RESUMO
In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolytic activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To resolve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via ion-exchange chromatography. After the noncovalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolytic activity and could efficiently catalyze inter-molecular protein ligation and intramolecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis.
Assuntos
Cisteína Endopeptidases , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/química , Engenharia de Proteínas/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Ligases/genética , Ligases/metabolismo , Ligases/química , Bambusa/genética , Bambusa/enzimologia , Mutagênese Sítio-Dirigida , Folhas de Planta/enzimologia , Folhas de Planta/genética , Sequência de AminoácidosRESUMO
The peptide hormone ghrelin (an agonist) and LEAP2 (an antagonist) play important functions in energy metabolism via their receptor GHSR, an A-class G protein-coupled receptor. Ghrelin, LEAP2, and GHSR are widely present from fishes to mammals. However, our recent study suggested that fish GHSRs have different binding properties to ghrelin: a GHSR from the lobe-finned fish Latimeria chalumnae (coelacanth) is efficiently activated by ghrelin, but GHSRs from the ray-finned fish Danio rerio (zebrafish) and Larimichthys crocea (large yellow croaker) have lost binding to ghrelin. Do fish GHSRs use another peptide as their agonist? In the present study we tested to two fish motilins from D. rerio and L. chalumnae because motilin is distantly related to ghrelin. In ligand binding and activation assays, the fish GHSRs from D. rerio and L. crocea displayed no detectable or very low binding to all tested motilins; however, the fish GHSR from L. chalumnae bound to its motilin with high affinity and was efficiently activated by it. Therefore, it seemed that motilin is not a ligand for GHSR in the ray-finned fish D. rerio and L. crocea, but is an efficient agonist for GHSR in the lobe-finned fish L. chalumnae, one of the closest fish relatives of tetrapods. The results of present study suggested that GHSR might have two efficient agonists, ghrelin and motilin, in ancient fishes; however, this feature might be only preserved in some extant fishes with ancient evolutionary origins.
RESUMO
Our recent study confirmed that the mature neuropeptide FAM237A, also known as neurosecretory protein GL (NPGL), is an efficient agonist for GPR83. The paralog FAM237B was previously reported as a weak agonist for GPR83. In the present study, we prepared mature human FAM237B via an intein-fusion approach and demonstrated that it could cause a significant activation effect at the nanomolar range (1â10 nM) in a NanoBiT-based ß-arrestin recruitment assay. Thus, FAM237B appears to be another endogenous agonist for GPR83 and future in vivo studies will be required to confirm this.
Assuntos
Neuropeptídeos , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
G protein-coupled receptor 83 (GPR83) is primarily expressed in the brain and is implicated in the regulation of energy metabolism and some anxiety-related behaviours. Recently, the PCSK1N/proSAAS-derived peptide PEN, the procholecystokinin-derived peptide proCCK56-63, and family with sequence similarity 237 member A (FAM237A) were all reported as efficient agonists of GPR83. However, these results have not yet been reproduced by other laboratories and thus GPR83 is still officially an orphan receptor. The peptide PEN and proCCK56-63 share sequence similarity; however, they are completely different from FAM237A. To identify its actual ligand(s), in the present study we developed NanoLuc Binary Technology (NanoBiT)-based ligand-binding assay, fluorescent ligand-based visualization, and NanoBiT-based ß-arrestin recruitment assay for human GPR83. Using these assays, we demonstrated that mature human FAM237A could bind to GPR83 with nanomolar range affinity, and could activate this receptor and induce its internalization with nanomolar range efficiency in transfected human embryonic kidney 293T cells. However, we did not detect any interaction of PEN and proCCK56-63 with GPR83 using these assays. Thus, our results confirmed that FAM237A is an efficient agonist of GPR83, but did not support PEN and proCCK56-63 as ligands of this receptor. Clarification of their pairing paves the way for further functional studies of the brain-specific receptor GPR83 and the so far rarely studied neuropeptide FAM237A in the future.
