RESUMO
Peptides presented by HLA-E, a molecule with very limited polymorphism, represent attractive targets for T cell receptor (TCR)-based immunotherapies to circumvent the limitations imposed by the high polymorphism of classical HLA genes in the human population. Here, we describe a TCR-based bispecific molecule that potently and selectively binds HLA-E in complex with a peptide encoded by the inhA gene of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans. We reveal the biophysical and structural bases underpinning the potency and specificity of this molecule and demonstrate its ability to redirect polyclonal T cells to target HLA-E-expressing cells transduced with mycobacterial inhA as well as primary cells infected with virulent Mtb. Additionally, we demonstrate elimination of Mtb-infected cells and reduction of intracellular Mtb growth. Our study suggests an approach to enhance host T cell immunity against Mtb and provides proof of principle for an innovative TCR-based therapeutic strategy overcoming HLA polymorphism and therefore applicable to a broader patient population.
Assuntos
Antígenos de Histocompatibilidade Classe I , Mycobacterium tuberculosis , Receptores de Antígenos de Linfócitos T , Linfócitos T , Mycobacterium tuberculosis/imunologia , Humanos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Linfócitos T/imunologia , Antígenos HLA-E , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Tuberculose/imunologiaRESUMO
Naturally occurring T cells that recognize microbial peptides via HLA-E, a nonpolymorphic HLA class Ib molecule, could provide the foundation for new universal immunotherapeutics. However, confidence in the biological relevance of putative ligands is crucial, given that the mechanisms by which pathogen-derived peptides can access the HLA-E presentation pathway are poorly understood. We systematically interrogated the HIV proteome using immunopeptidomic and bioinformatic approaches, coupled with biochemical and cellular assays. No HIV HLA-E peptides were identified by tandem mass spectrometry analysis of HIV-infected cells. In addition, all bioinformatically predicted HIV peptide ligands (>80) were characterized by poor complex stability. Furthermore, infected cell elimination assays using an affinity-enhanced T cell receptor bispecific targeted to a previously reported HIV Gag HLA-E epitope demonstrated inconsistent presentation of the peptide, despite normal HLA-E expression on HIV-infected cells. This work highlights the instability of the HIV HLA-E peptidome as a major challenge for drug development.
Assuntos
Infecções por HIV , Antígenos HLA-E , Humanos , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Epitopos , Infecções por HIV/terapia , Peptídeos/metabolismoRESUMO
PURPOSE: The goal of this systematic review was to examine the current literature on the urinary microbiome and its associations with noninfectious, nonmalignant, urologic diseases. Secondarily, we aimed to describe the most common bioinformatics used to analyze the urinary microbiome. METHODS: A comprehensive literature search of Ovid MEDLINE using the keywords "microbiota" AND "prostatic hyperplasia," "microbiota" AND "urinary bladder, overactive," "microbiota" AND "pelvic pain," and "microbiota" AND "urolithiasis" OR "nephrolithiasis" OR "urinary calculi" AND "calcium oxalate" was performed to identify relevant clinical microbiome studies associated with noninfectious benign urological conditions published from 2010 to 2022. We included human studies that evaluated the urinary, stone, or semen microbiota, or any combination of the above-mentioned locations. RESULTS: A total of 25 human studies met the inclusion criteria: 4 on benign prostatic hyperplasia (BPH), 9 on overactive bladder (OAB), 8 on calcium oxalate stones, and 4 on chronic pelvic pain syndrome (CPPS). Specific taxonomic profiles in the urine microbiome were associated with each pathology, and evaluation of alpha- and beta-diversity and relative abundance was accounted for most of the studies. Symptom prevalence and severity were also analyzed and showed associations with specific microbes. CONCLUSION: The study of the urogenital microbiome is rapidly expanding in urology. Noninfectious benign urogenital diseases, such as BPH, calcium oxalate stones, CPPS, and OAB were found to be associated with specific microbial taxonomies. Further research with larger study populations is necessary to solidify the knowledge of the urine microbiome in these conditions and to facilitate the creation of microbiome-based diagnostic and therapeutic approaches.
