RESUMO
NF-κB is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Several key steps in its activation are mediated by the ubiquitin (Ub) system. One uncharacterized step is limited proteasomal processing of the NF-κB1 precursor p105 to the p50 active subunit. Here, we identify KPC1 as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signaling. Overexpression of KPC1 inhibits tumor growth likely mediated via excessive generation of p50. Also, overabundance of p50 downregulates p65, suggesting that a p50-p50 homodimer may modulate transcription in place of the tumorigenic p50-p65. Transcript analysis reveals increased expression of genes associated with tumor-suppressive signals. Overall, KPC1 regulation of NF-κB1 processing appears to constitute an important balancing step among the stimulatory and inhibitory activities of the transcription factor in cell growth control.
Assuntos
Subunidade p50 de NF-kappa B/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Sistema Livre de Células , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Subunidade p50 de NF-kappa B/química , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Transdução de Sinais , Ubiquitina-Proteína Ligases/isolamento & purificação , UbiquitinaçãoRESUMO
We designed a platform for monitoring the degradation of exogenous proteins in live cells. We engineered a semi-synthetic platform, which consists of Enhanced Green Fluorescent Protein tagged with SpyCatcher to enable its conjugation to a SpyTag peptide bearing a Von Hippel-Lindau E3 ligand, which was delivered to live cells to promote its degradation. This platform lays the ground for studying the degradation of endogenous proteins equipped with SpyTag and for tracking the degradation of post-translationally modified proteins in live cells.
Assuntos
Proteólise , Peptídeos , Processamento de Proteína Pós-TraducionalRESUMO
Chemical manipulation of naturally occurring peptides offers a convenient route for generating analogs to screen against different therapeutic targets. However, the limited success of the conventional chemical libraries has urged chemical biologists to adopt alternative methods such as phage and mRNA displays and create libraries of a large number of variants for the screening and selection of novel peptides. Messenger RNA (mRNA) display provides great advantages in terms of the library size and the straightforward recovery of the selected polypeptide sequences. Importantly, the integration of the flexible in vitro translation (FIT) system with the mRNA display provides the basis of the random nonstandard peptides integrated discovery (RaPID) approach for the introduction of diverse nonstandard motifs, such as unnatural side chains and backbone modifications. This platform allows the discovery of functionalized peptides with tight binding against virtually any protein of interest (POI) and therefore shows great potential in the pharmaceutical industry. However, this method has been limited to targets generated by recombinant expression, excluding its applications to uniquely modified proteins, particularly those with post-translational modifications.Chemical protein synthesis allows a wide range of changes to the protein's chemical composition to be performed, including side chain and backbone modifications and access to post-translationally modified proteins, which are often inaccessible or difficult to achieve via recombinant expression methods. Notably, d-proteins can be prepared via chemical synthesis, which has been used in mirror image phase display for the discovery of nonproteolytic d-peptide binders.Combining chemical protein synthesis with the RaPID system allows the production of a library of trillions of cyclic peptides and subsequent selection for novel cyclic peptide binders targeting a uniquely modified protein to assist in studying its unexplored biology and possibly the discovery of new drug candidates.Interestingly, the small post-translational modifier protein ubiquitin (Ub), with its various polymeric forms, regulates directly or indirectly many biochemical processes, e.g., proteasomal degradation, DNA damage repair, cell cycle regulation, etc. In this Account, we discuss combining the RaPID approach against various synthetic Ub chains for selecting effective and specific macrocyclic peptide binders. This offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling. We highlight experimental approaches and conceptual adaptations required to design and modulate the activity of Lys48- and Lys63-linked Ub chains by macrocyclic peptides. We also present the applications of these approaches to shed light on related biological activities and ultimately their activity against cancer. Finally, we contemplate future developments still pending in this exciting multidisciplinary field.
