Academic literature on the topic 'C-di-AMP synthase'

Introduction: The aim of the study was to evaluate the endothelioprotective activity of 4-hydroxy-3,5-di-tret-butylcinnamic acid in conditions of experimental cerebral ischemia. Materials and Methods: The brain ischemia was reproduced by the method of irreversible right-sided thermocoagulation of the middle cerebral artery. As comparative drugs, mexidol (30 mg/kg) and sulodexide (30 U/kg) were used. The vasodilating function of the vascular endothelium was assessed by the change in the rate of cerebral blood flow when the synthesis of nitric oxide was modified. Antithrombotic function was assessed by changes in the concentration of thromboxane A2, fibrinogen, von Willebrand factor activity and platelet aggregation activity. Serum concentration of C-reactive protein served as a marker of the state of anti-inflammatory endothelial function. To determine the potential mechanism of endothelioprotective activity of 4-4-hydroxy-3,5-di-tret-butylcinnamic acid, the anti-radical activity of this compound toward superoxide and nitrosy-radicals was assessed; and the effect of the compound on the mitochondrial function was studied, by evaluating the functional activity of mitochondrial ATP synthetase and cytochrome-c-oxidase by ELISA. Results and Discussion: In the course of the study, a positive effect of 4-hydroxy-3,5-di-tret-butylcinnamic acid on the state of endothelial function in cerebral ischemia was established, which was expressed in the preservation of vasodilating (restoring the vascular reaction to acetylcholine, nitro-L-arginine methyl ether, L-arginine), antithrombotic (a decrease in the concentration of thromboxane A2, fibrinogen and von Willebrand factor activity by 241.9% (p <0.05), 73.5% (p <0.05), 20.4% (p <0.05), respectively, a decrease in the degree of aggregation and platelet aggregation rate by 56.7 % (p <0.05) and 52.8% (p <0.05), respectively, and anti-inflammatory vascular endothelial function (99.1% C-reactive protein reduction (p <0.05)). The 4-hydroxy-3,5-di-tret-butylcinnamic acid compound in vitro tests suppressed generation of superoxide (IC50 = 1.99 mg/ml) and nitrosyl radical (IC50 = 1.92 mg/ml), eliminated NO-synthase uncoupling, and restored the mitochondrial function (increase in mitochondrial ATP synthase and cytochrome-c-oxidase activity by 23.5% (p <0.05) and 110.8% (p <0.05), respectively). Conclusion: The study demonstrated the presence of endotheliotropic activity of 4-hydroxy-3,5-di-tret-butylcinnamic acid, which is expressed in the preservation of vasodilating, antithrombotic and anti-inflammatory functions of the vascular endothelium in conditions of cerebral ischemia. At the same time, the anti-radical properties of this compound, as well as the direct effect on the functional activity of the NO-synthase system and the improvement of the mitochondrial function, may underlie the endotheliotropic effects of 4-hydroxy-3,5-di-tret-butylcinnamic acid.

Hydroxybenzylidene-indolinones, newly identified inhibitors of c-di-AMP synthases, inhibit biofilm formation, Gram-positive bacterial growth and sensitize resistant bacteria to methicillin and vancomycin.

ABSTRACT The EAL domain (also known as domain of unknown function 2 or DUF2) is a ubiquitous signal transduction protein domain in the Bacteria. Its involvement in hydrolysis of the novel second messenger cyclic dimeric GMP (c-di-GMP) was demonstrated in vivo but not in vitro. The EAL domain-containing protein Dos from Escherichia coli was reported to hydrolyze cyclic AMP (cAMP), implying that EAL domains have different substrate specificities. To investigate the biochemical activity of EAL, the E. coli EAL domain-containing protein YahA and its individual EAL domain were overexpressed, purified, and characterized in vitro. Both full-length YahA and the EAL domain hydrolyzed c-di-GMP into linear dimeric GMP, providing the first biochemical evidence that the EAL domain is sufficient for phosphodiesterase activity. This activity was c-di-GMP specific, optimal at alkaline pH, dependent on Mg2+ or Mn2+, strongly inhibited by Ca2+, and independent of protein oligomerization. Linear dimeric GMP was shown to be 5′pGpG. The EAL domain from Dos was overexpressed, purified, and found to function as a c-di-GMP-specific phosphodiesterase, not as a cAMP-specific phosphodiesterase, in contrast to previous reports. The EAL domains can hydrolyze 5′pGpG into GMP, however, very slowly, thus implying that this activity is irrelevant in vivo. Therefore, c-di-GMP is the exclusive substrate of EAL. Multiple-sequence alignment revealed two groups of EAL domains hypothesized to correspond to enzymatically active and inactive domains. The domains in the latter group have mutations in residues conserved in the active domains. The enzymatic inactivity of EAL domains may explain their coexistence with GGDEF domains in proteins possessing c-di-GMP synthase (diguanulate cyclase) activity.