Assuntos
Neuropeptídeos , Receptores Acoplados a Proteínas G , Humanos , Ligantes , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Encéfalo/metabolismo , Metabolismo EnergéticoRESUMO
Recently, liver-expressed antimicrobial peptide 2 (LEAP2) was identified as an endogenous antagonist and an inverse agonist of the ghrelin receptor GHSR. However, its functions in lower vertebrates are not well understood. Our recent study demonstrated that both LEAP2 and ghrelin are functional towards a fish GHSR from Latimeria chalumnae, an extant coelacanth believed to be one of the closest ancestors of tetrapods. However, amino acid sequence alignment identified that the 6.58 position (Ballesteros-Weinstein numbering system) of most fish GHSRs are not occupied by an aromatic Phe residue, which is absolutely conserved in all known GHSRs from amphibians to mammals, and is responsible for human GHSR binding to its agonist, ghrelin. To test whether these unusual fish receptors are functional, we studied the ligand binding properties of three representative fish GHSRs, two from Danio rerio (zebrafish) and one from Larimichthys crocea (large yellow croaker). After overexpression in human embryonic kidney 293T cells, the three fish GHSRs retained normal binding to all tested LEAP2s, except for a second LEAP2 from L. crocea. However, they displayed almost no binding to all chemically synthesized n-octanoylated ghrelins, despite these ghrelins all retaining normal function towards human and coelacanth GHSRs. Thus, it seems that LEAP2 is a more conserved ligand than ghrelin towards fish GHSRs. Our results not only provided new insights into the interaction mechanism of GHSRs with LEAP2s and ghrelins, but also shed new light on the functions of LEAP2 and ghrelin in different fish species.
Assuntos
Grelina , Peixe-Zebra , Animais , Humanos , Grelina/metabolismo , Ligantes , Peixe-Zebra/metabolismo , Agonismo Inverso de Drogas , Receptores de Grelina/agonistas , Receptores de Grelina/metabolismo , Mamíferos/metabolismoRESUMO
The orexigenic peptide ghrelin exerts important functions in energy metabolism and has therapeutic potential to treat certain diseases. Native ghrelin carries an essential O-fatty acyl moiety; however, this post-translational modification is susceptible to hydrolysis by certain esterases in circulation, representing a major route of its in vivo inactivation. In the present study, we developed a novel approach to prepare various esterase-resistant ghrelin analogs via photoinduced thiol-ene click chemistry. A recombinant unacylated human ghrelin mutant was reacted with commercially available terminal alkenes; thus, various alkyl moieties were introduced to the side chain of its unique Cys3 residue via a thioether bond. Among 11 S-alkylated ghrelin analogs, analog 11, generated by reacting with 2-methyl-1-octene, not only acquired much higher stability in serum but also retained full activity compared with native human ghrelin. Thus, the present study provided an efficient approach to prepare highly stable and highly active ghrelin analogs with therapeutic potential.
RESUMO
BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a critical event contributing to more aggressive phenotypes in cancer cells. EMT is frequently activated in radiation-targeted cells during the course of radiotherapy, which often endows cancers with acquired radioresistance. However, the upstream molecules driving the signaling pathways of radiation-induced EMT have not been fully delineated. METHODS: In this study, RNA-seq-based transcriptome analysis was performed to identify the early responsive genes of HeLa cells to γ-ray irradiation. EMT-associated genes were knocked down by siRNA technology or overexpressed in HeLa cells and A549 cells, and the resulting changes in phenotypes of EMT and radiosensitivity were assessed using qPCR and Western blotting analyses, migration assays, colony-forming ability and apoptosis of flow cytometer assays. RESULTS: Through RNA-seq-based transcriptome analysis, we found that LPAR5 is downregulated in the early response of HeLa cells to γ-ray irradiation. Radiation-induced alterations in LPAR5 expression were further revealed to be a bidirectional dynamic process in HeLa and A549 cells, i.e., the early downregulating phase at 2 ~ 4 h and the late upregulating phase at 24 h post-irradiation. Overexpression of LPAR5 prompts EMT programing and migration of cancer cells. Moreover, increased expression of LPAR5 is significantly associated with IR-induced EMT and confers radioresistance to cancer cells. Knockdown of LPAR5 suppressed IR-induced EMT by attenuating the activation of ERK signaling and downstream Snail, MMP1, and MMP9 expression. CONCLUSIONS: LPAR5 is an important upstream regulator of IR-induced EMT that modulates the ERK/Snail pathway. This study provides further insights into understanding the mechanism of radiation-induced EMT and identifies promising targets for improving the effectiveness of cancer radiation therapy.
