(s)
Sustitución de Aminoácidos , Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Neuraminidasa/genética , Ácidos Carbocíclicos , Secuencia de Aminoácidos , Animales , Ensayos Clínicos Fase III como Asunto , Ciclopentanos/uso terapéutico , Perros , Guanidinas/uso terapéutico , Humanos , Virus de la Influenza B , Concentración 50 Inhibidora , Células de Riñón Canino Madin Darby , Simulación de Dinámica Molecular , Estudios Multicéntricos como Asunto , Mutación , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/química , Genética Inversa , Replicación Viral/efectos de los fármacos
12.
Emerg Infect Dis ; 25(4): 838-840, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30882323
13.
Bioorg Med Chem ; 26(22): 5845-5851, 2018 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-30420328

RESUMEN

Herein we report the anti-inflammatory activity of lobaric acid and pseudodepsidones isolated from the nordic lichen Stereocaulon paschale. Lobaric acid (1) and three compounds (2, 7 and 9) were found to inhibit the NF-κB activation and the secretion of pro-inflammatory cytokines (IL-1ß and TNF-α) in LPS-stimulated macrophages. Inhibition and docking simulation experiments provided evidence that lobaric acid and pseudodepsidones bind to PPAR-γ between helix H3 and the beta sheet, similarly to partial PPAR-γ agonists. These findings suggest that lobaric acid and pseudodepsidones reduce the expression of pro-inflammatory cytokines by blocking the NF-κB pathway via the activation of PPAR-γ.


Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Depsidos/farmacología , Lactonas/farmacología , Líquenes/química , FN-kappa B/antagonistas & inhibidores , PPAR gamma/agonistas , Salicilatos/farmacología , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/aislamiento & purificación , Supervivencia Celular/efectos de los fármacos , Depsidos/química , Depsidos/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Humanos , Lactonas/química , Lactonas/aislamiento & purificación , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Simulación del Acoplamiento Molecular , Estructura Molecular , FN-kappa B/metabolismo , PPAR gamma/metabolismo , Salicilatos/química , Salicilatos/aislamiento & purificación , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Células U937
14.
Steroids ; 140: 104-113, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30273695

RESUMEN

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a promising therapeutic target known to play a pivotal role in the progression of estrogen-dependent diseases such as breast cancer, and endometriosis. This enzyme is responsible for the last step in the biosynthesis of the most potent estrogen, estradiol (E2) and its inhibition would prevent the growth of estrogen-sensitive tumors. Based on molecular modeling with docking experiments, we identified two promising C3-oxiranyl/oxiranylmethyl-estrane derivatives that would bind competitively and irreversibly in the catalytic site of 17ß-HSD1. They have been synthesized in a short and efficient route and their inhibitory activities over 17ß-HSD1 have been assessed by an enzymatic assay. Compound 15, with an oxiranylmethyl group at position C3, was more likely to bind the catalytic site and showed an interesting, but weak, inhibitory activity with an IC50 value of 1.3 µM (for the reduction of estrone into E2 in T-47D cells). Compound 11, with an oxiranyl at position C3, produced a lower inhibition rate, and the IC50 value cannot be determined. When tested in estrogen-sensitive T-47D cells, both compounds were also slightly estrogenic, although much less than the estrogenic hormone E2.


Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Estranos/síntesis química , Estranos/farmacología , Simulación del Acoplamiento Molecular , 17-Hidroxiesteroide Deshidrogenasas/química , Línea Celular Tumoral , Técnicas de Química Sintética , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Estranos/química , Estranos/metabolismo , Humanos , Conformación Proteica
15.
Mol Ther ; 26(11): 2604-2616, 2018 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-30195724

RESUMEN

Duchenne muscular dystrophy (DMD), a severe hereditary disease affecting 1 in 3,500 boys, mainly results from the deletion of exon(s), leading to a reading frameshift of the DMD gene that abrogates dystrophin protein synthesis. Pairs of sgRNAs for the Cas9 of Staphylococcus aureus were meticulously chosen to restore a normal reading frame and also produce a dystrophin protein with normally phased spectrin-like repeats (SLRs), which is not usually obtained by skipping or by deletion of complete exons. This can, however, be obtained in rare instances where the exon and intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion) are at similar positions in the SLR. We used pairs of sgRNAs targeting exons 47 and 58, and a normal reading frame was restored in myoblasts derived from muscle biopsies of 4 DMD patients with different exon deletions. Restoration of the DMD reading frame and restoration of dystrophin expression were also obtained in vivo in the heart of the del52hDMD/mdx. Our results provide a proof of principle that SaCas9 could be used to edit the human DMD gene and could be considered for further development of a therapy for DMD.


