RESUMEN
Integral to the protein structure/function paradigm, oligomeric state is typically conserved along with function across evolution. However, notable exceptions such as the hemoglobins show how evolution can alter oligomerization to enable new regulatory mechanisms. Here, we examine this linkage in histidine kinases (HKs), a large class of widely distributed prokaryotic environmental sensors. While the majority of HKs are transmembrane homodimers, members of the HWE/HisKA2 family can deviate from this architecture as exemplified by our finding of a monomeric soluble HWE/HisKA2 HK (EL346, a photosensing light-oxygen-voltage [LOV]-HK). To further explore the diversity of oligomerization states and regulation within this family, we biophysically and biochemically characterized multiple EL346 homologs and found a range of HK oligomeric states and functions. Three LOV-HK homologs are primarily dimeric with differing structural and functional responses to light, while two Per-ARNT-Sim-HKs interconvert between differentially active monomers and dimers, suggesting dimerization might control enzymatic activity for these proteins. Finally, we examined putative interfaces in a dimeric LOV-HK, finding that multiple regions contribute to dimerization. Our findings suggest the potential for novel regulatory modes and oligomeric states beyond those traditionally defined for this important family of environmental sensors.
Asunto(s)
Proteínas Bacterianas , Histidina Quinasa , Multimerización de Proteína , Proteínas Bacterianas/metabolismo , Histidina Quinasa/metabolismo , Activación EnzimáticaRESUMEN
Although histidine kinases (HKs) are critical sensors of external stimuli in prokaryotes, the mechanisms by which their sensor domains control enzymatic activity remain unclear. Here, we report the full-length structure of a blue light-activated HK from Erythrobacter litoralis HTCC2594 (EL346) and the results of biochemical and biophysical studies that explain how it is activated by light. Contrary to the standard view that signaling occurs within HK dimers, EL346 functions as a monomer. Its structure reveals that the light-oxygen-voltage (LOV) sensor domain both controls kinase activity and prevents dimerization by binding one side of a dimerization/histidine phosphotransfer-like (DHpL) domain. The DHpL domain also contacts the catalytic/ATP-binding (CA) domain, keeping EL346 in an inhibited conformation in the dark. Upon light stimulation, interdomain interactions weaken to facilitate activation. Our data suggest that the LOV domain controls kinase activity by affecting the stability of the DHpL/CA interface, releasing the CA domain from an inhibited conformation upon photoactivation. We suggest parallels between EL346 and dimeric HKs, with sensor-induced movements in the DHp similarly remodeling the DHp/CA interface as part of activation.
Asunto(s)
Modelos Moleculares , Proteínas Quinasas/química , Transducción de Señal/fisiología , Sphingomonadaceae/enzimología , Cromatografía en Gel , Cromatografía Liquida , Biología Computacional , Cristalización , Dimerización , Escherichia coli , Histidina Quinasa , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Conformación Proteica , Transducción de Señal/genética , Difracción de Rayos XRESUMEN
N-Pyridinylthiophene carboxamide (compound 21) displays activity against peripheral nerve sheath cancer cells and mouse xenografts by an unknown mechanism. Through medicinal chemistry, we identified a more active derivative, compound 9, and found that only analogues with structures similar to nicotinamide retained activity. Genetic screens using compound 9 found that both NAMPT and NMNAT1, enzymes in the NAD salvage pathway, are necessary for activity. Compound 9 is metabolized by NAMPT and NMNAT1 into an adenine dinucleotide (AD) derivative in a cell-free system, cultured cells, and mice, and inhibition of this metabolism blocked compound activity. AD analogues derived from compound 9 inhibit IMPDH in vitro and cause cell death by inhibiting IMPDH in cells. These findings nominate these compounds as preclinical candidates for the development of tumor-activated IMPDH inhibitors to treat neuronal cancers.