Antimicrobial resistance (AMR) poses a huge threat to public health. The development of novel antibiotics is an effective strategy to tackle AMR. Cyclic diadenylate monophosphate (c-di-AMP) has recently been identified as an essential signal molecule for some important bacterial pathogens involved in various bacterial physiological processes, leading to its synthase diadenylate cyclase becoming an attractive antimicrobial drug target. In this study, based on the enzymatic activity of diadenylate cyclase of Streptococcus suis (ssDacA), we established a high-throughput method of screening for ssDacA inhibitors. Primary screening with a compound library containing 1133 compounds identified IPA-3 (2,2′-dihydroxy-1,1′-dinapthyldisulfide) as an ssDacA inhibitor. High-performance liquid chromatography (HPLC) analysis further indicated that IPA-3 could inhibit the production of c-di-AMP by ssDacA in vitro in a dose-dependent manner. Notably, it was demonstrated that IPA-3 could significantly inhibit the growth of several Gram-positive bacteria which harbor an essential diadenylate cyclase but not E. coli, which is devoid of the enzyme, or Streptococcus mutans, in which the diadenylate cyclase is not essential. Additionally, the binding site in ssDacA for IPA-3 was predicted by molecular docking, and contains residues that are relatively conserved in diadenylate cyclase of Gram-positive bacteria. Collectively, our results illustrate the feasibility of ssDacA as an antimicrobial target and consider IPA-3 as a promising starting point for the development of a novel antibacterial.

Abstract Background & Significance: Prostate cancer (PCa) is the second leading cause of cancer deaths (~34,000 in 2021 (ACS)) in American men. Castration resistance and resistance to the next-gen androgen receptor (AR) targeted drugs are major challenges. Castration resistance involves multiple mechanisms, including androgen-independent signaling by androgen receptor (AR)- or its variants, and lineage plasticity (LP) with AR-indifferent neuroendocrine (NE) differentiation. Identifying new vulnerabilities across these multitude mechanisms could provide new therapeutic avenues against castration-resistant PCa (CRPC). The cell-surface di-ganglioside GD2 is overexpressed in neural crest cell tumors such as neuroblastoma & melanoma and chimeric (Dinutuximab) or humanized (Naxitamab) anti-GD2 antibodies are now FDA-approved for high-risk neuroblastoma therapy. GD2 expression is reported in other cancers such as breast cancer and glioma and is linked to cancer stem cell behavior. While limited prior studies have detected GD2 expression in PCa cell lines or tumor tissues, nothing is known about the functional role of GD2 in PCa. Objectives: We hypothesized that GD2 overexpression in PCa could play a pro-tumorigenic role and that linkage of GD2 overexpression with CRPC progression may reveal the potential of targeting GD2 for CRPC therapy. Study Design & Results: Immunohistochemical analysis of PCa patient and patient-derived xenograft tissue microarrays (TMAs) revealed GD2 expression in a subset of tumor cells. Fluorescence-activated cell sorter analysis of PCa cell lines showed strong constitutive GD2 expression on murine CRPC cell line RM-1 (derived from mutant Ras and c-Myc overexpressing prostatic epithelial cells) and human PCa line 22Rv1 (overexpresses wild-type AR and ARv7 splice variant). GD2 expression was induced de novo upon induction of lineage plasticity in GD2-negative LNCaP C4-2 prostate adenocarcinoma cell line by shRNA knockdown (KD) of RB1 or TP53. High GD2 expression was also induced when C4-2B cells were made enzalutamide resistant (C4-2BER). Induction of GD2 expression correlated with increased expression of rate-limiting GD2 biosynthetic pathway enzyme GD3 synthase (GD3S). CRISPR-Cas9 mediated stable GD3S knockout (KO) in the RM1 cell line led to the loss of GD2 expression. The GD3S-KO RM1 cells exhibited reduced proliferation, migration, invasion, and tumor sphere forming ability compared to the control cells. Intratibial injections in castrated male C57BL/6 mice showed a significant reduction in tumor development by GD3S KO RM1 cells compared to control cells. Conclusions: Our studies demonstrate that GD2 is expressed in a subset of prostate cancers. Cell line-based studies show that GD2 expression promotes pro-tumorigenic traits. Future studies will assess the biological roles of GD2 in PCa and the potential of targeting GD2+ CRPC with antibody-based approved therapeutic agents. Citation Format: Aaqib M. Bhat, Bhopal C. Mohapatra, Insha Mushtaq, Sukanya Chakraborty, Samikshan Dutta, Sameer Mirza, Matthew D. Storck, Subodh M. Lele, Ming-Fong Lin, Bruce J. Trock, Karen S. Sfanos, Colm Morrissey, Eva Corey, Jonathan Melamed, Leah Cook, Kaustubh Datta, Jane Meza, Jawed Siddiqui, Surinder K. Batra, Vimla Band, Hamid Band. Di-ganglioside GD2 expression and role in promoting tumorigenicity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2411.

