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1.
Bioinformatics ; 39(8)2023 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-37527005

RESUMEN

MOTIVATION: Identifying peptides associated with the major histocompability complex class II (MHCII) is a central task in the evaluation of the immunoregulatory function of therapeutics and drug prototypes. MHCII-peptide presentation prediction has multiple biopharmaceutical applications, including the safety assessment of biologics and engineered derivatives in silico, or the fast progression of antigen-specific immunomodulatory drug discovery programs in immune disease and cancer. This has resulted in the collection of large-scale datasets on adaptive immune receptor antigenic responses and MHC-associated peptide proteomics. In parallel, recent deep learning algorithmic advances in protein language modeling have shown potential in leveraging large collections of sequence data and improve MHC presentation prediction. RESULTS: Here, we train a compact transformer model (AEGIS) on human and mouse MHCII immunopeptidome data, including a preclinical murine model, and evaluate its performance on the peptide presentation prediction task. We show that the transformer performs on par with existing deep learning algorithms and that combining datasets from multiple organisms increases model performance. We trained variants of the model with and without MHCII information. In both alternatives, the inclusion of peptides presented by the I-Ag7 MHC class II molecule expressed by nonobese diabetic mice enabled for the first time the accurate in silico prediction of presented peptides in a preclinical type 1 diabetes model organism, which has promising therapeutic applications. AVAILABILITY AND IMPLEMENTATION: The source code is available at https://github.com/Novartis/AEGIS.


Asunto(s)
Diabetes Mellitus Experimental , Humanos , Ratones , Animales , Ratones Endogámicos NOD , Antígenos de Histocompatibilidad Clase II/metabolismo , Péptidos/química , Antígenos , Unión Proteica
2.
Proc Natl Acad Sci U S A ; 118(31)2021 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-34326266

RESUMEN

Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.


Asunto(s)
Antibacterianos/metabolismo , Membrana Celular/fisiología , Nutrientes/metabolismo , Porinas/metabolismo , Pseudomonas aeruginosa/fisiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte Biológico/fisiología , Permeabilidad de la Membrana Celular
3.
Chembiochem ; 21(21): 3096-3111, 2020 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-32537808

RESUMEN

Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). Nitrogen-containing bisphosphonates, a current treatment for bone diseases, have been shown to block the growth of the T. brucei parasites by inhibiting farnesyl pyrophosphate synthase (FPPS); however, due to their poor pharmacokinetic properties, they are not well suited for antiparasitic therapy. Recently, an allosteric binding pocket was discovered on human FPPS, but its existence on trypanosomal FPPS was unclear. We applied NMR and X-ray fragment screening to T. brucei FPPS and report herein on four fragments bound to this previously unknown allosteric site. Surprisingly, non-bisphosphonate active-site binders were also identified. Moreover, fragment screening revealed a number of additional binding sites. In an early structure-activity relationship (SAR) study, an analogue of an active-site binder was unexpectedly shown to bind to the allosteric site. Overlaying identified fragment binders of a parallel T. cruzi FPPS fragment screen with the T. brucei FPPS structure, and medicinal chemistry optimisation based on two binders revealed another example of fragment "pocket hopping". The discovery of binders with new chemotypes sets the framework for developing advanced compounds with pharmacokinetic properties suitable for the treatment of parasitic infections by inhibition of FPPS in T. brucei parasites.


Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Geraniltranstransferasa/antagonistas & inhibidores , Trypanosoma brucei brucei/efectos de los fármacos , Sitios de Unión/efectos de los fármacos , Inhibidores Enzimáticos/química , Geraniltranstransferasa/metabolismo , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Trypanosoma brucei brucei/enzimología
4.
Environ Microbiol ; 18(3): 819-32, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26718479

RESUMEN

Previous studies have suggested that antibiotic vectorization by siderophores (iron chelators produced by bacteria) considerably increases the efficacy of such drugs. The siderophore serves as a vector: when the pathogen tries to take up iron via the siderophore, it also takes up the antibiotic. Catecholates are among the most common iron-chelating compounds used in synthetic siderophore-antibiotic conjugates. Using reverse transcription polymerase chain reaction and proteomic approaches, we showed that the presence of catecholate compounds in the medium of Pseudomonas aeruginosa led to strong activation of the transcription and expression of the outer membrane transporter PfeA, the ferri-enterobactin importer. Iron-55 uptake assays on bacteria with and without PfeA expression confirmed that catechol compounds imported iron into P. aeruginosa cells via PfeA. Uptake rates were between 0.3 × 10(3) and 2 × 10(3) Fe atoms/bacterium/min according to the used catechol siderophore in iron-restricted medium, and remained as high as 0.8 × 10(3) Fe atoms/bacterium/min for enterobactin, even in iron-rich medium. Reverse transcription polymerase chain reaction and proteomic approaches showed that in parallel to this switching on of PfeA expression, a repression of the expression of pyochelin (PCH) pathway genes (PCH being one of the two siderophores produced by P. aeruginosa for iron acquisition) was observed.


