RESUMO
Protein ubiquitination is a key post-translational modification in regulating many fundamental cellular processes and dysregulation of these processes can give rise to a vast array of diseases. Unravelling the molecular mechanisms of ubiquitination hence is an important area in current ubiquitin research with as aim to understand this enigmatic process. The complexity of ubiquitin (Ub) signaling arises from the large variety of Ub conjugates, where Ub is attached to other Ub proteins, Ub-like proteins, and protein substrates. The chemical preparation of such Ub conjugates in high homogeneity and in adequate amounts contributes greatly to the deciphering of Ub signaling. The strength of these chemically synthesized conjugates lies in the chemo-selectivity in which they can be created that are sometimes difficult to obtain using biochemical methodology. In this review, we will discuss the progress in the chemical protein synthesis of state-of-the-art Ub and Ub-like chemical probes, their unique concepts and related discoveries in the ubiquitin field.
Assuntos
Processamento de Proteína Pós-Traducional , Ubiquitina , Ubiquitina/metabolismo , Ubiquitinação , Proteínas/metabolismo , Transdução de SinaisRESUMO
Small ubiquitin-like modifiers (SUMOs) are conjugated to protein substrates in cells to regulate their function. The attachment of SUMO family members SUMO1-3 to substrate proteins is reversed by specific isopeptidases called SENPs (sentrin-specific protease). Whereas SENPs are SUMO-isoform or linkage type specific, comprehensive analysis is missing. Furthermore, the underlying mechanism of SENP linkage specificity remains unclear. We present a high-throughput synthesis of 83â isopeptide-linked SUMO-based fluorescence polarization reagents to study enzyme preferences. The assay reagents were synthesized via a native chemical ligation-desulfurization protocol between 11-mer peptides containing a γ-thiolysine and a SUMO3 thioester. Subsequently, five recombinantly expressed SENPs were screened using these assay reagents to reveal their deconjugation activity and substrate preferences. In general, we observed that SENP1 is the most active and nonselective SENP while SENP6 and SENP7 show the least activity. Furthermore, SENPs differentially process peptides derived from SUMO1-3, who form a minimalistic representation of diSUMO chains. To validate our findings, five distinct isopeptide-linked diSUMO chains were chemically synthesized and proteolysis was monitored using a gel-based read-out.
Assuntos
Corantes Fluorescentes , Ensaios de Triagem em Larga Escala , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Endopeptidases/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Proteólise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/síntese química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/químicaRESUMO
Chemical protein synthesis has proven to be a powerful tool to access homogenously modified proteins. The chemical synthesis of nanobodies (Nb) would create possibilities to design tailored Nbs with a range of chemical modifications such as tags, linkers, reporter groups, and subsequently, Nb-drug conjugates. Herein, we describe the total chemical synthesis of a 123 amino-acid Nb against GFP. A native chemical ligation- desulfurization strategy was successfully applied for the synthesis of this GFP Nb, modified with a propargyl (PA) moiety for on-demand functionalization. Biophysical characterization indicated that the synthetic GFP Nb-PA was correctly folded after internal disulfide bond formation. The synthetic Nb-PA was functionalized with a biotin or a sulfo-cyanine5 dye by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), resulting in two distinct probes used for functional inâ vitro validation in pull-down and confocal microscopy settings.
Assuntos
Azidas , Anticorpos de Domínio Único , Alcinos/química , Azidas/química , Biotina , Química Click , Cobre/química , Dissulfetos , Proteínas/químicaRESUMO
Spatiotemporal control over biochemical signaling processes involving G protein-coupled receptors (GPCRs) is highly desired for dissecting their complex intracellular signaling. We developed sixteen photoswitchable ligands for the human histamine H3 receptor (hH3 R). Upon illumination, key compound 65 decreases its affinity for the hH3 R by 8.5-fold and its potency in hH3 R-mediated Gi protein activation by over 20-fold, with the trans and cis isomer both acting as full agonist. In real-time two-electrode voltage clamp experiments in Xenopus oocytes, 65 shows rapid light-induced modulation of hH3 R activity. Ligand 65 shows good binding selectivity amongst the histamine receptor subfamily and has good photolytic stability. In all, 65 (VUF15000) is the first photoswitchable GPCR agonist confirmed to be modulated through its affinity and potency upon photoswitching while maintaining its intrinsic activity, rendering it a new chemical biology tool for spatiotemporal control of GPCR activation.