The endoplasmic-reticulum-resident protein STING (Stimulator of IFN genes) is a downstream signaling effector of cytosolic DNA sensor cGAS (cyclic GMP-AMP synthase). STING-mediated innate immune activation plays a key role in tumor- and self-DNA elicited anti-tumor immunity and autoimmunity, respectively, yet the mechanism remains largely unclear. We utilized murine models of allogeneic hematopoietic cell transplantation (allo-HCT) to study the biology of STING in antigen-presetting cells (APCs) and T cells. STING expression in donor T cells was dispensable for their ability to induce graft-versus-host disease (GVHD), a major complication of allo-HCT in the clinic. However, when STING-deficient mice were used as recipients, more severe disease was induced after allo-HCT. Using bone marrow (BM) chimeras where STING was absent in different compartments, we found that STING-deficiency on host hematopoietic cells (Fig. A), but not on non-hematopoietic cells, was primarily responsible for exacerbating the disease. Furthermore, STING expression on host CD11c+ cells played a dominant role in the regulation of allogeneic T-cell responses (Fig. B). Mechanistically, STING deficiency resulted in increased survival, activation and function of irradiated APCs, including macrophages and dendritic cells (DCs, fig. C-D). To further determine the role of STING in APCs, we generated a STING V154M knock-in mouse model, in which V154M mutation in TMEM173 causes constitutive activation of STING. Consistently, constitutive activation of STING attenuated the survival, activation and function of APCs isolated from STING V154M knock-in mice. In addition, STING-deficient APCs augmented donor T-cell expansion, chemokine receptor expression and migration into intestinal tissues (Fig. E), resulting in accelerated/exacerbated disease. Using pharmacologic approaches, we demonstrate that systemic administration of a STING agonist (c-di-GMP) to recipient mice before transplantation significantly reduced GVHD mortality (Fig. F). In conclusion, we report an inhibitory role of STING in regulating survival and T-cell priming function of hematopoietic APCs, especially CD11c+ cells, after allo-HCT. We validate that pharmacological activation of STING may serve as a potential therapeutic strategy to constrain APCs and induce immune tolerance. Figure Disclosures No relevant conflicts of interest to declare.