Assuntos
Peptídeos , Proteínas , Peptídeos/química , Peptídeos Cíclicos/química , Descoberta de Drogas , RNA Mensageiro/química , Biblioteca de PeptídeosRESUMO
Nuclear factor κB (NF-κB) is an important transcriptional regulator that is involved in numerous cellular processes, including cell proliferation, immune response, cell survival, and malignant transformation. It relies on the ubiquitin-proteasome system (UPS) for several of the steps in the concerted cascade of its activation. Previously, we showed that the ubiquitin (Ub) ligase KPC1 is involved in ubiquitination and limited proteasomal processing of the NF-κB1 p105 precursor to generate the p50 active subunit of the "canonical" heterodimeric transcription factor p50-p65. Overexpression of KPC1 with the generation of an excessive amount of p50 was shown to suppress tumors, an effect which is due to multiple mechanisms. Among them are suppression of expression of programmed cell death-ligand 1 (PD-L1), overexpression of a broad array of tumor suppressors, and secretion of cytokines which results in recruitment of suppressive immune cells into the tumor. Here, we show that the site of KPC1 to which p105 binds is exceptionally short and is made up of the seven amino acids WILVRLW. Attachment of this short stretch to a small residual part (â¼20%) of the ligase that also contains the essential Really Interesting New Gene (RING)-finger domain was sufficient to bind p105, conjugate to it Ub, and suppress tumor growth in an animal model. Fusion of the seven amino acids to a Von Hippel-Lindau protein (pVHL)-binding ligand (which serves as a "universal" ligase for many proteolysis-targeting chimeras; PROTACs) resulted in a compound that stimulated conjugation of Ub to p105 in a cell-free system and its processing to p50 in cells and restricted cell growth.
Assuntos
Subunidade p50 de NF-kappa B/metabolismo , NF-kappa B/metabolismo , Ubiquitina-Proteína Ligases/genética , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , NF-kappa B/genética , Neoplasias , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/fisiologia , Proteólise , Transdução de Sinais/fisiologia , Fator de Transcrição RelA/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genéticaRESUMO
Despite the great advances in discovering cyclic peptides against protein targets, their reduced aqueous solubility, cell permeability, and activity of the cyclic peptide restrict its utilization in advanced biological research and therapeutic applications. Here we report on a novel approach of structural alternation of the exocyclic and linker parts that led to a new derivative with significantly improved cell activity allowing us to dissect its mode of action in detail. We have identified an effective cyclic peptide (CP7) that induces approximately a 9-fold increase in DNA damage accumulation and a remarkable increase in apoptotic cancer cell death compared to the reported molecule. Notably, treating cells with CP7 leads to a dramatic decrease in the efficiency of non-homologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs), which is accompanied by an increase in homologous recombination (HR) repair. Interestingly, treating BRCA1-deficient cells with CP7 restores HR integrity, which is accompanied by increased resistance to CP7. Additionally, CP7 treatment increases the sensitivity of cancer cells to ionizing radiation. Collectively, our findings demonstrate that CP7 is a selective inhibitor of NHEJ, offering a potential strategy to enhance the effectiveness of radiation therapy.
