Protein degradation by a component of the chaperonin-linked protease ClpP.

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.

Probing for optimal photoaffinity linkers of benzophenone-based photoaffinity probes for adenylating enzymes.

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.

Recent progress in the reprogramming of nonribosomal peptide synthetases.

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.

Development of a chemical scaffold for inhibiting nonribosomal peptide synthetases in live bacterial cells.

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.

HMGA2 drives the IGFBP1/AKT pathway to counteract the increase in P27KIP1 protein levels in mtDNA/RNA-less cancer cells.

Recent comprehensive analyses of mtDNA and orthogonal RNA-sequencing data revealed that in numerous human cancers, mtDNA copy numbers and mtRNA amounts are significantly reduced, followed by low respiratory gene expression. Under such conditions (called mt-Low), cells encounter severe cell proliferation defects; therefore, they must acquire countermeasures against this fatal disadvantage during malignant transformation. This study elucidated a countermeasure against the mt-Low condition-induced antiproliferative effects in hepatocellular carcinoma (HCC) cells. The mechanism relied on the architectural transcriptional regulator HMGA2, which was preferably expressed in HCC cells of the mt-Low type in vitro and in vivo. Detailed in vitro analyses suggest that HMGA2 regulates insulin-like growth factor binding protein 1 (IGFBP1) expression, leading to AKT activation, which then phosphorylates the cyclin-dependent kinase inhibitor (CKI), P27KIP1, and facilitates its ubiquitin-mediated degradation. Accordingly, intervention in the HMGA2 function by RNAi resulted in an increase in P27KIP1 levels and an induction of senescence-like cell proliferation inhibition in mt-Low-type HCC cells. Conclusively, the HMGA2/IGFBP1/AKT axis has emerged as a countermeasure against P27KIP1 CKI upregulation under mt-Low conditions, thereby circumventing cell proliferation inhibition and supporting the tumorigenic state. Notably, similar to in vitro cell lines, HMGA2 was likely to regulate IGFBP1 expression in HCC in vivo, thereby contributing to poor patient prognosis. Considering the significant number of cases under mt-Low or the threat of CKI upregulation cancer-wide, the axis is noteworthy as a vulnerability of cancer cells or target for tumor-agnostic therapy inducing irreversible cell proliferation inhibition via CKI upregulation in a large population with cancer.

An indispensable role of TAZ in anoikis resistance promoted by OTUB1 deubiquitinating enzyme in basal-like triple-negative breast cancer cells.

Aggressive cancers, such as triple-negative breast cancer (TNBC), are mostly fatal because of their potential to metastasize to distant organs. Cancer cells acquire various abilities to metastasize, including resistance to anoikis, an apoptotic cell death induced by loss of anchorage to the extracellular matrix. Transcriptional coactivator with PDZ binding motif (TAZ) and Yes-associated protein (YAP), the downstream effectors of the Hippo pathway, regulate cell- and tissue-level architectures by responding to mechanical microenvironments of cells, including the cell-extracellular matrix interaction. The Hippo pathway is frequently disrupted in cancer cells, and TAZ and YAP are irrelevantly activated, potentially resulting in anchorage-independent survival/proliferation of cancer cells and metastatic progression. The study aims to investigate the roles of TAZ and YAP in anoikis resistance in basal-like (BL) TNBC cells, which comprise a major subtype (>70%) of TNBC. We found that TAZ and YAP had nonredundant roles in anchorage-independent cancer cell survival or anoikis resistance. Particularly, TAZ was indispensable for anoikis resistance in BL-TNBC cells but not for survival of non-transformed mammary epithelial cells (MECs). In contrast, YAP, a paralog of TAZ, was indispensable for survival of both non-transformed MECs and cancer cells. Therefore, TAZ might be a preferable therapeutic target against dissemination of aggressive cancer cells without killing normal cells. Interestingly, TAZ was abnormally stabilized in BL-TNBC cells under non-adherent conditions, which promoted anoikis resistance. Furthermore, OTUB1, a deubiquitinating enzyme, was responsible for the stabilization of TAZ in detached BL-TNBC cells. Importantly, simultaneous high expression of TAZ and OTUB1 was associated with poor prognosis in BC. Thus, OTUB1 has emerged as a potentially druggable target. Successful inhibition of OTUB1 enzymatic activity is expected to downregulate TAZ and eventually prevents metastasis of aggressive cancers, such as BL-TNBC.