Assuntos
Metaloproteinase 1 da Matriz , Neoplasias , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Células HeLa , Humanos , Metaloproteinase 9 da Matriz , RNA Interferente Pequeno , Receptores de Ácidos LisofosfatídicosRESUMO
Stimulated Raman scattering (SRS) microscopy in combination with innovative tagging strategies offers great potential as a universal high-throughput biomedical imaging tool. Here, we report rationally tailored small molecular monomers containing triple-bond units with large Raman scattering cross-sections, which can be polymerized at the nanoscale for enhancement of SRS contrast with smaller but brighter optical nanotags with artificial fingerprint output. From this, a class of triple-bond rich polymer nanoparticles (NPs) was engineered by regulating the relative dosages of three chemically different triple-bond monomers in co-polymerization. The bonding strategy allowed for 15 spectrally distinguishable triple-bond combinations. These accurately structured nano molecular aggregates, rather than long-chain macromolecules, could establish a universal method for generating small-sized biological SRS imaging tags with high sensitivity for high-throughput multi-color biomedical imaging.
Assuntos
Nanopartículas/química , Imagem Óptica , Polímeros/química , Humanos , Células MCF-7 , Estrutura Molecular , Análise Espectral RamanRESUMO
Recent studies have demonstrated that liver-expressed antimicrobial peptide 2 (LEAP2) antagonizes the ghrelin receptor GHSR1a in mammals. However, its antagonistic function in lower vertebrates has not yet been tested. LEAP2 orthologs have been identified from a variety of fish species; however, previous studies all focused on their antimicrobial activity. To test whether LEAP2 functions as a GHSR1a antagonist in the lowest vertebrates, we studied the antagonism of a fish LEAP2 from Latimeria chalumnae, an extant coelacanth that is one of the closest living fish relatives of tetrapods. Using binding assays, we demonstrated that the coelacanth LEAP2 and ghrelin bound to the coelacanth GHSR1a with IC50 values in the nanomolar range. Using activation assays, we demonstrated that the coelacanth ghrelin activated the coelacanth GHSR1a with an EC50 value in the nanomolar range, and this activation effect was efficiently antagonized by a nanomolar range of the coelacanth LEAP2. In addition, we also showed that the human LEAP2 and ghrelin were as effective as their coelacanth orthologs towards the coelacanth GHSR1a; however, the coelacanth peptides had moderately lower activity towards the human GHSR1a. Thus, LEAP2 serves as an endogenous antagonist of the ghrelin receptor GHSR1a in coelacanth and the ghrelin-LEAP2-GHSR1a system has evolved slowly since its emergence in ancient fish.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Proteínas Sanguíneas , Evolução Molecular , Proteínas de Peixes , Peixes , Receptores de Grelina , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Peixes/genética , Peixes/metabolismo , Células HEK293 , Humanos , Receptores de Grelina/antagonistas & inibidores , Receptores de Grelina/genética , Receptores de Grelina/metabolismoRESUMO
The gastric peptide ghrelin has important functions in energy metabolism and cellular homeostasis by activating growth hormone secretagogue receptor type 1a (GHSR1a). The N-terminal residues of ghrelin orthologs from all vertebrates are quite conserved; however, in orthologs from Cavia porcellus and Phyllostomus discolor, Ser2 and Leu5 are replaced by a smaller Ala and a positively charged Arg, respectively. In the present study, we first demonstrated that the hydrophobic Leu5 is essential for the function of human ghrelin, because Ala replacement caused an approximately 100-fold decrease in activity. However, replacement of Leu5 by an Arg residue caused much less disruption; further replacement of Ser2 by Ala almost restored full activity, although the [S2A] mutation itself showed slight detriments, implying that the positively charged Arg5 in the [S2A,L5R] mutant might form alternative interactions with certain receptor residues to compensate for the loss of the essential Leu5. To identify the responsible receptor residues, we screened GHSR1a mutants in which all conserved negatively charged residues in the extracellular regions and all aromatic residues in the ligand-binding pocket were mutated separately. According to the decrease in selectivity of the mutant receptors towards [S2A,L5R]ghrelin, we deduced that the positively charged Arg5 of the ghrelin mutant primarily interacts with the essential aromatic Phe286 at the extracellular end of the sixth transmembrane domain of GHSR1a by forming cation-π and π-π interactions. The present study provided new insights into the binding mechanism of ghrelin with its receptor, and thus would facilitate the design of novel ligands for GHSR1a.