RESUMO
There is a continuous demand to improve our understanding of fundamental processes that underlie human health and disease. Therefore, novel strategies that can assist in these efforts are required. For example, molecular biology and genetic approaches have revolutionized our understanding of protein-mediated processes by facilitating their direct visualization and analyses in living cells. Despite these developments, genetic manipulation has limitations in controlling events that occur after translation such as posttranslational modifications (PTMs), which are imperative regulatory elements. As a result, developing new methods to study PTMs in live cells is a major bottleneck in deciphering their exact roles in the myriad cellular processes.Synthetic and semisynthetic proteins are prepared by combining solid phase peptide synthesis (SPPS) and chemoselective ligation approaches with synthetic or recombinant peptides. Employing protein synthesis allows chemists to incorporate natural and unnatural modifications with virtually unlimited number of functional groups into the protein's sequence, such as PTMs and their mimics. In addition, synthetic proteins can include additional elements such as fluorescent tags, reactive groups, caged units, and enrichment handles. Therefore, harnessing the power of chemical protein synthesis offers great opportunities to study fundamental biological processes.Unfortunately, the low cell permeability of proteins limits their applications mainly to in vitro settings, excluding live cell studies. As a result, chemical biologists have been attempting to overcome these limitations by developing protein delivery methods that would enable the study of custom-made proteins in a biological context. Success with these strategies should enable accurate determination of protein localization, degradation, folding, interactions, and involvement in the assembly of membrane-less organelles formed by liquid-liquid phase separation inside cells. Importantly, protein delivery approaches are complementary to genetic manipulations, and combining these approaches should pave the way to new discoveries.In this Account, we describe recent developments in protein delivery methods, with emphasis on those most compatible with synthetic proteins. We highlight experimental approaches and conceptual adaptations required to design and study synthetic proteins in live cells, with or without genetic manipulation. In addition, we highlight the strength and weakness of these approaches for both the delivery and the subsequent studies. We also describe our endeavors to deliver synthetic proteins to cells via cell penetrating peptides (CPPs) and multiplexed bead loading (MBL), as showcases of the applications of these methods to shed light on biological processes. Lastly, we contemplate other future applications of synthetic proteins to answer questions that are currently unapproachable.
Assuntos
Peptídeos Penetradores de Células , Proteínas , Peptídeos Penetradores de Células/química , Humanos , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas/química , Técnicas de Síntese em Fase SólidaRESUMO
Targeting the cell nucleus remains a challenge for drug delivery. Here, we present a universal platform for the smart design of nanoparticle (NP) decoration that is based on: (i) a spacer polymer, commonly biotin-polyethylene-glycol-thiol, whose grafting density and molecular weight can be tuned for optimized performance, and (ii) protein binding peptides, such as cell penetrating peptides (CPPs), cancer-targeting peptides, or nuclear localization signal (NLS) peptides, that are linked to the PEG free-end by universal chemistry. We manifested our platform with two different bromo-acetamide (Br-Ac) modified NLSs. We used cell extract-based and live cell assays to demonstrate the recruitment of dynein motor proteins, which drive the NP active transport toward the nucleus, and the enhancement of cellular and nuclear entry, manifesting the properties of NLS as a CPP. Our control of the NP decoration scheme, and the modularity of our platform, carry great advantages for nano-carrier design for drug delivery applications.
Assuntos
Cinesinas , Nanopartículas , Polietilenoglicóis , PolímerosRESUMO
α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 ß2ß3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure-activity relationships of α-conotoxins, which may help in the design of more selective tools.
Assuntos
Conotoxinas , Caramujo Conus , Receptores Nicotínicos , Animais , Ratos , Conotoxinas/farmacologia , Conotoxinas/química , Caramujo Conus/química , Caramujo Conus/fisiologia , Antagonistas Nicotínicos/farmacologia , Caramujos , PolinésiaRESUMO
In fundamental research and drug discovery, there is still a need for effective and straightforward chemical approaches for generating cyclic peptides. The divergent synthesis of cyclic peptides remains a challenge, in particular when cyclization is carried out in the presence of unprotected side chains and a nonpeptidic component within the cycle is needed. Herein, we describe a novel and efficient strategy based on Au(I)-mediated cyclization of unprotected peptides through rapid (30-60 min) amine addition on a propargyl group to generate an imine linkage. Mechanistic insights reveal that the reaction proceeds via regioselective Markovnikov's addition of the amine on the Au(I)-activated propargyl. This strategy was successfully applied to prepare efficiently (56-94%) over 35 diverse cyclic peptides having different sequences and lengths. We have also achieved stereoselective reduction of cyclic imines employing chiral ligands. The practicality of our method was extended for the synthesis of cyclic peptides that bind Lys48-linked di-ubiquitin chains with high affinity, leading to apoptosis of cancer cells.