Total Synthesis of Calanthoside, a Potential Hair Growth Stimulant: A Facile Synthetic Approach via One-Pot S- and O-Glucosidic Bond Formation.

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 ∼84% yield.

Structure-activity relationship study of 4,5-didehydroguadiscine, an aporphine alkaloid showing potent melanogenesis-inhibitory activity in B16 melanoma cells.

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.

Rac1-mediated sustained ß4 integrin level develops reattachment ability of breast cancer cells after anchorage loss.

Previously, we reported that non-apoptotic cell death was induced in non-malignant mammary epithelial cells (HMECs) upon loss of anchorage during 48 h incubation in suspension. In this study, we examined HMECs in suspension at an earlier time point and found that most of them lost attachment ability to substrata when replated, although >80% were alive. This suggested that HMECs lost reattachment ability (RA) prior to cell death upon detachment. Concomitant with the loss of RA, a decrease in the levels of ß1 and ß4 integrin was observed. In sharp contrast, breast cancer cells retained integrin levels, reattached to substrata, and formed colonies after exposure to anchorage loss as efficiently as those maintained under adherent conditions. Such RA of cancer cells is essential for the metastatic process, especially for establishing adhesion contact with ECM in the secondary organ after systemic circulation. Further analysis suggested that sustained levels of ß4 integrin, which was mediated by Rac1, was critical for RA after anchorage loss and lung metastasis of breast cancer cells. In the cancer cells, persistent Rac1 activity enhanced escape of ß4 integrin from lysosomal degradation depending on actin-related protein 2/3 and TBC1D2, a GTPase-activating protein of Rab7 GTPase. Notably, simultaneous high expression of ITGB4 and RAC1 was associated with poor prognosis in patients with breast cancer. Therefore, ß4 integrin and Rac1 are attractive therapeutic targets to eliminate RA in cancer cells, thereby preventing the initial step of colonization at the secondary organ during metastasis.

Elongation of the side chain by linear alkyl groups increases the potency of salacinol, a potent α-glucosidase inhibitor from the Ayurvedic traditional medicine "Salacia," against human intestinal maltase.

Four chain-extended analogs (12a-12d) and two related de-O-sulfonated analogs (13a and 13c) by introducing alkyl groups (a: R = C3H7, b R = C6H13, c: R = C8H17, d: R = C10H21) to the side chains of salacinol (1), a natural α-glucosidase inhibitor from Ayurvedic traditional medicine "Salacia", were synthesized. The α-glucosidase inhibitory activities of all the synthesized analogs were evaluated in vitro. Against human intestinal maltase, the inhibitory activities of 12a and 13a with seven-carbon side chain were equal to that of 1. In contrast, analogs (12b-12d, and 13c) exhibited higher level of inhibitory activity against the same enzyme than 1 and had equal or higher potency than those of the clinically used anti-diabetics, voglibose, acarbose, and miglitol. Thus, elongation of the side chains of 1 was effective for specifically increasing the inhibitory activity against human intestinal maltase.

Inhibition of efflux pumps aids small-molecule probe-based fluorescence labeling and imaging in the Gram-negative bacterium Escherichia coli.

A major challenge in fluorescence imaging experiments, which are essential to determine protein activity, expression, and localization, is the penetration of small-molecule probes through the outer membrane permeability barrier of bacteria. Here, we describe a novel strategy for small-molecule probe-based fluorescence protein labeling and imaging in the Gram-negative bacterium Escherichia coli. We targeted a siderophore enterobactin biosynthetic enzyme EntE in E. coli. When coupled with an efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone, small-molecule probes were able to efficiently enter the cells, leading to the fluorescence labeling and imaging of overproduced EntE in E. coli. This study demonstrates that the combination of small-molecule probes with appropriate efflux pump inhibitors may substantially enhance their interaction with the target proteins in live bacteria.

High expression of FOXM1 critical for sustaining cell proliferation in mitochondrial DNA-less liver cancer cells.

