ABSTRACT
Protein degradation is a tightly regulated biological process that maintains bacterial proteostasis. ClpPs are a highly conserved family of serine proteases that associate with the AAA + ATPase (an ATPase associated with diverse cellular activities) to degrade protein substrates. Identification and biochemical characterization of protein substrates for the AAA + ATPase-dependent ClpP degradation systems are considered essential for gaining an understanding of the molecular operation of the complex ClpP degradation machinery. Consequently, expanding the repertoire of protein substrates that can be degraded in vitro and within bacterial cells is necessary. Here, we report that AAA + ATPase-ClpP proteolytic complexes promote degradation of the secondary metabolite surfactin synthetases SrfAA, SrfAB, and SrfAC in Bacillus subtilis. On the basis of in vitro and in-cell studies coupled with activity-based protein profiling of nonribosomal peptide synthetases, we showed that SrfAC is targeted to the ClpC-ClpP proteolytic complex, whereas SrfAA is hydrolyzed not only by the ClpC-ClpP proteolytic complex but also by different ClpP proteolytic complexes. Furthermore, SrfAB does not appear to be a substrate for the ClpC-ClpP proteolytic complex, thereby implying that other ClpP proteolytic complexes are involved in the degradation of this surfactin synthetase. Natural product biosynthesis is regulated by the AAA + ATPase-ClpP degradation system, indicating that protein degradation plays a role in the regulatory stages of biosynthesis. However, few studies have examined the regulation of protein degradation levels. Furthermore, SrfAA, SrfAB, and SrfAC were identified as protein substrates for AAA + ATPase-ClpP degradation systems, thereby contributing to a better understanding of the complex ClpP degradation machinery.
Subject(s)
Bacillus subtilis , Bacterial Proteins , Biological Products , Endopeptidase Clp , Proteolysis , Endopeptidase Clp/metabolism , Biological Products/metabolism , Biological Products/chemistry , Bacillus subtilis/enzymology , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Peptide Synthases/metabolism , Adenosine Triphosphatases/metabolismABSTRACT
In cells, proteins are synthesized, function, and degraded (dead). Protein synthesis (spring) is important for the life of proteins. However, how proteins die is equally important for organisms. Proteases are secreted from cells and used as nutrients to break down external proteins. Proteases degrade unwanted and harmful cellular proteins. In eukaryotes, a large enzyme complex called the proteasome is primarily responsible for cellular protein degradation. Prokaryotes, such as bacteria, have similar protein degradation systems. In this review, we describe the structure and function of the ClpXP complex in the degradation system, which is an ATP-dependent protease in bacterial cells, with a particular focus on ClpP.
ABSTRACT
The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5'-O-N-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes 2-8 for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes 2-8 tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe 2 with a 4,4'-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.
Subject(s)
Benzophenones , Peptide Synthases , Photoaffinity Labels , Benzophenones/chemistry , Benzophenones/chemical synthesis , Benzophenones/pharmacology , Benzophenones/metabolism , Photoaffinity Labels/chemistry , Photoaffinity Labels/chemical synthesis , Peptide Synthases/metabolism , Peptide Synthases/chemistry , Molecular StructureABSTRACT
The extracellular signal-regulated kinase (ERK) MAPK pathway is dysregulated in various human cancers and is considered an attractive therapeutic target for cancer. Therefore, several inhibitors of this pathway are being developed, and some are already used in the clinic. We have previously identified an anticancer compound, ACA-28, with a unique property to preferentially induce ERK-dependent apoptosis in melanoma cells. To comprehensively understand the biological cellular impact induced by ACA-28, we performed a global gene expression analysis of human melanoma SK-MEL-28 cells exposed to ACA-28 using a DNA microarray. The transcriptome analysis identified nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcription factor that combats oxidative stress, as the most upregulated genetic pathway after ACA-28 treatment. Consistently, ACA-28 showed properties to increase the levels of reactive oxygen species (ROS) as well as Nrf2 protein, which is normally repressed by proteasomal degradation and activated in response to oxidative stresses. Furthermore, the ROS scavenger N-acetyl cysteine significantly attenuated the anticancer activity of ACA-28. Thus, ACA-28 activates Nrf2 signaling and exerts anticancer activity partly via its ROS-stimulating property. Interestingly, human A549 cancer cells with constitutively high levels of Nrf2 protein showed resistance to ACA-28, as compared with SK-MEL-28. Transient overexpression of Nrf2 also increased the resistance of cells to ACA-28, while knockdown of Nrf2 exerted the opposite effect. Thus, upregulation of Nrf2 signaling protects cancer cells from ACA-28-mediated cell death. Notably, the Nrf2 inhibitor ML385 substantially enhanced the cell death-inducing property of ACA-28 in pancreatic cancer cells, T3M4 and PANC-1. Our data suggest that Nrf2 plays a key role in determining cancer cell susceptibility to ACA-28 and provides a novel strategy for cancer therapy to combine the Nrf2 inhibitor and ACA-28.