Asunto(s)
Antibacterianos/metabolismo , Catecoles/metabolismo , Enterobactina/metabolismo , Fenoles/metabolismo , Pseudomonas aeruginosa/metabolismo , Sideróforos/metabolismo , Tiazoles/metabolismo , Hierro/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Pseudomonas aeruginosa/genética
5.
Nat Commun ; 14(1): 5497, 2023 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-37679328

RESUMEN

Human interleukin-1ß (hIL-1ß) is a pro-inflammatory cytokine involved in many diseases. While hIL-1ß directed antibodies have shown clinical benefit, an orally available low-molecular weight antagonist is still elusive, limiting the applications of hIL-1ß-directed therapies. Here we describe the discovery of a low-molecular weight hIL-1ß antagonist that blocks the interaction with the IL-1R1 receptor. Starting from a low affinity fragment-based screening hit 1, structure-based optimization resulted in a compound (S)-2 that binds and antagonizes hIL-1ß with single-digit micromolar activity in biophysical, biochemical, and cellular assays. X-ray analysis reveals an allosteric mode of action that involves a hitherto unknown binding site in hIL-1ß encompassing two loops involved in hIL-1R1/hIL-1ß interactions. We show that residues of this binding site are part of a conformationally excited state of the mature cytokine. The compound antagonizes hIL-1ß function in cells, including primary human fibroblasts, demonstrating the relevance of this discovery for future development of hIL-1ß directed therapeutics.


Asunto(s)
Citocinas , Delgadez , Humanos , Interleucina-1beta , Peso Molecular , Sitios de Unión , Biofisica
6.
Bioinformatics ; 26(1): 1-5, 2010 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-19850757

RESUMEN

MOTIVATION: Prochlorococcus possesses the smallest genome of all sequenced photoautotrophs. Although the number of regulatory proteins in the genome is very small, the relative number of small regulatory RNAs is comparable with that of other bacteria. The compact genome size of Prochlorococcus offers an ideal system to search for targets of small RNAs (sRNAs) and to refine existing target prediction algorithms. RESULTS: Target predictions for the cyanobacterial sRNA Yfr1 were carried out with INTARNA in Prochlorococcus MED4. The ultraconserved Yfr1 sequence motif was defined as the putative interaction seed. To study the impact of Yfr1 on its predicted mRNA targets, a reporter system based on green fluorescent protein (GFP) was applied. We show that Yfr1 inhibits the translation of two predicted targets. We used mutation analysis to confirm that Yfr1 directly regulates its targets by an antisense interaction sequestering the ribosome binding site, and to assess the importance of interaction site accessibility.


Asunto(s)
Marcación de Gen/métodos , Genoma Bacteriano/genética , Proteínas Fluorescentes Verdes/genética , Prochlorococcus/genética , ARN Bacteriano/genética , ARN Mensajero/genética , Análisis de Secuencia de ARN/métodos , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular
7.
Biochemistry ; 48(26): 6166-74, 2009 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-19485419

RESUMEN

BaP1, a zinc-dependent endopeptidase belonging to the P-I class of snake venom metalloproteinases, exerts multiple tissue-damaging activities, leading to hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. Interestingly, this metalloproteinase shows a high degree of structural homology with the catalytic domain of human adamalysins and matrix metalloproteinases, especially at the strictly conserved zinc binding motif and the so-called Met turn. This highlights BaP1 as an interesting model concerning inhibitor design for several medicinally important metalloproteinases, such as tumor necrosis factor alpha converting enzyme. Here, we report the first crystal structure of BaP1 complexed with a peptidomimetic inhibitor. Suitable crystals were obtained at four different pH values (4.6, 6.5, 7.5, and 8.0), and four high-resolution structures (1.46, 1.14, 1.08, and 1.05 A) were established. These structures and the detailed analysis of the structure-activity relationship of the bound inhibitor form a basis for the design of potent BaP1 inhibitors. The latter can be used for the treatment of local pathological effects caused by snake bites, mainly due to metalloproteinases such as BaP1. Besides, the high-resolution structure is an excellent starting point for the rational development of inhibitors for human metalloproteinases. The finding of a flexible loop region may have a great impact on further studies as to date little is known about the structural dependencies of the hemorrhagic activity of snake venom metalloproteinases.