The copy number of mitochondrial DNA (mtDNA) is decreased in most cancer types, including hepatocellular carcinoma (HCC), compared to normal counterparts. However, a decrease in mtDNA usually leads to defects in cell proliferation, which contradicts the robustness of cancer cell proliferation. In this study, we found that four out of seven HCC cell lines were of the mtDNA-less type. Interestingly, FOXM1, a member of the FOX transcription factor family, was highly expressed in a subset of them with proliferative potential maintained. B-MYB, a partner of FOXM1, was also expressed in the same cell lines. RNAi-mediated experiments demonstrated that when FOXM1/B-MYB was silenced in the cell lines, cell cycle-related genes were downregulated, while p21Cip1 was induced with senescence-associated ß-galactosidase, resulting in G1/S cell cycle arrest. These results suggest that high expression of FOXM1/B-MYB is critical for sustaining cell proliferation in mtDNA-less cells. In addition, we found that high expression of FOXM1 was mediated by the deubiquitinating enzyme, OTUB1, in one cell line. Thus, interference with FOXM1/B-MYB expression, such as through OTUB1 inhibition, may induce a dormant state of senescence-like proliferation arrest in mtDNA-less cancer cells. This finding may be utilized for the development of precision medicine for relevant cancers.

Activity, Binding, and Modeling Studies of a Reprogrammed Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket.

The gatekeeping adenylation (A) domain of the non-ribosomal peptide synthetase (NRPS) selectively incorporates specific proteinogenic/non-proteinogenic amino acid into a growing peptide chain. The EntE of the enterobactin NRPS is a discrete aryl acid A-domain with 2,3-dihydroxybenzoic acid (DHB) substrate specificity. Reprogrammed EntE N235G variant possesses an enlarged substrate recognition site, and is capable of accepting non-native aryl acids. Biochemical characterization of this unique substrate recognition site should provide a better understanding of activi-site microenvironments. Here, we synthesized a non-hydrolysable adenylate analogue with 2-aminobenzoic acid (2-ABA), 3-aminobenzoic acid (3-ABA), and 4-aminobenzoic acid (4-ABA) and used them to calculate the apparent inhibition constants (Kiapp.). Dose-response experiments using 3-ABA-sulfamoyladenosine (AMS) provided Kiapp. values of 596 nM for wild-type EntE and 2.4 nM for the N235G variants. These results suggest that 3-amino group of benzoic acid plays an important role in substrate recognition by the N235G variant. These findings would help designing aryl acid substrates with substituents at the 2- and 3-positions.

Precise Probing of Residue Roles by NRPS Code Swapping: Mutation, Enzymatic Characterization, Modeling, and Substrate Promiscuity of Aryl Acid Adenylation Domains.

Aryl acids are most commonly found in iron-scavenging siderophores but are not limited to them. The nonribosomal peptide synthetase (NRPS) codes of aryl acids remain poorly elucidated relative to those of amino acids. Here, we defined more precisely the role of active-site residues in aryl acid adenylation domains (A-domains) by gradually grafting the NRPS codes used for salicylic acid (Sal) into an archetypal aryl acid A-domain, EntE [specific for the substrate 2,3-dihydroxybenzoic acid (DHB)]. Enzyme kinetics and modeling studies of these EntE variants demonstrated that the NRPS code residues at positions 236, 240, and 339 collectively regulate the substrate specificity toward DHB and Sal. Furthermore, the EntE variants exhibited the ability to activate the non-native aryl acids 3-hydroxybenzoic acid, 3-aminobenzoic acid, 3-fluorobenzoic acid, and 3-chlorobenzoic acid. These studies enhance our knowledge of the NRPS codes of aryl acids and could be exploited to reprogram aryl acid A-domains for non-native aryl acids.

Probing the Compatibility of an Enzyme-Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains.

An important challenge in natural product biosynthesis is the biosynthetic design and production of artificial peptides. One of the most promising strategies is reprogramming adenylation (A) domains to expand the substrate repertoire of nonribosomal peptide synthetases (NRPSs). Therefore, the precise detection of subtle structural changes in the substrate binding pockets of A domains might accelerate their reprogramming. Here we show that an enzyme-linked immunosorbent assay (ELISA) using a combination of small-molecule probes can detect the effects of substrate binding pocket residue substitutions in A-domains. When coupled with a set of aryl acid A-domain variants (total of nine variants), the ELISA can analyze the subtle differences in their active-site architectures. Furthermore, the ELISA-based screening was able to identify the variants with substrate binding pockets that accepted a non-cognate substrate from an original pool of 45. These studies demonstrate that ELISA is a reliable platform for providing insights into the active-site properties of A-domains and can be applied for the reprogramming of NRPS A-domains.

Activity-Based Protein Profiling of Non-ribosomal Peptide Synthetases.