Assuntos
Ouro , Iminas , Aminas , Ciclização , Peptídeos/química , Peptídeos Cíclicos/químicaRESUMO
Ubiquitin (Ub) and its related small Ub like modifier (SUMO) are among the most influential protein post-translational modifications in eukaryotes. Unfortunately, visualizing these modifications in live cells is a challenging task. Chemical protein synthesis offers great opportunities in studying and further understanding Ub and SUMO biology. Nevertheless, the low cell permeability of proteins limits these studies mainly for inâ vitro applications. Here, we introduce a multiplexed protein cell delivery approach, termed MBL (multiplexed bead loading), for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We applied MBL to visualize ubiquitination and SUMOylation events in live and untransfected cells without fluorescent protein tags or perturbation to their endogenous levels. Our study reveals unprecedented involvements of Ub and SUMO2 in lysosomes depending on conjugation states. We envision that this approach will improve our understanding of dynamic cellular processes such as formation and disassembly of membraneless organelles.
Assuntos
Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Ubiquitina , Sobrevivência Celular , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K48-based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.
RESUMO
Peptides and proteins can be either synthesized using solid-phase peptide synthesis (SPPS) or by applying a combination of SPPS and ligation approaches to address fundamental questions related to human health and disease, among others. The demand for their production either by chemical or biological methods continues to raise significant interests from the synthetic community. In this context, transition metals such as Pd, Ag, Hg, Tl, Au, Zn, Ni, and Cu have also contributed to the field of peptide and protein synthesis such as in peptide conjugation, extending native chemical ligation (NCL), and for regioselective disulfide bonds formation. In this review, we highlight, summarize, and evaluate the use of various transition metals in the chemical synthesis of peptides and proteins with emphasis on recent developments in this exciting research area.
Assuntos
Peptídeos/síntese química , Proteínas/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Sequência de Aminoácidos , Catálise , Ciclização , Dissulfetos/química , Humanos , Metais/química , Solventes/química , Propriedades de Superfície , Elementos de Transição/químicaRESUMO
Deciphering the protein posttranslational modification (PTM) code is one of the greatest biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in their regulation leads to various disease. Chemical protein synthesis allows preparation of ubiquitinated and phosphorylated proteins to study their biochemical properties in great detail. However, monitoring these modifications in intact cells or in cell extracts mostly depends on antibodies, which often have off-target binding. Here, we report that the most widely used antibody for ubiquitin (Ub) phosphorylated at serine 65 (pUb) has significant off-targets that appear during mitosis. These off-targets are connected to polo-like kinase 1 (PLK1) mediated phosphorylation of cell cycle-related proteins and the anaphase promoting complex subunit 1 (APC1).
Assuntos
Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase , Proteínas de Ciclo Celular , Mitose , Processamento de Proteína Pós-Traducional , Ubiquitina , Anticorpos/genética , Anticorpos/metabolismo , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Mitose/genética , Mitose/fisiologia , Fosforilação , Ligação Proteica/genética , Ligação Proteica/fisiologia , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Serina/genética , Serina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação , Quinase 1 Polo-LikeRESUMO
Modifying cyclic cell-penetrating deca-arginine (cR10) peptides with 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) improves the uptake efficiency of synthetic ubiquitin (Ub) cargoes into living cells. To probe the role of the DABCYL moiety, we performed time-lapse microscopy and fluorescence lifetime imaging microscopy (FLIM) of fluorescent DABCYL-R10 to evaluate the impact on cell entry by the formation of nucleation zones. Furthermore, we performed a structure-uptake relationship study with 13 DABCYL derivatives coupled to CPP to examine their effect on the cell-uptake efficiency when conjugated to mono-Ub through disulfide linkages. Our results show that through structure variations of the DABCYL moiety alone we could reach, at nanomolar concentration, an additional threefold increase in the cytosolic delivery of Ub, which will enable studies on various intracellular processes related to Ub signaling.