Assuntos
Agonistas dos Receptores Histamínicos/farmacologia , Receptores Histamínicos H3/metabolismo , Agonistas dos Receptores Histamínicos/síntese química , Agonistas dos Receptores Histamínicos/química , Humanos , Estrutura Molecular , Processos FotoquímicosRESUMO
Autophagy is a conserved cellular process involved in the degradation of intercellular materials. During this process, double-membrane vesicles called autophagosomes engulf cytoplasmic components ready for degradation. A key component in the formation of autophagosomes are the autophagy-related (Atg) proteins, including microtubule-associated protein light chain 3A (LC3A) and 3B (LC3B). After the C-terminus of LC3 is conjugated to a phospholipid, it promotes the elongation of the phagosome and provides a docking station for the delivery of proteins ready for degradation. Since dysregulation of the autophagy pathway has been associated with a variety of human diseases, components of this process have been considered as potential therapeutic targets. However, the mechanistic details of LC3-specific ligases and deconjugation enzymes are far from unraveled and chemical tools for activity profiling could aid in affording more insights into this process. Herein, we describe a native chemical ligation approach for the synthesis of two LC3 activity-based probes (ABPs). Initial studies show that the probes covalently interact with the cysteine protease ATG4B, showcasing the potential of these probes to unravel mechanistic and structural details.
RESUMO
Solid-phase peptide synthesis (SPPS) enables the synthesis of chemically modified peptides and proteins. Chemically synthesized ubiquitin(-like) proteins containing a fluorescent tag or reactive warhead have proven to be important tools in elucidating biological processes. Here, we describe the first fully synthetic method for the linear synthesis of two LC3 ubiquitin-like proteins using disaggregating building blocks and heated synthesis. Both LC3A and LC3B were synthesized and equipped with a fluorescent rhodamine tag, followed by folding of the proteins and liquid chromatography-mass spectrometry and SDS-PAGE analysis to prove that the quality of the synthetic material is comparable to expressed material.
Assuntos
Corantes , Temperatura Alta , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Cromatografia Líquida , UbiquitinasRESUMO
The human histamine H3 receptor (hH3R) is predominantly expressed in the CNS, where it regulates the synthesis and release of histamine and other neurotransmitters. Due to its neuromodulatory role, the hH3R has been associated with various CNS disorders, including Alzheimer's and Parkinson's disease. Markedly, the hH3R gene undergoes extensive splicing, resulting in 20 isoforms, of which 7TM isoforms exhibit variations in the intracellular loop 3 (IL3) and/or C-terminal tail. Particularly, hH3R isoforms that display variations in IL3 (e.g., hH3R-365) are shown to differentially signal via Gαi-dependent pathways upon binding of biased agonists (e.g., immepip, proxifan, imetit). Nevertheless, the mechanisms underlying biased agonism at hH3R isoforms remain unknown. Using a structure-function relationship study with a broad range of H3R agonists, we thereby explored determinants underlying isoform bias at hH3R isoforms that exhibit variations in IL3 (i.e., hH3R-445, -415, -365, and -329) in a Gαi-dependent pathway (cAMP inhibition). Hence, we systematically characterized hH3R isoforms on isoform bias by comparing various ligand properties (i.e., structural and molecular) to the degree of isoform bias. Importantly, our study provides novel insights into the structural and molecular basis of receptor isoform bias, highlighting the importance to study GPCRs with multiple isoforms to better tailor drugs.