Asunto(s)
NAD , Niacinamida , Tiofenos , Animales , NAD/metabolismo , Humanos , Ratones , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/farmacología , Niacinamida/química , Tiofenos/farmacología , Tiofenos/química , Tiofenos/metabolismo , Línea Celular Tumoral , IMP Deshidrogenasa/antagonistas & inhibidores , IMP Deshidrogenasa/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Nicotinamida Fosforribosiltransferasa/metabolismo , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Neoplasias de la Vaina del Nervio/tratamiento farmacológico , Neoplasias de la Vaina del Nervio/metabolismo , Neoplasias de la Vaina del Nervio/patología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa/antagonistas & inhibidoresRESUMEN
Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and in some cases, new therapeutic leads. In select cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.
RESUMEN
Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and new therapeutic leads. In selected cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.
Asunto(s)
Antineoplásicos , Neoplasias del Colon , Humanos , Reparación de la Incompatibilidad de ADN , Antineoplásicos/farmacología , Mutagénesis , CitotoxinasRESUMEN
Light-oxygen-voltage (LOV) domains serve as the photosensory modules for a wide range of plant and bacterial proteins, conferring blue light-dependent regulation to effector activities as diverse as enzymes and DNA binding. LOV domains can also be engineered into a variety of exogenous targets, allowing similar regulation for new protein-based reagents. Common to these proteins is the ability for LOV domains to reversibly form a photochemical adduct between an internal flavin chromophore and the surrounding protein, using this to trigger conformational changes that affect output activity. Using the Erythrobacter litoralis protein EL222 model system that links LOV regulation to a helix-turn-helix (HTH) DNA binding domain, we demonstrated that the LOV domain binds and inhibits the HTH domain in the dark, releasing these interactions upon illumination [Nash, A. I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 9449-9454]. Here we combine genomic and in vitro selection approaches to identify optimal DNA binding sites for EL222. Within the bacterial host, we observe binding at several genomic sites using a 12 bp sequence consensus that is also found by in vitro selection methods. Sequence-specific alterations in the DNA consensus reduce EL222 binding affinity in a manner consistent with the expected binding mode, a protein dimer binding to two repeats. Finally, we demonstrate the light-dependent activation of transcription of two genes adjacent to an EL222 binding site. Taken together, these results shed light on the native function of EL222 and provide useful reagents for further basic and applications research of this versatile protein.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Secuencias Hélice-Giro-Hélice , Unión Proteica/efectos de la radiación , Factores de Transcripción Activadores/metabolismo , Sitios de Unión , Inmunoprecipitación de Cromatina , ADN/metabolismo , Mononucleótido de Flavina/química , Luz , Técnica SELEX de Producción de AptámerosRESUMEN
A series of N-acyl benzothiazoles shows selective and potent cytotoxicity against cancer cell lines expressing cytochrome P450 4F11. A prodrug form is metabolized by cancer cells into an active inhibitor of stearoyl-CoA desaturase (SCD). Substantial variation on the acyl portion of the inhibitors allowed the identification of (R)-27, which balanced potency, solubility, and lipophilicity to allow proof-of-concept studies in mice. The prodrugs were activated inside the tumor, where they can arrest tumor growth. Together, these observations offer promise that a tumor-activated prodrug strategy might exploit the essentiality of SCD for tumor growth, while avoiding toxicity associated with systemic SCD inhibition.
Asunto(s)
Benzotiazoles/farmacología , Inhibidores Enzimáticos/farmacología , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Animales , Benzotiazoles/farmacocinética , Línea Celular Tumoral , Familia 4 del Citocromo P450/metabolismo , Femenino , Humanos , Ratones , Profármacos/metabolismo , Distribución TisularRESUMEN
Pathogens find diverse niches for survival including inside a host cell where replication occurs in a relatively protective environment. Vibrio parahaemolyticus is a facultative intracellular pathogen that uses its type 3 secretion system 2 (T3SS2) to invade and replicate inside host cells. Analysis of the T3SS2 pathogenicity island encoding the T3SS2 appeared to lack a mechanism for egress of this bacterium from the invaded host cell. Using a combination of molecular tools, we found that VPA0226, a constitutively secreted lipase, is required for escape of V. parahaemolyticus from the host cells. This lipase must be delivered into the host cytoplasm where it preferentially uses fatty acids associated with innate immune response to esterify cholesterol, weakening the plasma membrane and allowing egress of the bacteria. This study reveals the resourcefulness of microbes and the interplay between virulence systems and host cell resources to evolve an ingenious scheme for survival and escape.