Adipogenesis is a complex process in which cell commitment and mitotic clonal expansion (MCE) are in-sequence crucial events leading to terminal adipocyte differentiation. The molecules able to block some key signals in this cascade can hamper adipogenesis becoming promising agents to counteract hyperplasia and hypertrophy of adipose tissue. Mono- and di-caffeoylquinic acid isomers are biologically active polyphenols, displaying in vitro and in vivo antioxidant, hepatoprotective, anti-diabetic and anti-obesity properties. Among these isomers, 3,5-dicaffeoylquinic acid (DCQA) has been reported to inhibit lipid accumulation in adipose cells more successfully than others. Thus, we investigated DCQA effects and molecular mechanisms on 3T3-L1 pre-adipocytes induced to differentiate with a hormonal cocktail (MDI). Oil Red O incorporation assessed that DCQA pre-treatment inhibited lipid accumulation in 3T3-L1 cells induced to differentiate for 10 days. At this time, an increased phosphorylation of both AMP-activated kinase and acetyl-CoA carboxylase, as well as a strong decrease in fatty acid synthase protein level, were registered by immunoblotting, thereby suggesting that DCQA treatment can reduce fatty acid anabolism in 3T3-L1 adipocytes. Furthermore, BrdU incorporation assay, performed 48 h after hormonal stimulation, revealed that DCQA treatment was also able to hinder the 3T3-L1 cell proliferation during the MCE, which is an essential step in the adipogenic process. Thus, we focused our attention on early signals triggered by the differentiation stimuli. In the first hours after hormonal cocktail administration, the activation of ERK1/2 and Akt kinases, or CREB and STAT3 transcription factors, was not affected by DCQA pre-treatment. Whereas 24 h after MDI induction, DCQA pre-treated cells showed increased level of the transcription factor Nrf2, that induced the expression of the antioxidant enzyme heme oxygenase 1 (HO-1). In control samples, the expression level of HO-1 was reduced 24 h after MDI induction in comparison with the higher amount of HO-1 protein found at 2 h. The HO-1 decrease was functional by allowing reactive oxygen species to boost and allowing cell proliferation induction at the beginning of MCE phase. Instead, in DCQA-treated cells the HO-1 expression was maintained at high levels for a further 24 h; in fact, its expression decreased only 48 h after MDI stimulation. The longer period in which HO-1 expression remained high led to a delay of the MCE phase, with a subsequent inhibition of both C/EBP-α expression and adipocyte terminal differentiation. In conclusion, DCQA counteracting an excessive adipose tissue expansion may become an attractive option in obesity treatment.