Asunto(s)
Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Venenos de Víboras/enzimología , Animales , Biocatálisis , Bothrops , Dominio Catalítico , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Cinética , Metaloendopeptidasas/antagonistas & inhibidores , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/farmacología , Unión Proteica , Conformación Proteica , Agua/química , Zinc/química
8.
Structure ; 27(2): 268-280.e6, 2019 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-30554842

RESUMEN

Research efforts to discover potential new antibiotics for Gram-negative bacteria suffer from high attrition rates due to the synergistic action of efflux systems and the limited permeability of the outer membrane (OM). One strategy to overcome the OM permeability barrier is to identify small molecules that are natural substrates for abundant OM channels and use such compounds as scaffolds for the design of efficiently permeating antibacterials. Here we present a multidisciplinary approach to identify such potential small-molecule scaffolds. Focusing on the pathogenic bacterium Acinetobacter baumannii, we use OM proteomics to identify DcaP as the most abundant channel during infection in rodents. The X-ray crystal structure of DcaP reveals a trimeric, porin-like structure and suggests that dicarboxylic acids are potential transport substrates. Electrophysiological experiments and all-atom molecular dynamics simulations confirm this notion and provide atomistic information on likely permeation pathways and energy barriers for several small molecules, including a clinically relevant ß-lactamase inhibitor.


Asunto(s)
Infecciones por Acinetobacter/metabolismo , Acinetobacter baumannii/metabolismo , Porinas/química , Porinas/metabolismo , Sulbactam/farmacología , Inhibidores de beta-Lactamasas/farmacología , Acinetobacter baumannii/efectos de los fármacos , Animales , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cristalografía por Rayos X , Ácidos Dicarboxílicos/metabolismo , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Multimerización de Proteína , Proteómica , Ratas
9.
J Mol Biol ; 367(2): 409-18, 2007 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-17275835

RESUMEN

The enzyme 2,6-dihydroxy-pseudo-oxynicotine hydrolase from the nicotine-degradation pathway of Arthrobacter nicotinovorans was crystallized and the structure was determined by an X-ray diffraction analysis at 2.1 A resolution. The enzyme belongs to the alpha/beta-hydrolase family as derived from the chain-fold and from the presence of a catalytic triad with its oxyanion hole at the common position. This relationship assigns a pocket lined by the catalytic triad as the active center. The asymmetric unit contains two C(2)-symmetric dimer molecules, each adopting a specific conformation. One dimer forms a more spacious active center pocket and the other a smaller one, suggesting an induced-fit. All of the currently established C-C bond cleaving alpha/beta-hydrolases are from bacterial meta-cleavage pathways for the degradation of aromatic compounds and cover their active center with a 40 residue lid placed between two adjacent strands of the beta-sheet. In contrast, the reported enzyme shields its active center with a 110 residue N-terminal domain, which is absent in the meta-cleavage hydrolases. Since neither the substrate nor an analogue could be bound in the crystals, the substrate was modeled into the active center using the oxyanion hole as a geometric constraint. The model was supported by enzymatic activity data of 11 point mutants and by the two dimer conformations suggesting an induced-fit. Moreover, the model assigned a major role for the large N-terminal domain that is specific to the reported enzyme. The proposal is consistent with the known data for the meta-cleavage hydrolases although it differs in that the reaction does not release alkenes but a hetero-aromatic compound in a retro-Friedel-Crafts acylation. Because the hydrolytic water molecule can be assigned to a geometrically suitable site that can be occupied in the presence of the substrate, the catalytic triad may not form a covalent acyl-enzyme intermediate but merely support a direct hydrolysis.


Asunto(s)
Arthrobacter/enzimología , Proteínas Bacterianas/química , Hidrolasas/química , Modelos Moleculares , Nicotina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dimerización , Hidrolasas/genética , Hidrolasas/metabolismo , Datos de Secuencia Molecular , Mutación , Estructura Secundaria de Proteína , Agua/química
10.
ACS Infect Dis ; 4(10): 1519-1528, 2018 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-30039960

RESUMEN

Understanding molecular properties of outer membrane channels of Gram-negative bacteria is of fundamental significance as they are the entry point of polar antibiotics into bacteria. Outer membrane proteomics revealed OccK8 (OprE) to be among the five most expressed substrate specific channels of the clinically important Pseudomonas aeruginosa. The high-resolution X-ray structure and electrophysiology highlighted a very narrow pore. However, experimental in vitro methods showed the transport of natural amino acids and antibiotics, among them ceftazidime. We used molecular dynamics simulations to reveal the importance of the physicochemical properties of ceftazidime in modulating the translocation through OccK8, proposing a structure-function relationship. As in general porins, the internal electric field favors the translocation of polar molecules by gainful energy compensation in the central constriction region. Importantly, the comparatively narrow OccK8 pore can undergo a substrate-induced expansion to accommodate relatively large-sized substrates.