Subject(s)
Melanoma , Pancreatic Neoplasms , Humans , Extracellular Signal-Regulated MAP Kinases/metabolism , Reactive Oxygen Species/metabolism , Melanoma/drug therapy , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Pancreatic Neoplasms/drug therapyABSTRACT
The adenylation (A) domain is essential for non-ribosomal peptide synthetases (NRPSs), which synthesize various peptide-based natural products, including virulence factors, such as siderophores and genotoxins. Hence, the inhibition of A-domains could attenuate the virulence of pathogens. 5'-O-N-(Aminoacyl or arylacyl)sulfamoyladenosine (AA-AMS) is a bisubstrate small-molecule inhibitor of the A-domains of NRPSs. However, the bacterial cell permeability of AA-AMS is typically a problem owing to its high hydrophilicity. In this study, we investigated the influence of a modification of 2'-OH in the AMS scaffold with different functional groups on binding to target enzymes and bacterial cell penetration. The inhibitor 7 with a cyanomethyl group at 2'-OH showed desirable inhibitory activity against both recombinant and intracellular gramicidin S synthetase A (GrsA) in the gramicidin S-producer Aneurinibacillus migulanus ATCC 9999, providing an alternative scaffold to develop novel A-domain inhibitors.
ABSTRACT
Herein, ring-cleaved (24) and truncated (25) analogues of an azasugar, 1-deoxynojirimycin (23), exhibited inhibitory activity (Ki = 4-10 µM) equal to that of the parent compound (1, Ki = 14 µM). Based on this structure-activity relationship (SAR), four ring-cleaved (26a-26c and 27c) and three truncated (28a-28c) analogues of salacinol (1), a potent thiosugar-ring-containing α-glucosidase inhibitor, were synthesised. Bioassay results revealed that all the synthetics were inactive, indicating that the 5-membered thiosugar ring of 1 played an essential role in the potent activities of sulfonium-type inhibitors. The present findings are interesting and important in understanding the function of salacinol, considering that the observed inhibitory activity trend was contrary to the SAR observed in aza-compounds (23, 24, and 25) in a previous study, which suggested that the cyclic structure did not contribute to their strong inhibitory activity.
ABSTRACT
Proteins in the cells are born (synthesized), work, and die (decomposed). In the life of a protein, its birth is obviously important, but how it dies is equally important in living organisms. Proteases secreted into the outside of cells are used to decompose the external proteins and the degradation products are taken as the nutrients. On the other hand, there are also proteases that decompose unnecessary or harmful proteins which are generated in the cells. In eukaryotes, a large enzyme complex called the proteasome is primarily responsible for degradation of such proteins. Bacteria, which are prokaryotes, have a similar system as the proteasome. We would like to explain the bacterial degradation system of proteins or the death of proteins, which is performed by ATP-dependent protease Clp, with a particular focus on the ClpXP complex, and with an aspect as a target for antibiotics against bacteria.
Subject(s)
Bacteria , Proteasome Endopeptidase Complex , Proteolysis , Proteasome Endopeptidase Complex/metabolism , ATP-Dependent Proteases/metabolism , Bacteria/metabolism , Biological Transport , Bacterial Proteins/metabolismABSTRACT
In this study, we discover a good NO/HNO precursor, N-hydroxypyridinesulfonamide, and the regioselective radical nitrososulfonylation reaction of propargyl alcohols. Direct and unique isoxazole synthesis afforded a good-to-high yield of 5-alkyl-3-aryl-4-pyridinesulfonylisoxazoles. Copper-catalyzed aerobic oxidation could efficiently proceed in the presence of thiazolidine-2,4-dione. This work provides a powerful method for the synthesis and functionalization of alkyl-substituted isoxazoles and explores a new investigation route for drug-drug discovery.
ABSTRACT
Nonribosomal peptide synthetases (NRPSs) biosynthesize nonribosomal peptide (NRP) natural products, which belong to the most promising resources for drug discovery and development because of their wide range of therapeutic applications. The results of genetic, biochemical, and bioinformatics analyses have enhanced our understanding of the mechanisms of the NRPS machinery. A major goal in NRP biosynthesis is to reprogram the NRPS machinery to enable the biosynthetic production of designed peptides. Reprogramming strategies for the NRPS machinery have progressed considerably in recent years, thereby increasing the yields and generating modified peptides. Here, the recent progress in NRPS reprogramming and its application in peptide synthesis are described.