Assuntos
Microbiota , Hiperplasia Prostática , Bexiga Urinária Hiperativa , Cálculos Urinários , Masculino , Humanos , Hiperplasia Prostática/tratamento farmacológico , Oxalato de Cálcio , Bexiga Urinária Hiperativa/tratamento farmacológico , Dor PélvicaRESUMO
The soluble methane monooxygenase receives electrons from NADH via its reductase MmoC for oxidation of methane, which is itself an attractive C1 building block for a future bioeconomy. Herein, we present biochemical and spectroscopic insights into the reductase from the marine methanotroph Methylomonas methanica MC09. The presence of a flavin adenine dinucleotide (FAD) and [2Fe2S] cluster as its prosthetic group were revealed by reconstitution experiments, iron determination and electron paramagnetic resonance spectroscopy. As a true halotolerant enzyme, MmoC still showed 50 % of its specific activity at 2â M NaCl. We show that MmoC produces only trace amounts of superoxide, but mainly hydrogen peroxide during uncoupled turnover reactions. The characterization of a highly active reductase is an important step for future biotechnological applications of a halotolerant sMMO.
Assuntos
Oxirredutases , Oxigenases , Espectroscopia de Ressonância de Spin Eletrônica , Flavina-Adenina Dinucleotídeo/metabolismo , Metano , Methylomonas , Oxirredução , Oxigenases/metabolismoRESUMO
Methane is a widespread energy source and can serve as an attractive C1 building block for a future bioeconomy. The soluble methane monooxygenase (sMMO) is able to break the strong C-H bond of methane and convert it to methanol. The high structural complexity, multiplex cofactors, and unfamiliar folding or maturation procedures of sMMO have hampered the heterologous production and thus biotechnological applications. Here, we demonstrate the heterologous production of active sMMO from the marine Methylomonas methanica MC09 in Escherichia coli by co-synthesizing the GroES/EL chaperonin. Iron determination, electron paramagnetic resonance spectroscopy, and native gel immunoblots revealed the incorporation of the non-heme diiron centre and homodimer formation of active sMMO. The production of recombinant sMMO will enable the expansion of the possibilities of detailed studies, allowing for a variety of novel biotechnological applications.
Assuntos
Proteínas de Escherichia coli , Methylomonas , Chaperoninas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Metano/metabolismo , Methylomonas/metabolismo , Oxigenases/metabolismoRESUMO
Production of an extracellular matrix is essential for biofilm formation, as this matrix both secures and protects the cells it encases. Mechanisms underlying production and assembly of matrices are poorly understood. Vibrio cholerae, relies heavily on biofilm formation for survival, infectivity, and transmission. Biofilm formation requires Vibrio polysaccharide (VPS), which is produced by vps gene-products, yet the function of these products remains unknown. Here, we demonstrate that the vps gene-products vpsO and vpsU encode respectively for a tyrosine kinase and a cognate tyrosine phosphatase. Collectively, VpsO and VpsU act as a tyrosine phosphoregulatory system to modulate VPS production. We present structures of VpsU and the kinase domain of VpsO, and we report observed autocatalytic tyrosine phosphorylation of the VpsO C-terminal tail. The position and amount of tyrosine phosphorylation in the VpsO C-terminal tail represses VPS production and biofilm formation through a mechanism involving the modulation of VpsO oligomerization. We found that tyrosine phosphorylation enhances stability of VpsO. Regulation of VpsO phosphorylation by the phosphatase VpsU is vital for maintaining native VPS levels. This study provides new insights into the mechanism and regulation of VPS production and establishes general principles of biofilm matrix production and its inhibition.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Polissacarídeos Bacterianos/biossíntese , Multimerização Proteica , Proteínas Tirosina Fosfatases/metabolismo , Vibrio cholerae/fisiologia , Proteínas de Bactérias/genética , Fosforilação/fisiologia , Polissacarídeos Bacterianos/genética , Proteínas Tirosina Fosfatases/genéticaRESUMO