Asunto(s)
Proteínas Bacterianas/metabolismo , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Lipasa/metabolismo , Vibrio parahaemolyticus/metabolismo , Esterificación , Islas Genómicas , Sistemas de Secreción Tipo III , Vibrio parahaemolyticus/enzimologíaRESUMEN
Medical treatments and personal hygiene lead to the steady release of pharmaceutical and personal care products (PPCPs) into the environment. Some of these PPCPs have been shown to have detrimental environmental effects and could potentially impact human health. Understanding the biological transformation of PPCPs is essential for accurately determining their ultimate environmental fate, conducting accurate risk assessments, and improving PPCP removal. We summarize the current literature concerning the biological transformation of PPCPs in wastewater treatment plants, the environment, and by pure cultures of bacterial isolates. Although some PPCPs, such as ibuprofen, are readily degraded under most studied conditions, others, such as carbamazepine, tend to be recalcitrant. This variation in the biodegradability of PPCPs can be attributed to structural differences, because PPCPs are classified by application, not chemical structure. The degradation pathways of octylphenol by Sphingomonas sp. strain PWE1, ibuprofen by Sphingomonas sp. strain Ibu-2, and DEET by Pseudomonas putida DTB are discussed in more detail.
Asunto(s)
Bacterias Gramnegativas/metabolismo , Productos Domésticos , Preparaciones Farmacéuticas/metabolismo , Eliminación de Residuos Líquidos/métodos , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Biodegradación Ambiental , DEET/química , DEET/metabolismo , Ibuprofeno/química , Ibuprofeno/metabolismo , Repelentes de Insectos/química , Repelentes de Insectos/metabolismo , Fenoles/química , Fenoles/metabolismoRESUMEN
Enteric pathogens employ sophisticated strategies to colonize and infect mammalian hosts. Gram-negative bacteria, such as Escherichia coli, Salmonella, and Campylobacter jejuni, are among the leading causes of gastrointestinal tract infections worldwide. The virulence strategies of many of these Gram-negative pathogens rely on type III secretion systems (T3SSs), which are macromolecular syringes that translocate bacterial effector proteins directly into the host cytosol. However, synthesis of T3SS proteins comes at a cost to the bacterium in terms of growth rate and fitness, both in the environment and within the host. Therefore, expression of the T3SS must be tightly regulated to occur at the appropriate time and place during infection. Enteric pathogens have thus evolved regulatory mechanisms to control expression of their T3SSs in response to specific environmental and host cues. These regulatory cascades integrate multiple physical and chemical signals through complex transcriptional networks. Although the power of bacterial genetics has allowed elucidation of many of these networks, the biochemical interactions between signal and sensor that initiate the signaling cascade are often poorly understood. Here, we review the physical and chemical signals that Gram-negative enteric pathogens use to regulate T3SS expression during infection. We highlight the recent structural and functional studies that have elucidated the biochemical properties governing both the interaction between sensor and signal and the mechanisms of signal transduction from sensor to downstream transcriptional networks.