Subject(s)
Biological Products , Peptide Synthases , Peptide Synthases/genetics , Peptide Synthases/analysis , Peptide Synthases/metabolism , Peptide Biosynthesis, Nucleic Acid-Independent , PeptidesABSTRACT
The first total synthesis of calanthoside (1), which exhibits potent proliferative activity against human hair follicle dermal papilla cells, has been achieved in seven steps with an overall yield of 43% on a gram scale starting from anthranilic acid (11). The synthetic strategy features a one-pot process involving thioglucoside bond formation via nucleophilic substitution reaction and enol-glucosylation for building the S-,O-bisdesmoside structure of 1. Moreover, the one-pot reaction showed broad substrate adaptability to several sugar donors other than d-glucose, thus affording S,O-bisglycoside intermediates in â¼84% yield.
Subject(s)
Glucosides , Hair , Humans , Glucosides/chemistry , Glycosides/pharmacology , Hair FollicleABSTRACT
We describe activity-based protein profiling for analyzing the adenylation domains of non-ribosomal peptide synthetases (ABPP-NRPS) in bacterial proteomes. Using a range of non-proteoinogenic amino acid sulfamoyladenosines, the competitive format of ABPP-NRPS provided substrate tolerance toward non-proteinogenic amino acids. When coupled with precursor-directed biosynthesis, a non-proteinogenic amino acid (O-allyl-L-serine) was successfully incorporated into gramicidin S.
Subject(s)
Amino Acids , Peptides , Bacteria/metabolism , Gramicidin , Peptide Synthases/chemistry , Substrate SpecificityABSTRACT
Many amino acid-containing natural products are biosynthesized by large, multifunctional enzymes known as non-ribosomal peptide synthetases (NRPSs). Adenylation (A) domains in NRPSs are responsible for the incorporation of amino acid building blocks and can be considered as engineering domains; therefore, advanced techniques are required to not only rapidly verify expression and folding, but also accelerate the functional prediction of the A-domains in lysates from native and heterologous systems. We recently developed activity-based protein profiling (ABPP) of NRPSs that offers a simple and robust analytical platform for A-domains and provides insights into their enzyme-substrate specificity. In this chapter, we describe the design and synthesis of these ABPP probes and provide a summary of our work on the development of a series of protocols for labeling, visualizing, and analyzing endogenous NRPSs in complex biological systems.
Subject(s)
Gramicidin , Peptide Synthases , Peptide Synthases/chemistry , Substrate Specificity , Amino AcidsABSTRACT
4'-Phosphopantetheinylation is an essential posttranslational modification of the primary and secondary metabolic pathways in prokaryotes and eukaryotes. Several peptide-based natural products are biosynthesized by large, multifunctional enzymes known as nonribosomal peptide synthetases (NRPSs), responsible for producing virulence factors and many pharmaceuticals. The thiolation (T) domain serves as a covalent tether for substrates and intermediates in nonribosomal peptide biosynthesis and must be posttranslationally modified with a 4'-phosphopantetheinyl group. To detect 4'-phosphopantetheinylation of NRPS in bacterial proteomes, we developed a 5'-(vinylsulfonylaminodeoxy)adenosine scaffold with a clickable functionality, enabling effective chemical labeling of 4'-phosphopantethylated NRPSs. In this chapter, we describe the design and synthesis of an activity-based protein profiling probe and summarize our work toward developing a series of protocols for the labeling and visualization of 4'-phosphopantetheinylation of endogenous NRPSs in complex proteomes.
Subject(s)
Adenosine , Proteome , Adenosine/chemistry , Bacteria/metabolism , Peptide Synthases/chemistryABSTRACT
The extracellular-signal-regulated-kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT-007a (GT-7), an anti-cancer compound, stimulates ERK phosphorylation, thereby inducing growth inhibition and apoptosis in T3M4 pancreatic cancer cells. However, how GT-7 stimulates ERK phosphorylation and induces apoptosis in ERK-active T3M4 cells remains unclear. To look into the mechanism, we performed a spatiotemporal analysis of ERK phosphorylation mediated by GT-7 in T3M4 cells. The immunoblotting showed that GT-7 stimulates ERK phosphorylation within 1 h, which was more remarkable after 2 h. Importantly, apoptosis induction as evaluated by the cleaved Caspase-3 was observed only after 2-h incubation with GT-7. The immunofluorescence staining revealed the enrichment of phosphorylated ERK (phospho-ERK) in the nucleus upon 1-h incubation with GT-7. Fractionation experiments showed that GT-7 increases phospho-ERK levels in the cytoplasm within 1 h, whereas nuclear phospho-ERK accumulation is observed after 2-h incubation with GT-7. MEK inhibition by U0126 significantly diminishes nuclear phospho-ERK distribution and apoptosis induction stimulated by GT-7. Thus, GT-7 may initiate the induction of ERK phosphorylation in the cytoplasm, which leads to phospho-ERK enrichment in the nucleus. This nuclear phospho-ERK accumulation by GT-7 precedes and may underlie apoptosis induction in T3M4.
