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1.
Methods Mol Biol ; 2718: 11-21, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37665452

RESUMEN

A detailed study of the cellular surfaceome poses major challenges for mass spectrometry analysis. Surface proteins are low abundant compared to intracellular proteins, and their inefficient extraction in aqueous medium leads to their aggregation and precipitation. To tackle such problems, surface biotinylation is frequently used to tag surface proteins with biotin, allowing for their enrichment, leading to a more sensitive mapping of surface proteomes. We here detail a new surface biotinylation protocol based on furan-biotin affinity purification to enrich plasma membrane proteins for proteomics. This protocol involves biotinylation of cell surface membrane proteins on viable cells, followed by affinity enrichment using streptavidin beads, trypsin digestion, peptide cleanup, and LC-MS/MS analysis.


Asunto(s)
Biotina , Espectrometría de Masas en Tándem , Biotinilación , Cromatografía Liquida , Proteínas de la Membrana , Furanos
2.
Angew Chem Int Ed Engl ; 62(20): e202302688, 2023 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-36917014

RESUMEN

The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.


Asunto(s)
Islotes Pancreáticos , Kisspeptinas , Ratones , Animales , Humanos , Kisspeptinas/química , Kisspeptinas/metabolismo , Péptidos/química , Islotes Pancreáticos/diagnóstico por imagen , Islotes Pancreáticos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Imagen Óptica , Aminoácidos/metabolismo
3.
Angew Chem Weinheim Bergstr Ger ; 135(20): e202302688, 2023 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38516305

RESUMEN

The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.

4.
Chem Commun (Camb) ; 57(49): 6054-6057, 2021 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-34036992

RESUMEN

We describe furan as a triggerable 'warhead' for site-specific cross-linking using the actin and thymosin ß4 (Tß4)-complex as model of a weak and dynamic protein-protein interaction (PPI) with known 3D structure and with application potential in disease contexts. The identified cross-linked residues demonstrate that lysine is a target for the furan warhead. The presented in vitro validation of covalently acting 'furan-armed' Tß4-variants provides initial proof to further exploit furan-technology for covalent drug design targeting lysines.


Asunto(s)
Reactivos de Enlaces Cruzados/química , Furanos/química , Timosina/química , Actinas/química , Modelos Moleculares , Unión Proteica
5.
Front Chem ; 9: 799706, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35252125

RESUMEN

Using a coiled-coil peptide dimer as a model system to explore furan reactivity, we describe novel cross-link partners of furan warheads for site-specific cross-linking. We demonstrate that replacement of weak interhelical ionic contacts with a furan moiety and its potential cross-link partner affords covalently connected coiled-coil motifs upon furan activation. We describe for the first time the reaction of the activated furan warhead with cysteine and tyrosine, besides the previously reported lysine, thus enhancing the versatility of the furan cross-link methodology by the possibility to target different amino acids. The present in vitro validation of "furan-armed" α-helices provides further grounds for exploiting furan technology in the development of furan-modified ligands/proteins to target proteins in a covalent way through various amino acid side chains.

6.
Cell Chem Biol ; 25(3): 268-278.e4, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29290623

RESUMEN

Mitochondria are dynamic organelles that produce most of the cellular ATP, and are involved in many other cellular functions such as Ca2+ signaling, differentiation, apoptosis, cell cycle, and cell growth. One key process of mitochondrial dynamics is mitochondrial fusion, which is catalyzed by mitofusins (MFN1 and MFN2) and OPA1. The outer mitochondrial membrane protein MFN2 plays a relevant role in the maintenance of mitochondrial metabolism, insulin signaling, and mutations that cause neurodegenerative disorders. Therefore, modulation of proteins involved in mitochondrial dynamics has emerged as a potential pharmacological strategy. Here, we report the identification of small molecules by high-throughput screen that promote mitochondrial elongation in an MFN1/MFN2-dependent manner. Detailed analysis of their mode of action reveals a previously unknown connection between pyrimidine metabolism and mitochondrial dynamics. Our data indicate a link between pyrimidine biosynthesis and mitochondrial dynamics, which maintains cell survival under stress conditions characterized by loss of pyrimidine synthesis.


Asunto(s)
Mitocondrias/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Pirimidinas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Dihidroorotato Deshidrogenasa , Doxorrubicina/farmacología , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Complejo III de Transporte de Electrones/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Leflunamida/farmacología , Ratones , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/agonistas , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/agonistas , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Porinas/genética , Porinas/metabolismo , Pirimidinas/biosíntesis , ARN Mensajero/metabolismo , Transcriptoma/efectos de los fármacos
7.
ACS Omega ; 3(12): 17057-17069, 2018 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-31458325

RESUMEN

Serious infections caused by bacteria that are resistant to commonly used antibiotics have become a major global healthcare problem in the 21st century. Multidrug-resistant bacteria causing severe infections mainly grow in complex bacterial communities known as biofilms, in which bacterial resistance to antibacterial agents and to the host immune system is strengthened. As drug resistance is becoming a threatening problem, it is necessary to develop new antimicrobial agents with novel mechanisms of action. Here, we designed and synthesized a small library of N-substituted hydroxylamine (N-HA) compounds with antibacterial activity. These compounds, acting as radical scavengers, inhibit the bacterial ribonucleotide reductase (RNR) enzyme. RNR enzyme is essential for bacterial proliferation during infection, as it provides the building blocks for DNA synthesis and repair. We demonstrate the broad antimicrobial effect of several drug candidates against a variety of Gram-positive and Gram-negative bacteria, together with low toxicity toward eukaryotic cells. Furthermore, the most promising compounds can reduce the biomass of an established biofilm on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. This study settles the starting point to develop new N-hydroxylamine compounds as potential effective antibacterial agents to fight against drug-resistant pathogenic bacteria.

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