Quorum sensing allows bacterial cells to communicate through the release of soluble signaling molecules into the surrounding medium. It plays a pivotal role in controlling bacterial conjugation in Gram-positive cells, a process that has tremendous impact on health. Intracellular regulatory proteins of the RRNPP family are common targets of these signaling molecules. The RRNPP family of gene regulators bind signaling molecules at their C-terminal domain (CTD), but have highly divergent functionalities at their N-terminal effector domains (NTD). This divergence is also reflected in the functional states of the proteins, and is highly interesting from an evolutionary perspective. RappLS20 is an RRNPP encoded on the Bacillus subtilis plasmid pLS20. It relieves the gene repression effectuated by RcopLS20 in the absence of the mature pLS20 signaling peptide Phr*pLS20. We report here an in-depth structural study of apo and Phr*pLS20-bound states of RappLS20 at various levels of atomic detail. We show that apo-RappLS20 is dimeric and that Phr*pLS20-bound Rap forms NTD-mediated tetramers. In addition, we show that RappLS20 binds RcopLS20 directly in the absence of Phr*pLS20 and that addition of Phr*pLS20 releases RcopLS20 from RappLS20. This allows RcopLS20 to bind the promotor region of crucial conjugation genes blocking their expression.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon , Multimerização Proteica , Transativadores/metabolismo , Bacillus subtilis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Conjugação Genética/genética , Peptídeos/metabolismo , Regiões Promotoras Genéticas , Repetições de Tetratricopeptídeos , Transativadores/química , Transativadores/genéticaRESUMO
Quorum sensing plays crucial roles in bacterial communication including in the process of conjugation, which has large economical and health-related impacts by spreading antibiotic resistance. The conjugative Bacillus subtilis plasmid pLS20 uses quorum sensing to determine when to activate the conjugation genes. The main conjugation promoter, Pc, is by default repressed by a regulator RcopLS20 involving DNA looping. A plasmid-encoded signalling peptide, Phr*pLS20, inactivates the anti-repressor of RcopLS20, named RappLS20, which belongs to the large group of RRNPP family of regulatory proteins. Here we show that DNA looping occurs through interactions between two RcopLS20 tetramers, each bound to an operator site. We determined the relative promoter strengths for all the promoters involved in synthesizing the regulatory proteins of the conjugation genes, and constructed an in vivo system uncoupling these regulatory genes to show that RappLS20 is sufficient for activating conjugation in vivo. We also show that RappLS20 actively detaches RcopLS20 from DNA by preferentially acting on the RcopLS20 molecules involved in DNA looping, resulting in sequestration but not inactivation of RcopLS20. Finally, results presented here in combination with our previous results show that activation of conjugation inhibits competence and competence development inhibits conjugation, indicating that both processes are mutually exclusive.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Conjugação Genética , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Plasmídeos/genética , Regiões Promotoras GenéticasRESUMO
Coordinated dynamics of individual components in active matter are an essential aspect of life on all scales. Establishing a comprehensive, causal connection between intracellular, intercellular, and macroscopic behaviors has remained a major challenge due to limitations in data acquisition and analysis techniques suitable for multiscale dynamics. Here, we combine a high-throughput adaptive microscopy approach with machine learning, to identify key biological and physical mechanisms that determine distinct microscopic and macroscopic collective behavior phases which develop as Bacillus subtilis swarms expand over five orders of magnitude in space. Our experiments, continuum modeling, and particle-based simulations reveal that macroscopic swarm expansion is primarily driven by cellular growth kinetics, whereas the microscopic swarming motility phases are dominated by physical cell-cell interactions. These results provide a unified understanding of bacterial multiscale behavioral complexity in swarms.