Assuntos
Grelina/química , Receptores de Grelina/química , Animais , Quirópteros , Grelina/genética , Grelina/metabolismo , Cobaias , Células HEK293 , Humanos , Ligação Proteica , Domínios Proteicos , Receptores de Grelina/genética , Receptores de Grelina/metabolismoRESUMO
Relaxin family peptide receptor 3 (RXFP3) is a G protein-coupled receptor implicated in the regulation of food intake and stress response upon activation by the neuropeptide relaxin-3. In recent studies, interactions of RXFP3 with some natural or synthetic ligands have been investigated. In the present study, we identified the hydrophobic interactions of human RXFP3 with the chimeric agonist R3/I5 and the chimeric antagonist R3(ΔB23-27)R/I5 using a newly developed NanoBiT-based homogenous binding assay. We first demonstrated that the conserved large aliphatic B15Ile and B19Ile were important for the binding of the agonist and antagonist to RXFP3, because alanine replacement significantly decreased their receptor-binding potency. Thereafter, we demonstrated that the conserved large aliphatic Leu246 and Leu248 in extracellular loop 2 were important for RXFP3 binding to the agonist and antagonist, because alanine replacement significantly decreased the binding affinity of RXFP3 for both ligands. Finally, we deduced probable hydrophobic interactions based on the ability of RXFP3 mutants to distinguish the wild-type and mutant ligands: Leu246 of RXFP3 interacted with B15Ile of both ligands, while Leu248 of RXFP3 interacted with both B15Ile and B19Ile of the agonist and antagonist. The present results not only provided new insights into the interaction mechanism of RXFP3 with agonists and antagonists, but also demonstrated usefulness of the NanoBiT-based homogenous binding assay to study the interaction mechanism of certain receptors with their ligands.
Assuntos
Medições Luminescentes/métodos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Estruturais , Ligação Proteica/genética , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relaxina/química , Relaxina/genética , Relaxina/metabolismoRESUMO
Liver-expressed antimicrobial peptide 2 (LEAP2) was recently identified as a competitive antagonist for the G protein-coupled receptor GHSR1a, the cognate receptor for the gastric peptide ghrelin. LEAP2 plays important functions in energy metabolism by tuning the ghrelin-GHSR1a system. However, the molecular mechanism by which LEAP2 binds to GHSR1a is largely unknown. In the present study, we first conducted alanine-scanning mutagenesis on the N-terminal fragment of human LEAP2 and demonstrated that the positively charged Arg6 and the aromatic Phe4 are essential for LEAP2 binding to GHSR1a. To identify the receptor residues interacting with the essential Arg6 and Phe4 of LEAP2, we conducted extensive site-directed mutagenesis on GHSR1a. After all conserved negatively charged residues in the extracellular regions of human GHSR1a were mutated, only mutation of Asp99 caused much more detriments to GHSR1a binding to LEAP2 than binding to ghrelin, suggesting that the absolutely conserved Asp99 of GHSR1a probably interacts with the essential Arg6 of LEAP2. After five conserved Phe residues in the predicted ligand-binding pocket of human GHSR1a were mutated, three of them were identified as important for GHSR1a binding to LEAP2. According to a structural model of GHSR1a, we deduced that the adjacent Phe279 and Phe312 might interact with the essential Phe4 of LEAP2, while Phe119 might interact with the aromatic Trp5 of LEAP2. The present study provided new insights into the interaction of LEAP2 with its receptor, and would facilitate the design of novel ligands for GHSR1a in future studies.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Proteínas Sanguíneas/química , Receptores de Grelina/química , Substituição de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ligação Proteica , Domínios Proteicos , Receptores de Grelina/genética , Receptores de Grelina/metabolismoRESUMO