Asunto(s)
Sistemas CRISPR-Cas/genética , Distrofina/genética , Terapia Genética , Distrofia Muscular de Duchenne/genética , Animales , Proteína 9 Asociada a CRISPR/genética , Modelos Animales de Enfermedad , Distrofina/uso terapéutico , Exones/genética , Mutación del Sistema de Lectura/genética , Edición Génica , Regulación de la Expresión Génica , Humanos , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/terapia , Mioblastos , Eliminación de Secuencia , Staphylococcus aureus/enzimología
16.
Chembiochem ; 2018 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-29858881

RESUMEN

Peptidomimetic HIV protease inhibitors are an important class of drugs used in the treatment of AIDS. The synthesis of a new type of diol-based peptidomimetics is described. Our route is flexible, uses d-glucal as an inexpensive starting material, and makes minimal use of protection/deprotection cycles. Binding affinities from molecular docking simulations suggest that these compounds are potential inhibitors of HIV protease. Moreover, the antiproliferative activities of compounds 33 a, 35 a, and 35 b on HT-29, M21, and MCF7 cancer cell lines are in the low micromolar range. The results provide a platform that could facilitate the development of medically relevant asymmetrical diol-based peptidomimetics.

18.
Eur J Med Chem ; 147: 130-149, 2018 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-29427906

RESUMEN

The ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) was recently shown to promote mineralization of the aortic valve, hence, its inhibition represents a significant target. A quinazoline-4-piperidine sulfamide compound (QPS1) has been described as a specific and non-competitive inhibitor of NPP1. We report herein the synthesis and in vitro inhibition studies of novel quinazoline-4-piperidine sulfamide analogues using QPS1 as the lead compound. Of the 26 derivatives prepared, four compounds were found to have Ki < 105 nM against human NPP1.


Asunto(s)
Amidas/farmacología , Inhibidores Enzimáticos/farmacología , Piperidinas/farmacología , Pirofosfatasas/antagonistas & inhibidores , Quinazolinas/farmacología , Amidas/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Hidrolasas Diéster Fosfóricas/metabolismo , Piperidinas/química , Pirofosfatasas/metabolismo , Quinazolinas/química , Relación Estructura-Actividad
19.
Mol Cell Proteomics ; 17(2): 373-383, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29203496

RESUMEN

Understanding the function of cellular systems requires describing how proteins assemble with each other into transient and stable complexes and to determine their spatial relationships. Among the tools available to perform these analyses on a large scale is Protein-fragment Complementation Assay based on the dihydrofolate reductase (DHFR PCA). Here we test how longer linkers between the fusion proteins and the reporter fragments affect the performance of this assay. We investigate the architecture of the RNA polymerases, the proteasome and the conserved oligomeric Golgi (COG) complexes in living cells and performed large-scale screens with these extended linkers. We show that longer linkers significantly improve the detection of protein-protein interactions and allow to measure interactions further in space than the standard ones. We identify new interactions, for instance between the retromer complex and proteins related to autophagy and endocytosis. Longer linkers thus contribute an enhanced additional tool to the existing toolsets for the detection and measurements of protein-protein interactions and protein proximity in living cells.


Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Tetrahidrofolato Deshidrogenasa/metabolismo , Bioensayo , Escherichia coli/genética , Tetrahidrofolato Deshidrogenasa/genética , Levaduras/genética
20.
Biochem Mol Biol Educ ; 46(1): 31-38, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-28902453

RESUMEN

A recent scientific discipline, bioinformatics, defined as using informatics for the study of biological problems, is now a requirement for the study of biological sciences. Bioinformatics has become such a powerful and popular discipline that several academic institutions have created programs in this field, allowing students to become specialized. However, biology students who are not involved in a bioinformatics program also need a solid toolbox of bioinformatics software and skills. Therefore, we have developed a completely online bioinformatics course for non-bioinformaticians, entitled "BIF-1901 Introduction à la bio-informatique et à ses outils (Introduction to bioinformatics and bioinformatics tools)," given by the Department of Biochemistry, Microbiology, and Bioinformatics of Université Laval (Quebec City, Canada). This course requires neither a bioinformatics background nor specific skills in informatics. The underlying main goal was to produce a completely online up-to-date bioinformatics course, including practical exercises, with an intuitive pedagogical framework. The course, BIF-1901, was conceived to cover the three fundamental aspects of bioinformatics: (1) informatics, (2) biological sequence analysis, and (3) structural bioinformatics. This article discusses the content of the modules, the evaluations, the pedagogical framework, and the challenges inherent to a multidisciplinary, fully online course. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):31-38, 2018.


Asunto(s)
Biología Computacional/educación , Internet , Enseñanza , Humanos , Programas Informáticos , Estudiantes , Universidades
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