Assuntos
Bacillus subtilis/fisiologia , Movimento/fisiologia , Comunicação Celular/fisiologia , Proliferação de Células/fisiologia , Cinética , Aprendizado de MáquinaRESUMO
Vibrio cholerae is the causative agent of the diarrheal disease cholera, for which biofilm communities are considered to be environmental reservoirs. In endemic regions, and after algal blooms, which may result from phosphate enrichment following agricultural runoff, the bacterium is released from biofilms resulting in seasonal disease outbreaks. However, the molecular mechanism by which V. cholerae senses its environment and switches lifestyles from the biofilm-bound state to the planktonic state is largely unknown. Here, we report that the major biofilm scaffolding protein RbmA undergoes autocatalytic proteolysis via a phosphate-dependent induced proximity activation mechanism. Furthermore, we show that RbmA mutants that are defective in autoproteolysis cause V. cholerae biofilms to grow larger and mechanically stronger, correlating well with the observation that RbmA stability directly affects microbial community homeostasis and rheological properties. In conclusion, our biophysical study characterizes a novel phosphate-dependent breakdown pathway of RbmA, while microbiological data suggest a new, sensory role of this biofilm scaffolding element.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Compostos de Magnésio/farmacologia , Fosfatos/farmacologia , Proteólise , Vibrio cholerae/metabolismo , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/crescimento & desenvolvimentoRESUMO
BACKGROUND AND AIMS: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV-specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity-enhanced T Cell receptor with an anti-CD3 T Cell-activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus-derived peptides presented by human leukocyte antigen (HLA). APPROACH AND RESULTS: ImmTAV molecules specific for HLA-A*02:01-restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV-Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging-based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV-Env can redirect T cells from healthy and HBV-infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV-Env redirection of T cells induced cytolysis of antigen-positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA. CONCLUSIONS: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non-HBV-specific T cells, bypassing exhausted HBV-specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials.
Assuntos
Vírus da Hepatite B/imunologia , Hepatite B Crônica/tratamento farmacológico , Receptores de Antígenos de Linfócitos T/uso terapêutico , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/efeitos dos fármacos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Complexo CD3/antagonistas & inibidores , Linhagem Celular Tumoral , Epitopos/imunologia , Antígeno HLA-A2/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Hepatócitos , Humanos , Imunoconjugados/genética , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Linfócitos T/imunologiaRESUMO
BACKGROUND: Patients with preeclampsia admitted to the intensive care unit (ICU) may have risk factors for acute kidney injury (AKI). Although the use of neutrophil gelatinase-associated lipocalcin (NGAL) to predict AKI is previously validated, we could locate only scanty data regarding the epidemiology of AKI and role of NGAL in preeclamptic patients admitted to ICU. METHODS: Patients with preeclampsia admitted to our ICU were included. The incidence and severity of AKI during the entire ICU stay were assessed using kidney disease improving global outcomes criteria, while the a priori risk factors and serum NGAL were also evaluated. RESULTS: A total of 52 preeclamptic patients admitted to ICU were included, among whom the majority had eclampsia (75%). AKI developed in 25 (48.1%) patients with stages 1, 2, and 3 in 56, 36, and 8%, respectively. The incidence of sepsis (16 vs 0%), shock (40 vs 7.4%), and anemia (84 vs 59.3%) was significantly greater in patients with AKI (p < 0.05). ICU mortality (28 vs 3.7%), duration of ICU, and hospital stay were significantly higher in patients who developed AKI (p < 0.05). There was no association of serum NGAL [274 (240-335) ng/mL] with AKI or the mortality (p = 0.725, 0.861); there was, however, a significant discriminatory value for eclampsia [p = 0.019; area under curve = 0.736 (95% confidence interval: 0.569-0.904)]. CONCLUSIONS: Although AKI is common among patients with preeclampsia admitted to ICU, serum NGAL does not predict its occurrence. HOW TO CITE THIS ARTICLE: Tyagi A, Yadav P, Salhotra R, Das S, Singh PK, Garg D. Acute Kidney Injury in Severe Preeclamptic Patients Admitted to Intensive Care Unit: Epidemiology and Role of Serum Neutrophil Gelatinase-associated Lipocalcin. Indian J Crit Care Med 2021;25(9):1013-1019.