Non-ribosomal peptide (NRP) natural products are one of the most promising resources for drug discovery and development because of their wide-ranging of therapeutic potential, and their behavior as virulence factors and signaling molecules. The NRPs are biosynthesized independently of the ribosome by enzyme assembly lines known as the non-ribosomal peptide synthetase (NRPS) machinery. Genetic, biochemical, and bioinformatics analyses have provided a detailed understanding of the mechanism of NRPS catalysis. However, proteomic techniques for natural product biosynthesis remain a developing field. New strategies are needed to investigate the proteomes of diverse producer organisms and directly analyze the endogenous NRPS machinery. Advanced platforms should verify protein expression, protein folding, and activities and also enable the profiling of the NRPS machinery in biological samples from wild-type, heterologous, and engineered bacterial systems. Here, we focus on activity-based protein profiling strategies that have been recently developed for studies aimed at visualizing and monitoring the NRPS machinery and also for rapid labeling, identification, and biochemical analysis of NRPS enzyme family members as required for proteomic chemistry in natural product sciences.

Discovery of new benzhydrol biscarbonate esters as potent and selective apoptosis inducers of human melanomas bearing the activated ERK pathway: SAR studies on an ERK MAPK signaling modulator, ACA-28.

The recent discovery that an ERK signaling modulator [ACA-28 (2a)] preferentially kills human melanoma cell lines by inducing ERK-dependent apoptosis has generated significant interest in the field of anti-cancer therapy. In the first SAR study on 2a, here, we successfully developed candidates (2b, 2c) both of which induce more potent and selective apoptosis towards ERK-active melanoma cells than 2a, thus revealing the structural basis for inducing the ERK-dependent apoptosis and proposing the therapeutic prospect of these candidates against ERK-dependent cancers represented by melanoma.

Trapping the dynamic acyl carrier protein in fatty acid biosynthesis.

Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis. Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain. ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway. The transient nature of ACP-enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein-protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 Å crystal structure of the crosslinked AcpP-FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4'-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. These techniques, in combination with molecular dynamics simulations, show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. Here the foundation is laid for defining the dynamic action of carrier-protein activity in primary and secondary metabolism, providing insight into pathways that can have major roles in the treatment of cancer, obesity and infectious disease.

Chemical Strategies for Visualizing and Analyzing Endogenous Nonribosomal Peptide Synthetase (NRPS) Megasynthetases.

Nonribosomal peptide (NRP) natural products are among the most promising resources for drug discovery and development, owing to their wide range of biological activities and therapeutic applications. These peptide metabolites are biosynthesized by large multienzyme machinery known as NRP synthetases (NRPSs). The structural complexity of a number of NRPs poses an enormous challenge in their synthesis. A major issue in this field is reprogramming NRPS machineries to allow the biosynthetic production of artificial peptides. NRPS adenylation (A) domains are responsible for the incorporation of a wide variety of amino acids and can be considered as reprogramming sites; therefore, advanced methods to accelerate the functional prediction and assessment of A-domains are required. This Concept article demonstrates that activity-based protein profiling of NRPSs offers a simple, rapid, and robust analytical platform for A-domains and provides insights into enzyme-substrate candidates and active-site microenvironments. It also describes the background associated with the development and application of a method to analyze endogenous NRPS machinery in its natural environment.

Ginnalin B induces differentiation markers and modulates the proliferation/differentiation balance via the upregulation of NOTCH1 in human epidermal keratinocytes.

The red maple and sugar maple (Acer rubrum and A. saccharum, respectively) contain acertannins (ginnalins and maplexins), galloylated derivatives of 1,5-anhydro-d-glucitol (1,5-AG, 1). These compounds have a variety of potential medicinal properties and we have shown that some of them promote the expression of ceramide synthase 3. We now report on the beneficial effects of ginnalin B, (6-O-galloyl-1,5-AG, 5), leading to acceleration of skin metabolism and reduction of the turnover time. Ginnalin B dose-dependently increased the relative amount of keratin 10, keratin 1, and filaggrin gene, with maximal increase of 1.7-, 2.9, and 5.2-fold at 100 µM, respectively. The validation study showed that it had superior capacity to induce multiple stages of keratinocyte differentiation and significantly elevated the immunostaining site of keratin 10 and filaggrin in a 3-dimensional cultured human skin model, by 1.2 and 2.8-fold, respectively. Furthermore, ginnalin B caused the arrest of proliferation at the G0/G1 phase but it did not induce apoptotic cell death in normal human keratinocytes. Molecular studies revealed that ginnalin B up-regulated the levels of NOTCH1 and a concomitant increase p21 expression. Ginnalin B, therefore, represents a new class of promising functional and medical cosmetic compound and it could contribute to the maintenance of homeostasis of the epidermis.