Subject(s)
Extracellular Signal-Regulated MAP Kinases , Pancreatic Neoplasms , Humans , Extracellular Signal-Regulated MAP Kinases/metabolism , Phosphorylation , Signal Transduction , Pancreatic Neoplasms/drug therapy , Apoptosis , MAP Kinase Signaling System , Pancreatic NeoplasmsABSTRACT
We report the first attempt of double-spot structural modification on a side-chain moiety of sulfonium-type α-glucosidase inhibitors isolated from genus Salacia. A series of sulfonium salts with benzylidene acetal linkage at the C3' and C5' positions were designed and synthesized. In vitro enzyme inhibition evaluation showed that compounds with a strong electron-withdrawing group attached at the ortho position on the phenyl ring present stronger inhibitory activities. Notably, the most potent inhibitor 21b (1.0 mpk) can exhibit excellent hypoglycemic effects in mice, which can still compete with those of acarbose (20.0 mpk). Molecular docking of 21b demonstrated that besides conventional interacting patterns, the newly introduced benzylidene acetal moiety plays an important role in anchoring the whole molecule in a concave pocket of the enzyme. The successful identification of 21b as a lead compound for new drug discovery may provide a means for structure modification and diversification of the distinguished sulfonium-type α-glucosidase inhibitors.
Subject(s)
Glycoside Hydrolase Inhibitors , Hypoglycemic Agents , Mice , Animals , Hypoglycemic Agents/pharmacology , Hypoglycemic Agents/therapeutic use , Hypoglycemic Agents/chemistry , Structure-Activity Relationship , Molecular Docking Simulation , Acetals , alpha-Glucosidases/metabolism , Molecular StructureABSTRACT
Non-ribosomal peptide synthetases (NRPSs) biosynthesize many pharmaceuticals and virulence factors. The biosynthesis of these natural peptide products from biosynthetic gene clusters depends on complex regulations in bacteria. However, our current knowledge of NRPSs is based on enzymological studies using full NRPS systems and/or a single NRPS domain in heterologous hosts. Chemical and/or biochemical strategies to capture the endogenous activities of NRPSs facilitate studies on NRPS cell biology in bacterial cells. Here, we describe a chemical scaffold for the rapid and selective photoaffinity labelling of NRPSs in purified systems, crude biological samples and living bacterial cells. We synthesized photoaffinity labelling probes coupled with 5'-O-N-(phenylalanyl)sulfamoyladenosine with clickable alkyl diazirine or trifluoromethyl phenyl diazirine. We found that a trifluoromethyl phenyl diazirine-based probe cross-linked the Phe-activating domain of a GrsA-NRPS with high selectivity and sensitivity at shorter ultraviolet (UV) irradiation times (less than 5 min) relative to a prototypical benzophenone-based probe. Our results demonstrated that this quick labelling protocol can prevent damage to proteins and cells caused by long UV irradiation times, providing a mild photoaffinity labelling method for biological samples. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.