RESUMO
Biofilms are a ubiquitous mode of microbial life and display an increased tolerance to different stresses. Inside biofilms, cells may experience both externally applied stresses and internal stresses that emerge as a result of growth in spatially structured communities. In this review, we discuss the spatial scales of different stresses in the context of biofilms, and if cells in biofilms respond to these stresses as a collection of individual cells, or if there are multicellular properties associated with the response. Understanding the organizational level of stress responses in microbial communities can help to clarify multicellular functions of biofilms.
Assuntos
Bactérias/metabolismo , Biofilmes , Bactérias/citologia , Humanos , Estresse FisiológicoRESUMO
Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named relLS20, is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the RelLS20 shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research.
Assuntos
Proteínas de Bactérias/genética , Conjugação Genética , Farmacorresistência Bacteriana/genética , Endodesoxirribonucleases/genética , Firmicutes/enzimologia , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , DNA de Cadeia Simples/genética , Endodesoxirribonucleases/isolamento & purificação , Firmicutes/genética , Microbioma Gastrointestinal/genética , Transferência Genética Horizontal , Humanos , Plasmídeos/genéticaRESUMO
Bacterial biofilms represent a major form of microbial life on Earth and serve as a model active nematic system, in which activity results from growth of the rod-shaped bacterial cells. In their natural environments, ranging from human organs to industrial pipelines, biofilms have evolved to grow robustly under significant fluid shear. Despite intense practical and theoretical interest, it is unclear how strong fluid flow alters the local and global architectures of biofilms. Here, we combine highly time-resolved single-cell live imaging with 3D multiscale modeling to investigate the mechanisms by which flow affects the dynamics of all individual cells in growing biofilms. Our experiments and cell-based simulations reveal three quantitatively different growth phases in strong external flow and the transitions between them. In the initial stages of biofilm development, flow induces a downstream gradient in cell orientation, causing asymmetrical dropletlike biofilm shapes. In the later developmental stages, when the majority of cells are sheltered from the flow by the surrounding extracellular matrix, buckling-induced cell verticalization in the biofilm core restores radially symmetric biofilm growth, in agreement with predictions of a 3D continuum model.
Assuntos
Biofilmes/crescimento & desenvolvimento , Modelos Biológicos , Vibrio cholerae/fisiologia , MicrofluídicaRESUMO
Plasmid conjugation plays a significant role in the dissemination of antibiotic resistance and pathogenicity determinants. Understanding how conjugation is regulated is important to gain insights into these features. Little is known about regulation of conjugation systems present on plasmids from Gram-positive bacteria. pLS20 is a native conjugative plasmid from the Gram-positive bacterium Bacillus subtilis. Recently the key players that repress and activate pLS20 conjugation have been identified. Here we studied in detail the molecular mechanism regulating the pLS20 conjugation genes using both in vivo and in vitro approaches. Our results show that conjugation is subject to the control of a complex genetic switch where at least three levels of regulation are integrated. The first of the three layers involves overlapping divergent promoters of different strengths regulating expression of the conjugation genes and the key transcriptional regulator RcoLS20. The second layer involves a triple function of RcoLS20 being a repressor of the main conjugation promoter and an activator and repressor of its own promoter at low and high concentrations, respectively. The third level of regulation concerns formation of a DNA loop mediated by simultaneous binding of tetrameric RcoLS20 to two operators, one of which overlaps with the divergent promoters. The combination of these three layers of regulation in the same switch allows the main conjugation promoter to be tightly repressed during conditions unfavorable to conjugation while maintaining the sensitivity to accurately switch on the conjugation genes when appropriate conditions occur. The implications of the regulatory switch and comparison with other genetic switches involving DNA looping are discussed.