Asunto(s)
Adaptación Fisiológica , Campylobacter jejuni/metabolismo , Señales (Psicología) , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Salmonella/metabolismo , Sistemas de Secreción Tipo III/biosíntesis , Campylobacter jejuni/genética , Escherichia coli/genética , Salmonella/genéticaRESUMEN
Bile salts act as a stressor to bacteria that transit the intestinal tract. Enteric pathogens have hijacked bile as an intestinal signal to regulate virulence factors. We recently demonstrated that Vibrio parahemolyticus senses bile salts via a heterodimeric receptor formed by the periplasmic domains of inner-membrane proteins VtrA and VtrC. Crystal structures of the periplasmic complex reveal that VtrA and VtrC form a ß-barrel that binds bile salts in its hydrophobic interior to activate the VtrA cytoplasmic DNA-binding domain. Proteins with the same domain arrangement as VtrA and VtrC are widespread in Vibrio and related bacteria, where they are involved in regulating virulence and other unknown functions. Here we discuss our findings and review current knowledge on VtrA and VtrC homologs. We propose that signaling by these membrane-bound transcription factors can be advantageous for the regulation of membrane and secretory proteins.
Asunto(s)
Proteínas Bacterianas/genética , Ácidos y Sales Biliares/metabolismo , Vibriosis/microbiología , Vibrio parahaemolyticus/genética , Factores de Virulencia/genética , Animales , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Vibriosis/metabolismo , Vibrio parahaemolyticus/metabolismo , Factores de Virulencia/metabolismoRESUMEN
Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered that Vibrio parahaemolyticus VtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a ß-barrel with a hydrophobic inner chamber. A co-crystal structure of VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Fármacos Gastrointestinales/metabolismo , Sistemas de Secreción Tipo III , Vibrio parahaemolyticus/efectos de los fármacos , Vibrio parahaemolyticus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Análisis Mutacional de ADN , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Factores de Virulencia/metabolismoRESUMEN
The initial step in the biodegradation pathway of N,N-diethyl-m-toluamide (DEET) in Pseudomonas putida strain DTB is catalyzed by DEET hydrolase (DthA), which hydrolyzes the amide bond to yield 3-methylbenzoic acid and diethylamine. In order to extend our understanding of DthA, the enzyme was purified and characterized. The enzyme is most active at pH 7.9, and is probably a tetramer in its native state. The kinetic parameters of the wild-type enzyme are K(m) = 10.2 ± 0.8 µm, k(cat) = 5.53 ± 0.09 s(-1) , and k(cat) /K(m) = (5.4 ± 0.4) × 10(5) m(-1) ·s(-1) . Mild substrate inhibition was observed with DEET concentrations over 500 µm. A homology model of DthA was used to guide mutational analysis of the active site, confirming that the catalytic triad is formed by Ser166, Ap292, and His320. The oxyanion hole is formed by the side chain OH of Tyr84 and the backbone amide of Trp167, with the Tyr84 OH being essential for enzyme activity. The DthA model also revealed a hydrophobic substrate-binding pocket comprosed of Trp167, Met170, and Trp214. W167A and M170A mutations decreased enzymatic activity and exacerbated substrate inhibition, whereas Trp214, which probably plays a role in substrate recognition, was essential for enzymatic activity. The pH rate profile of DthA was fitted to two ionizable groups (pK(a1) = 6.1 and pK(a2) = 9.9) that probably correspond to Nε of His320 and the OH of Tyr84, respectively. In addition to catalyzing the hydrolysis of DEET, DthA hydrolyzed a variety of esters and amides.
Asunto(s)
Proteínas Bacterianas/química , Hidrolasas/química , Pseudomonas putida/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Dominio Catalítico , DEET/metabolismo , Glutamina/genética , Glutamina/metabolismo , Concentración de Iones de Hidrógeno , Hidrolasas/genética , Hidrolasas/metabolismo , Hidrólisis , Cinética , Mutagénesis Sitio-Dirigida , Especificidad por SustratoRESUMEN
Pseudomonas putida DTB grew aerobically with N,N-diethyl-m-toluamide (DEET) as a sole carbon source, initially breaking it down into 3-methylbenzoate and diethylamine. The former was further metabolized via 3-methylcatechol and meta ring cleavage. A gene from DTB, dthA, was heterologously expressed and shown to encode the ability to hydrolyze DEET into 3-methylbenzoate and diethylamine.