Asunto(s)
Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Ceftazidima/metabolismo , Bacterias Gramnegativas/metabolismo , Porinas/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Liposomas/metabolismo , Potenciales de la Membrana , Simulación de Dinámica Molecular , Porinas/química , Porinas/clasificación , Estructura Secundaria de Proteína , Transporte de Proteínas , Electricidad Estática
11.
J Mol Biol ; 364(4): 705-15, 2006 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-17027031

RESUMEN

C2 toxin from Clostridium botulinum is composed of the enzyme component C2-I, which ADP-ribosylates actin, and the binding and translocation component C2-II, responsible for the interaction with eukaryotic cell receptors and the following endocytosis. Three C2-I crystal structures at resolutions of up to 1.75 A are presented together with a crystal structure of C2-II at an appreciably lower resolution and a model of the prepore formed by fragment C2-IIa. The C2-I structure was determined at pH 3.0 and at pH 6.1. The structural differences are small, indicating that C2-I does not unfold, even at a pH value as low as 3.0. The ADP-ribosyl transferase activity of C2-I was determined for alpha and beta/gamma-actin and related to that of Iota toxin and of mutant S361R of C2-I that introduced the arginine observed in Iota toxin. The substantial activity differences between alpha and beta/gamma-actin cannot be explained by the protein structures currently available. The structure of the transport component C2-II at pH 4.3 was established by molecular replacement using a model of the protective antigen of anthrax toxin at pH 6.0. The C-terminal receptor-binding domain of C2-II could not be located but was present in the crystals. It may be mobile. The relative orientation and positions of the four other domains of C2-II do not differ much from those of the protective antigen, indicating that no large conformational changes occur between pH 4.3 and pH 6.0. A model of the C2-IIa prepore structure was constructed based on the corresponding assembly of the protective antigen. It revealed a surprisingly large number of asparagine residues lining the pore. The interaction between C2-I and C2-IIa and the translocation of C2-I into the target cell are discussed.


Asunto(s)
Toxinas Botulínicas/química , Clostridium botulinum/química , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/metabolismo , Actinas/metabolismo , Antígenos , Asparagina , Sitios de Unión , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Estructura Molecular
12.
FEBS Lett ; 580(30): 6943-7, 2006 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-17157838

RESUMEN

Natural bioactive compounds are of general interest for pharmaceutical research because they may serve as leads in drug development campaigns. Among them, microginins are linear peptides known to inhibit various exopeptidases. The crystal structure of microginin FR1 from Microcystis sp. bound to bovine lens leucine aminopeptidase was established at 1.73 Angstrom resolution. The observed binding structure could be beneficial for the design of potent aminopeptidase inhibitors.


Asunto(s)
Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Leucil Aminopeptidasa/antagonistas & inhibidores , Microcystis/química , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína
13.
Chem Biol ; 10(10): 997-1001, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-14583266

RESUMEN

Natural bioactive compounds are of general interest to pharmaceutical research because they may be used as leads in drug development campaigns. Among them, scyptolin A and B from Scytonema hofmanni PCC 7110 are known to inhibit porcine pancreatic elastase, which in turn resembles the attractive drug target neutrophil elastase. The crystal structure of scyptolin A as bound to pancreatic elastase was solved at 2.8 A resolution. The inhibitor occupies the most prominent subsites S1 through S4 of the elastase and prevents a hydrolytic attack by covering the active center with its rigid ring structure. The observed binding structure may help to design potent elastase inhibitors.


Asunto(s)
Cianobacterias/metabolismo , Depsipéptidos , Elastasa Pancreática/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Sitios de Unión , Cristalografía por Rayos X , Cianobacterias/química , Inhibidores Enzimáticos/farmacología , Elastasa Pancreática/metabolismo , Péptidos Cíclicos/química , Relación Estructura-Actividad , Tripsina/química
14.
Chem Biol ; 16(1): 28-35, 2009 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-19171303

RESUMEN

As glycosyltransferases found in nature often show distinct substrate specificity, glycosyltransferase engineering is an important research field. In this work, we were able to introduce an activity into a glycosyltransferase involved in natural product (landomycin E) biosynthesis. This was achieved by recognizing hot spot amino acids in glycosyltransferases which are strongly involved in determining substrate specificity.


Asunto(s)
Glicosiltransferasas/genética , Secuencia de Aminoácidos , Aminoglicósidos/biosíntesis , Glicosiltransferasas/metabolismo , Datos de Secuencia Molecular , Mutación , Ingeniería de Proteínas , Homología de Secuencia de Aminoácido , Especificidad por Sustrato
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