Assuntos
Peptídeos Penetradores de Células , Peptídeos Penetradores de Células/química , Proteínas , p-Dimetilaminoazobenzeno , Microscopia de Fluorescência , UbiquitinaRESUMO
The removal of ubiquitin (Ub) from a modified protein or Ub chain is a process that occurs regularly by the ubiquitin-proteasome system. This process is known to be mediated by various deubiquitinating enzymes (DUBs) in order to control the protein's half-life and its expression levels among many other signaling processes. Since the function of DUBs is also involved in numerous human diseases, such as cancer, there is an obvious need for an effective diagnostic probe that can monitor the activity of these enzymes. We have developed the first chemiluminescence probe for detection of DUBs activity. The probe was prepared by conjugation of the chemically synthesized C-terminally activated Ub(1-75) with a Gly-enolether precursor. Subsequent oxidation, under aqueous conditions, of the enolether conjuagate with singlet-oxygen furnished the dioxetane probe Ub-CL. This synthesis provides the first example of a dioxetane-luminophore protein conjugate. The probe's ability to detect deubiquitinating activity was successfully validated with three different DUBs. In order to demonstrate the advantage of our new probe, comparison measurements for detection of DUB UCH-L3 activity were performed between the chemiluminescent probe Ub-CL and the well-known Ub-AMC probe. The obtained data showed significantly higher S/N, for probe Ub-CL (>93-fold) in comparison to that observed for Ub-AMC (1.5-fold). We anticipate that the successful design and synthesis of the turn-ON protein-dioxetane conjugate probe, demonstrated in this work, will provide the insight and motivation for preparation of other relevant protein-dioxetane conjugates.
Assuntos
Endopeptidases , Processamento de Proteína Pós-Traducional , Humanos , UbiquitinaRESUMO
Live-cell delivery of a fully synthetic protein having selectivity towards a particular target is a promising approach with potential applications for basic research and therapeutics. Cell-penetrating peptides (CPPs) allow the cellular delivery of proteins but mostly result in endosomal entrapment, leading to lack of bioavailability. Herein, we report the design and synthesis of a CPP fused to 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL) to enhance cellular uptake of fluorescently labelled synthetic protein analogues in low micromolar concentration. The attachment of cyclic deca-arginine (cR10) modified with a single lysine linked to DABCYL to synthetic ubiquitin (Ub) and small ubiquitin-like modifier-2 (SUMO-2) scaffolds resulted in a threefold higher uptake efficacy in live cells compared to the unmodified cR10. We could also achieve cR10DABCYL-assisted delivery of Ub and a Ub variant (Ubv) based activity-based probes for functional studies of deubiquitinases in live cells.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina/metabolismo , p-Dimetilaminoazobenzeno/análogos & derivados , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Fluorescência , Humanos , Estrutura Molecular , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/síntese química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Ubiquitina/síntese química , Ubiquitina/química , p-Dimetilaminoazobenzeno/química , p-Dimetilaminoazobenzeno/metabolismoRESUMO
Development of modulators targeting specific interactions of ubiquitin-based conjugates with their partners is a formidable task since it requires a suitable screening assay and homogeneous ubiquitin conjugates. We developed a novel high-throughput strategy for screening ligands for Lys48-linked tetraubiquitin chain in a relatively simple, fast, and affordable manner. This approach combined with a state-of-the-art toolbox of chemical protein synthesis and a specially optimized Cys deprotection protocol enabled us to design highly potent, Lys48-linked tetraubiquitin chain selective "next generation" dimeric peptide modulators. The dimeric peptide exhibited cancer cell permeability and induced cell death with higher efficiency compared to its monocyclic analogue. These features make our dimeric peptide a promising candidate for further studies using in vivo models. Our assay can be adopted for other various ubiquitin chains in their free or anchored forms as well as conjugates for Ub-like modifiers.