Liver-expressed antimicrobial peptide 2 (LEAP2) is a highly conserved secretory peptide first isolated in 2003. However, its exact biological functions remained elusive until a recent study identified it as an endogenous antagonist for the growth hormone secretagogue receptor (GHSR1a), a G protein-coupled receptor for which the gastric peptide ghrelin is the endogenous agonist. By tuning the ghrelin-GHSR1a system, LEAP2 has an important function in energy metabolism. In the present study, we first demonstrated that LEAP2 and ghrelin actually bound to GHSR1a in a competitive manner, rather than in a non-competitive manner as previously reported, by binding assays and activation assays. Subsequently, we demonstrated that the antagonistic function of LEAP2 was drastically affected by the manner of its addition. LEAP2 primarily affected the maximal activation effect when added before ghrelin, whereas it primarily affected half-maximal effective concentration when added at the same time as ghrelin. Thus, LEAP2 behaved as a competitive antagonist if added at the same time as the agonist and a non-competitive antagonist if added before the agonist. This unusual property of LEAP2 might be caused by its slow dissociation from receptor GHSR1a. We also found that the N-terminal fragment of LEAP2 was important for receptor binding. Our present study revealed an antagonistic mechanism for LEAP2, and will facilitate the design of novel antagonists for receptor GHSR1a in future studies.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Grelina/metabolismo , Receptores de Grelina/metabolismo , Ligação Competitiva , Cálcio/metabolismo , Humanos , Cinética , Ligação Proteica , Receptores de Grelina/antagonistas & inibidoresRESUMO
Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely relaxin family peptide receptor 1-4 (RXFP1-4). We recently disclosed electrostatic interactions of the homologous RXFP3 and RXFP4 with some agonists based on activation complementation. However, this activation assay-based approach cannot be applied to antagonists that do not activate receptors. Herein, we propose a general approach suitable for both agonists and antagonists based on our newly-developed NanoBiT-based binding assay. We first validated the binding assay-based approach using the agonist relaxin-3, then applied it to the chimeric antagonist R3(ΔB23-27)R/I5. Three positively charged B-chain Arg residues of the agonist and antagonist were respectively replaced by a negatively charged Glu residue; meanwhile, the negatively charged Glu and Asp residue in the essential WxxExxxD motif of both receptors were respectively replaced by a positively charged Arg residue. Based on binding complementation of mutant ligands towards mutant receptors, we deduced possible electrostatic interactions of the agonist and antagonist with both RXFP3 and RXFP4: their B-chain C-terminal Arg residue interacts with the deeply buried Glu residue in the WxxExxxD motif of both receptors, and one or two of their B-chain central Arg residues interact with the shallowly buried Asp residue in the WxxExxxD motif of both receptors. Our present work shed new light on the interaction mechanism of RXFP3 and RXFP4 with agonists and antagonists, and also provided a novel approach for interaction studies of some plasma membrane receptors with their ligands.
Assuntos
Receptores Acoplados a Proteínas G/química , Receptores de Peptídeos/química , Relaxina/química , Motivos de Aminoácidos , Humanos , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relaxina/genética , Relaxina/metabolismo , Eletricidade EstáticaRESUMO
Relaxin family peptides perform a variety of biological functions by binding and activating relaxin family peptide receptor 1-4 (RXFP1-4), four A-class G protein-coupled receptors. In the present work, we developed a novel ligand binding assay for RXFP3 and RXFP4 based on NanoLuc complementation technology (NanoBiT). A synthetic ligation version of the low-affinity small complementation tag (SmBiT) was efficiently ligated to the A-chain N terminus of recombinant chimeric agonist R3/I5 using recombinant circular sortase A. After the ligation product R3/I5-SmBiT was mixed with human RXFP3 or RXFP4 genetically fused with a secretory large NanoLuc fragment (sLgBiT) at the N terminus, NanoLuc complementation was induced by high-affinity ligand-receptor binding. Binding kinetics and affinities of R3/I5-SmBiT with sLgBiT-fused RXFP3 and RXFP4 were conveniently measured according to the complementation-induced bioluminescence. Using R3/I5-SmBiT and the sLgBiT-fused receptor as a complementation pair, binding potencies of various ligands with RXFP3 and RXFP4 were quantitatively measured without the cumbersome washing step. The novel NanoBiT-based ligand binding assay is convenient for use and suitable for automation, thus will facilitate interaction studies of RXFP3 and RXFP4 with ligands in future. This assay can also be applied to some other plasma membrane receptors for pharmacological characterization of ligands in future studies.