RESUMEN
Asparaginyl endopeptidases (AEPs) have recently been widely utilized for peptide and protein modification. Labeling is however restricted to protein termini, severely limiting flexibility and scope in creating diverse conjugates as needed for therapeutic and diagnostic applications. Here, we use genetic code expansion to site-specifically modify target proteins with an isopeptide-linked glycylglycine moiety that serves as an acceptor nucleophile in AEP-mediated transpeptidation with various probes containing a tripeptidic recognition motif. Our approach allows simple and flexible labeling of recombinant proteins at any internal site and leaves a minimal, entirely peptidic footprint (NGG) in the conjugation product. We show site-specific labeling of diverse target proteins with various biophysical probes, including dual labeling at an internal site and the N-terminus. Furthermore, we harness AEP-mediated transpeptidation for generation of ubiquitin- and ubiquitin-like-modifier conjugates bearing a native isopeptide bond and only one point mutation in the linker region.
Asunto(s)
Cisteína Endopeptidasas , Péptidos , Cisteína Endopeptidasas/metabolismo , Proteínas Recombinantes/metabolismo , Ubiquitina/genéticaRESUMEN
Post-translational modification of proteins with ubiquitin and ubiquitin-like proteins (Ubls) is central to the regulation of eukaryotic cellular processes. Our ability to study the effects of ubiquitylation, however, is limited by the difficulty to prepare homogenously modified proteins in vitro and by the impossibility to selectively trigger specific ubiquitylation events in living cells. Here we combine genetic-code expansion, bioorthogonal Staudinger reduction and sortase-mediated transpeptidation to develop a general tool to ubiquitylate proteins in an inducible fashion. The generated ubiquitin conjugates display a native isopeptide bond and bear two point mutations in the ubiquitin C terminus that confer resistance toward deubiquitinases. Nevertheless, physiological integrity of sortase-generated diubiquitins in decoding cellular functions via recognition by ubiquitin-binding domains is retained. Our approach allows the site-specific attachment of Ubls to nonrefoldable, multidomain proteins and enables inducible and ubiquitin-ligase-independent ubiquitylation of proteins in mammalian cells, providing a powerful tool to dissect the biological functions of ubiquitylation with temporal control.
Asunto(s)
Ingeniería de Proteínas/métodos , Ubiquitinación/genética , Ubiquitinación/fisiología , Código Genético , Unión Proteica , Procesamiento Proteico-Postraduccional/genética , Proteínas , Especificidad por Sustrato/genética , Sumoilación/genética , Ubiquitina , UbiquitinasRESUMEN
Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio-orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence-based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse-chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis.
Asunto(s)
Colorantes Fluorescentes/química , Interleucinas/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Células HEK293 , Semivida , Humanos , Interleucinas/química , Interleucinas/genética , Cinética , Pliegue de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
Protein immobilization has gained high interest in recent years for its valuable applications in life sciences involving drug delivery and protein arrays. Herein, we combine sortase-mediated protein immobilization with the versatility of magnetic nanoparticles and a sensitive GFP-based quantification system. Using this method, we successfully immobilized and quantified the amount of coupled enzymes by fluorescence spectroscopy and assessed their activity by kinetic measurements. We show that sortase-mediated coupling of enzymes enables preparation of biological samples with a high demand of purity as demonstrated by single-molecule FRET. Here, we report that sortase-mediated protein ligation allows both N- and C-terminal site-specific protein immobilization. Additionally, we demonstrate that sortase-mediated protein immobilization is suitable for direct protein immobilization from complex lysates. Direct immobilization from lysate allows study of enzyme functionality without the need of time-consuming enzyme purification, while magnetic nanoparticles permit easy addition and removal of coupled enzymes to and from a reaction mixture.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Enzimas Inmovilizadas/química , Hierro/química , Fenómenos Magnéticos , Nanopartículas del Metal/química , Aminoaciltransferasas/química , Proteínas Bacterianas/química , Cisteína Endopeptidasas/química , Enzimas Inmovilizadas/metabolismo , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión GénicaRESUMEN
The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here, we present a method to stabilize transient protein complexes inâ vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low-affinity protein complexes between BrCnK-containing proteins and their binding partners can be stabilized inâ vivo in bacterial and mammalian cells. Using this approach, we determined the crystal structure of a transient GDP-bound complex between a small G-protein and its nucleotide exchange factor. We envision that this approach will prove valuable as a general tool for validating and characterizing protein-protein interactions inâ vitro and inâ vivo.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Reguladores de Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Reguladores de Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/química , Proteínas Fluorescentes Verdes/química , Células HEK293 , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Estabilidad ProteicaRESUMEN
Due to its unique role as a hydrogen-bond donor and its positive charge, the guanidine group is an important pharmacophoric group and often used in synthetic ligands. The chemical modification of the guanidine group is often considered to destroy its function. Herein, we show that the N-methylation, N-alkylation, or N-acylation of the guanidine group can be used to modify the receptor subtype specificity of the integrin ligand cilengitide. Using the αvß6/α5ß1-biselective ligand c(isoDGRkphg) and the αvß6-specific ligand c(FRGDLAFp(NMe)K(Ac) as examples, we show that the binding affinities of the ligands can be fine-tuned by this method to enhance the selectivity for αvß6. Furthermore, we describe a new strategy for the functionalization of integrin ligands. By introducing longer N-alkylguanidine and N-acylguanidine groups, we are able to simultaneously identify a hitherto unknown anchoring point and enhance the subtype selectivity of the ligand.