Assuntos
Desenvolvimento de Medicamentos , Fluorescência , Peptídeos Cíclicos/química , Ubiquitina/química , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Ubiquitina/farmacologiaRESUMO
Disulfide-rich peptides and proteins are among the most fascinating bioactive molecules. The difficulties associated with the preparation of these targets have prompted the development of various chemical strategies. Nevertheless, the production of these targets remains very challenging or elusive. Recently, we introduced a strategy for one-pot disulfide bond formation, tackling most of the previous limitations. However, the effect of the order of oxidation remained an underexplored issue. Herein we report on the complete synthetic flexibility of the approach with respect to the order of oxidation of three disulfide bonds in targets that lack the knot motif. In contrast, our study reveals an essential order of disulfide bond formation in the EETI-II knotted miniprotein. This synthetic strategy was applied for the synthesis of novel analogues of the plectasin antimicrobial peptide with enhanced activities against methicillin-resistant Staphylococcus aureus (MRSA), a notorious human pathogen.
Assuntos
Peptídeos Antimicrobianos/química , Cucurbitaceae/química , Dissulfetos/síntese química , Proteínas de Plantas/química , Dissulfetos/química , HumanosRESUMO
Chemists have been interested in the N-alkylation of a peptide bond because such a modification alters the conformation of the amide bond, interferes with hydrogen bond formation, and changes other properties of the peptide (e.g., solubility). This modification also opens the door for attaching functional groups for various applications. Nonetheless, the irreversibility of some of these modifications and the harsh conditions required for their removal currently limits the wide utility of this approach. Herein, we report applying a propargyl group for peptide bond modification at diverse junctions, which can be removed under mild and aqueous conditions via treatment with gold(I). Considering the straightforward conditions for both the installation and removal of this group, the propargyl group provides access to the benefits of backbone N-alkylation, while preserving the ability for on-demand depropargylation and full recovery of the native amide bond. This reversible modification was found to improve solid-phase peptide synthesis as demonstrated in the chemical synthesis of NEDD8 protein, without the use of special dipeptide analogues. Also, the reported approach was found to be useful in decaging a broad range of propargyl-based protecting groups used in chemical protein synthesis. Remarkably, reversing the order of the two residues in the propargylation site resulted in rapid amide bond cleavage, which extends the applicability of this approach beyond a removable backbone modification to a cleavable linker. The easy attach/detach of this functionality was also examined in loading and releasing of biotinylated peptides from streptavidin beads.
Assuntos
Dipeptídeos/química , Ouro/química , Proteína NEDD8/síntese química , Dipeptídeos/síntese química , Humanos , Ligação de Hidrogênio , Estrutura Molecular , Proteína NEDD8/química , Água/químicaRESUMO
The maleimide group is a widely used reagent for bioconjugation of peptides, proteins, and oligonucleotides employing Michael addition and Diels-Alder cycloaddition reactions. However, the utility of this functionality in chemical synthesis of peptides and proteins remains unexplored. We report, for the first time that PdII complexes can mediate the efficient removal of various succinimide derivatives in aqueous conditions. Succinimide removal by PdII was applied for the synthesis of two ubiquitin activity-based probes (Ub-ABPs) employing solid phase chemical ligation (SPCL). SPCL was achieved through a sequential three segment ligation on a polymer support via a maleimide anchor. The obtained probes successfully formed the expected covalent complexes with deubiquitinating enzymes (DUBs) USP2 and USP7, highlighting the use of our new method for efficient preparation of unique synthetic proteins. Importantly, we demonstrate the advantages of our newly developed method for the protection and deprotection of native cysteine with a succinimide group in a peptide fragment derived from thioredoxin-1 (Trx-1) obtained via intein based expression to enable ligation/desulfurization and subsequent disulfide bond formation in a one-pot process.