Subject(s)
Bacteria , Diazomethane , Diazomethane/metabolism , Bacteria/genetics , Peptide Synthases/chemistry , Peptide Synthases/genetics , Peptide Synthases/metabolism , Multigene FamilyABSTRACT
Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132), an organogermanium, is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP) in aqueous solutions, and reduces inflammation, pain and cancer, whereas the underlying mechanisms remain unknown. Sulfides including H2S, a gasotransmitter, generated from l-cysteine by some enzymes including cystathionine-γ-lyase (CSE), are pro-nociceptive, since they enhance Cav3.2 T-type Ca2+ channel activity expressed in the primary afferents, most probably by canceling the channel inhibition by Zn2+ linked via coordinate bonding to His191 of Cav3.2. Given that germanium is reactive to sulfur, we tested whether THGP would directly trap sulfide, and inhibit sulfide-induced enhancement of Cav3.2 activity and sulfide-dependent pain in mice. Using mass spectrometry and 1H NMR techniques, we demonstrated that THGP directly reacted with sulfides including Na2S and NaSH, and formed a sulfur-containing reaction product, which decreased in the presence of ZnCl2. In Cav3.2-transfected HEK293 cells, THGP inhibited the sulfide-induced enhancement of T-type Ca2+ channel-dependent membrane currents. In mice, THGP, administered systemically or locally, inhibited the mechanical allodynia caused by intraplantar Na2S. In the mice with cyclophosphamide-induced cystitis and cerulein-induced pancreatitis, which exhibited upregulation of CSE in the bladder and pancreas, respectively, systemic administration of THGP as well as a selective T-type Ca2+ channel inhibitor suppressed the cystitis-related and pancreatitis-related visceral pain. These data suggest that THGP traps sulfide and inhibits sulfide-induced enhancement of Cav3.2 activity, leading to suppression of Cav3.2-dependent pain caused by sulfide applied exogenously and generated endogenously.
Subject(s)
Calcium Channels, T-Type , Cystitis , Hydrogen Sulfide , Pancreatitis , Visceral Pain , Mice , Humans , Animals , HEK293 Cells , Calcium Channels, T-Type/physiology , Sulfides/pharmacology , Cystitis/chemically induced , Hydrogen Sulfide/metabolismABSTRACT
Although 4,5-didehydroguadiscine (12a), an alkaloid with potent melanogenesis-inhibitory activity isolated from Hornschuchia obliqua (Annonaceae), consists of an aporphine nucleus with an aromatized B-ring, to date, it has not been utilized as a template for structure-activity relationship (SAR) studies of pharmacological activities because of its exceptional structure. Accordingly, herein, five analogs (12b-12f) of 12a and five benzylisoquinoline analogs (13b-13f) lacking the C11a-C11b bond of 12b-12f were prepared. The inhibitory effects of 12b-12f and 13b-13f on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells were examined and compared with those of 12a. Melanogenesis-inhibitory activities of 12b-12f were the same as that of 12a, whereas the melanogenesis-inhibitory activities of 13b-13f were significantly inferior to those of 12a and 12b-12f. These results suggest that the C11a-C11b bond plays an essential role in the melanogenesis-inhibitory activities of 12a-12e.
Subject(s)
Alkaloids , Antineoplastic Agents , Aporphines , Melanoma, Experimental , Animals , Melanoma, Experimental/drug therapy , Melanins , Molecular Structure , Aporphines/pharmacology , Structure-Activity Relationship , Alkaloids/chemistry , Antineoplastic Agents/pharmacology , Cell Line, TumorABSTRACT
We present an in vitro and in-cell activity-based protein profiling (ABPP) protocol for endogenous nonribosomal peptide synthetases (NRPSs). This protocol enables the fluorescence labeling and imaging of an endogenous SrfAB-NRPS with high selectivity and sensitivity in the surfactin producer Bacillus subtilis. While we optimized this protocol for use with B. subtilis, the protocol can be applied to Aneurinibacillus migulanus and Escherichia coli. For complete details on the use and execution of this protocol, please refer to Ishikawa et al. (2022).
Subject(s)
Bacillus subtilis , Peptide Synthases , Bacillus subtilis/metabolism , Escherichia coli/genetics , Peptide Synthases/chemistryABSTRACT
Nonribosomal peptide synthetases (NRPSs) are complex multi-modular enzymes containing catalytic domains responsible for the loading and incorporation of amino acids into natural products. These unique molecular factories can produce peptides with nonproteinogenic d-amino acids in which the epimerization (E) domain catalyzes the conversion of l-amino acids to d-amino acids, but its mechanism remains not fully understood. Here, we describe the development of pantetheine crosslinking probes that mimic the natural substrate l-Phe of the initiation module of tyrocidine synthetase, TycA, to elucidate and study the catalytic residues of the E domain. Mechanism-based crosslinking assays and MALDI-TOF MS were used to identify both H743 and E882 as the crosslinking site residues, demonstrating their roles as catalytic bases. Mutagenesis studies further validated these results and allowed the comparison of reactivity between the catalytic residues, concluding that glutamate acts as the dominant nucleophile in the crosslinking reaction, resembling the deprotonation of the Cα-H of amino acids in the epimerization reaction. The crosslinking probes employed in these studies provide new tools for studying the molecular details of E domains, as well as the potential to study C domains. In particular, they would elucidate key information for how these domains function and interact with their substrates in nature, further enhancing the knowledge needed to assist combinatorial biosynthetic efforts of NRPS systems to produce novel compounds.