Asunto(s)
Guanidina/química , Integrinas/química , Oligopéptidos/química , Sitios de UniónRESUMEN
Post-translational modifications (PTMs) dynamically regulate cellular processes. Lysine undergoes a range of acylations, including malonylation, succinylation (SucK) and glutarylation (GluK). These PTMs increase the size of the lysine side chain and reverse its charge from +1 to -1 under physiological conditions, probably impacting protein structure and function. To understand the functional roles of these PTMs, homogeneously modified proteins are required for biochemical studies. While the site-specific encoding of PTMs and their mimics via genetic code expansion has facilitated the characterization of the functional roles of many PTMs, negatively charged lysine acylations have defied this approach. Here we describe site-specific incorporation of SucK and GluK into proteins via temporarily masking their negative charge through thioester derivatives. We prepare succinylated and glutarylated bacterial and mammalian target proteins, including non-refoldable multidomain proteins. This allows us to study how succinylation and glutarylation impact enzymatic activity of metabolic enzymes and regulate protein-DNA and protein-protein interactions in biological processes from replication to ubiquitin signalling.
Asunto(s)
Código Genético , Procesamiento Proteico-Postraduccional , Ácido Succínico , Ácido Succínico/metabolismo , Ácido Succínico/química , Humanos , Lisina/química , Lisina/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteínas/genética , Glutaratos/metabolismo , Glutaratos/químicaRESUMEN
The post-translational modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) represents one of the most important regulators in eukaryotic biology. Polymeric Ub/Ubl chains of distinct topologies control the activity, stability, interaction and localization of almost all cellular proteins and elicit a variety of biological outputs. Our ability to characterize the roles of distinct Ub/Ubl topologies and to identify enzymes and receptors that create, recognize and remove these modifications is however hampered by the difficulty to prepare them. Here we introduce a modular toolbox (Ubl-tools) that allows the stepwise assembly of Ub/Ubl chains in a flexible and user-defined manner facilitated by orthogonal sortase enzymes. We demonstrate the universality and applicability of Ubl-tools by generating distinctly linked Ub/Ubl hybrid chains, and investigate their role in DNA damage repair. Importantly, Ubl-tools guarantees straightforward access to target proteins, site-specifically modified with distinct homo- and heterotypic (including branched) Ub chains, providing a powerful approach for studying the functional impact of these complex modifications on cellular processes.
Asunto(s)
Polímeros/química , Ubiquitina/metabolismo , Daño del ADN/genética , Daño del ADN/fisiología , Humanos , Unión Proteica , Procesamiento Proteico-Postraduccional , Ubiquitina/genética , Ubiquitinación/genética , Ubiquitinación/fisiologíaRESUMEN
Specific targeting of the integrin subtype α5ß1 possesses high potential in cancer diagnosis and therapy. Through sequential N-methylation, we successfully converted the biselective α5ß1/αvß6 peptide c(phg- isoDGR-k) into a potent peptidic RGD binding α5ß1 subtype selective ligand c(phg- isoDGR-( NMe)k). Nuclear magnetic resonance spectroscopy and molecular modeling clarified the molecular basis of its improved selectivity profile. To demonstrate its potential in vivo, c(phg- isoDGR-( NMe)k) was trimerized with the chelator TRAP and used as a positron-emission tomography tracer for monitoring α5ß1 integrin expression in a M21 mouse xenograft.
Asunto(s)
Integrina alfa5beta1/efectos de los fármacos , Neoplasias/diagnóstico por imagen , Péptidos/síntesis química , Péptidos/farmacología , Animales , Femenino , Radioisótopos de Galio , Humanos , Integrina alfa5beta1/biosíntesis , Ligandos , Espectroscopía de Resonancia Magnética , Melanoma Experimental/diagnóstico por imagen , Metilación , Ratones , Ratones SCID , Modelos Moleculares , Estructura Molecular , Tomografía de Emisión de Positrones , Unión Proteica , Trazadores Radiactivos , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The guanidine group is one of the most important pharmacophoric groups in medicinal chemistry. The only amino acid carrying a guanidine group is arginine. In this article, an easy method for the modification of the guanidine group in peptidic ligands is provided, with an example of RGD-binding integrin ligands. It was recently demonstrated that the distinct modification of the guanidine group in these ligands allows for the selective modulation of the subtype (e.g., between the subtypes αv and α5). Moreover, a formerly unknown strategy for the functionalization via the guanidine group was demonstrated, and the synthetic approach is reviewed in this document. The modifications described here involve terminally (Nω) alkylated and acetylated guanidine groups. For the synthesis, tailor-made precursor molecules are synthesized, which are then subjected to a reaction with an orthogonally deprotected amine to transfer the pre-modified guanidine group. For the synthesis of alkylated guanidines, precursors based on N,N'-Di-Boc-1H-pyrazole-1-carboxamidine are used to synthesize acylated compounds, the precursor of choice being a correspondingly acylated derivative of N-Boc-S-methylisothiourea, which can be obtained in one- and two-step reactions.