Assuntos
Conjugação Genética , Resistência Microbiana a Medicamentos/genética , Plasmídeos/genética , Transcrição Gênica , Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras GenéticasRESUMO
The diacylglycerol lipases (DAGLα and DAGLß) hydrolyze DAG to generate 2-arachidonoylglycerol (2-AG), the principal endocannabinoid and main precursor of arachidonic acid (AA). The DAGLs make distinct tissue specific contributions toward 2-AG and AA levels, and therefore, selective modulators for these enzymes could play crucial roles toward harnessing their therapeutic potential. Relatively high-throughput assays have recently been reported for DAGLα and have proven useful toward the characterization of inhibitors of this enzyme. Similar assays are also warranted for DAGLß which was the aim of this study. We first adapted previously reported DAGLα membrane assays (using PNPB and DiFMUO as substrates) to measure recombinant DAGLß activity in membranes. In contrast to results with DAGLα, both substrates provided a relatively limited signal window for measuring DAGLß activity, however, an improved window was obtained when employing a third commercially available substrate, EnzChek. In order to further improve on the assay parameters, we successfully purified the glutathione S-transferase (GST) tagged catalytic domain of DAGLß. Activity of the enzyme was confirmed using EnzChek as well as two DAGL inhibitors (THL and OMDM-188). The purified DAGLß catalytic domain assay described here provides the basis for a relatively clean and convenient assay with the potential to be adapted for high-throughput drug discovery efforts.
Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Isoleucina/análogos & derivados , Lactonas/química , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/química , Membranas Artificiais , Humanos , Isoleucina/química , Lipase Lipoproteica/genética , Lipase Lipoproteica/isolamento & purificação , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Drought stress tolerance for crop improvement is an important goal worldwide. Drought is a complex trait, and it is vital to understand the complex physiological, biochemical, and molecular mechanisms of drought tolerance to tackle it effectively. Osmotic adjustment, oxidative stress management (OSM), and cell membrane stability (CMS) are major components of cellular tolerance under drought stress. In the current study, we explored the molecular basis of OSM in the drought tolerant rice variety, Nagina 22 and compared it with the popular drought sensitive rice variety, IR 64, under drought imposed at the reproductive stage, to understand how the parental polymorphisms correlate with the superiority of Nagina 22 and tolerant bulk populations under drought. RESULTS: We generated recombinant inbred lines (RIL) from contrasting parents Nagina 22 and IR 64 and focussed on spikelet fertility (SF), in terms of its correlation with OSM, which is an important component of drought tolerance in Nagina 22. Based on SF under drought stress and its correlations with other yield related traits, we used superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) activity assays to establish the relationship between SF and OSM genes in the tolerant and sensitive lines. Among the OSM enzymes studied, GR had a significant and positive correlation with single plant yield (SPY) under drought stress. GR was also positively correlated with APX but negatively so with SOD. Interestingly, none of the enzyme-morphology correlations were significant under irrigated control (IC). Through genome-wide SNP analysis of the 21 genes encoding for OSM enzymes, we identified the functional polymorphisms between the parents and identified superior alleles. By using network analysis of OSM genes in rice, we identified the genes that are central to the OSM network. CONCLUSIONS: From the biochemical and morphological data and the SNP analysis, the superiority of Nagina 22 in spikelet fertility under drought stress is because of its superior alleles for SOD (SOD2, SODCC1, SODA) and GR (GRCP2) rather than for APX, for which IR 64 had the superior allele (APX8). Nagina 22 can bypass APX8 by directly interacting with SODA. For nine of the 11 genes present in the central network, Nagina 22 had the superior alleles. We propose that Nagina 22 tolerance could mainly be because of SODA which is a reactive oxygen scavenger in mitochondria which is directly associated with spikelet fertility.
Assuntos
Adaptação Biológica/genética , Secas , Genótipo , Oryza/genética , Oryza/metabolismo , Estresse Oxidativo/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Endogamia , Polimorfismo de Nucleotídeo Único , Estresse Fisiológico , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default "OFF" state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.