In bacteria, cyclic-di-nucleotide based second messengers regulate various physiological processes including the stress response. For the past decades, cyclic diadenosine monophosphate (c-di-AMP) has emerged as a crucial second messenger in the bacterial world. It is an essential molecule implicated in fatty acid metabolism, antibiotic resistance, biofilm formation, virulence, DNA repair, potassium and osmotic homeostasis, sporulation etc. The level of c-di-AMP is maintained within the cell by the action of two opposing enzymes, namely diadenylate cyclases and phosphodiesterases. In mycobacteria, this molecule is essential for its regulatory role in bacterial physiology and host-pathogen interactions. However, such modulation of c-di-AMP remains to be explored in Mycobacterium smegmatis. Here, we systematically investigated the c-di-AMP synthase (MsDisA) and a hydrolase (MsPDE) from M. smegmatis at different pH and osmolytic conditions in vitro. Our biochemical assays show that the MsDisA activity is enhanced during the alkaline stress and c-di-AMP is readily produced without any intermediates. At pH 9.4, the MsDisA promoter activity in vivo increases significantly, strengthening this observation. However, under physiological conditions, the activity of MsDisA was moderate with the formation of intermediates. We also observed that the size of MsDisA is significantly increased upon incubation with the substrate. To get further insights into the structural characteristics, we solved a 3.02 Å cryo-EM structure of the MsDisA, revealing some of interesting properties. Analysis of individual domains shows that the N-terminal minimal region alone can form a functional octamer. Altogether, our results reveal the biochemical and structural regulation of mycobacterial c-di-AMP in response to various environmental stresses. Chapter 1 reviews the available literature in the field of second messengers and provide rational behind this study. The discovery of c-di-AMP was accidental, which later had known to regulate plethora of functions. This chapter stresses upon the need to investigate the significance of c-di-AMP homeostasis in Mycobacteria and the scope of the current study. Chapter 2 reports the homeostasis of c-di-AMP by DisA and PDE in M. smegmatis and the substrate-induced inhibitory mechanism of MsDisA. Promoter study suggested MsDisA is expressed in all the growth phases and the changes in the extracellular pH during mycobacterial growth give valuable hints about the trans-membrane pH regulation by the c-di-AMP molecule. Chapter 3 further elaborates the enzyme-substrate reaction and domain movement. The substrate-induced change of the MsDisA structure, which is demonstrated using biophysical characterization and Transmission Electron Microscopy (TEM) image analysis. The details on the structural characteristics of the MsDisA were obtained by cryo-EM. We reconstructed a 3.02 Å structure of MsDisA by Electron Cryomicroscopy (Cryo-EM). We observed an open-complex formation of this protein, which gives insight into the enzyme's active site. Chapter 4 describes the importance of individual domain of MsDisA in c-di-AMP synthesis and critical residues at catalytic core. This study provides a few interesting observations about the oligomerization and activity of the mutant proteins. Further, this chapter also talks about the second activity of DisA protein which binds to 4-way junctions DNA, resulting in an allosteric inhibition of c-di-AMP synthesis activity in Bacillus subtilis. Interestingly, the cryo-EM open structure of MsDisA shows a unique feature where two monomers stay apart to break D4 symmetry, preventing the protein from directly interacting with branched DNA which helps mycobacterial cell for the continuous synthesis of c-di-AMP under stress. Chapter 5 summarizes the results of the study and points out the future directions for the work. Appendix Chapters includes the work which I have carried out in my first three years. In appendix chapter 1, we worked on bacterial RNA polymerase and its smallest subunit ω. Here the emphasis of the work was to understand the mechanistic details of lethality by silent mutant of ω. Wild type ω shows a predominantly unstructured circular dichroic profile and becomes α-helical in the enzyme complex. This structural transition is perhaps the reason for the lack of function. We generated several silent mutants of ω to investigate the role of codon bias and the effect of rare codons with respect to their position in rpoZ. Not all silent mutations affect the structure. RNA polymerase when reconstituted with structurally altered silent mutants of ω is transcriptionally inactive. The Codon Plus strain, which has surplus tRNA, was used to assess for the rescue of the phenotype in lethal silent mutants. In appendix chapter 2, we have utilized 7-Aza-tryptophan to investigate whether there is binding coupled folding for omega. The photo physical properties of free 7-Aza-tryptophan give a strong red-shifted fluorescence (403 nm), which would allow us to trap the conformational changes that occur during the ω-β' interaction and sigma attachment to the core RNAP. The incorporation of non-natural analogue 7-Aza-Trp in ω was achieved by way of using an E. coli Trp auxotrophic strain (RF12) where we have overcharged the system with Tryptophanyl-tRNA synthetase (trpS) gene for proper incorporation. The interaction of 7-Aza-Trp incorporated ω with the core1 (α2ββ') RNAP is being studied here.

碩士
國立中興大學
生物化學研究所
102
【Theme one】 Recently, it was found that cyclic diadenosine monophosphate (c-di-AMP), which is one of the cyclic nucleotide second messengers, can exist in Bacillus subtilis (Bsu), in charge of adjusting spore formation and cell wall metabolism. The c-di-AMP concentration in Bacillus subtilis is chiefly adjusted by diadenylate cyclases (DAC) and phosphodiesterases (PDE). It is synthesized from two molecules of ATP by diadenylyl cyclase (DAC) enzymes which have a DGA-domain or RHR- domain, and is degraded to pApA by PDE, which have a DHH-domain or DHHA-domain. The research aim is to study the structure and the function of DAC protein in Bacillus subtilis str. 168 (BsuDacA). Currently, the crystal structure was determined to a resolution of 2.85A. We further used Differential Scanning Calorimetry (DSC) detect the interaction between the DAC domain BsuDacA(105-268) protein and ATP. From the result, the Tm of BsuDacA(105-268) in its apo-form and ATP-binding form are shown to be 45℃ and 50℃, respectively. The result indicate that ATP bind with BsuDacA to stabilize its conformation. Enzyme Kinetic assay indicates that BsuDacA(105-268) exhibits a Vmax and Km of 2.13 umol min－1 and 56.97 uM, respectively. To sum up, BsuDacA(105-268) can interact with ATP, and synthesize c-di-AMP to potentially assist cell wall formation. 【Theme two】 Pattern recognition receptors (PRRs) are essential for detecting invading Pathogens via the Pathogen-Associated Molecular Patterns (PAMPs). PRRs are located in the membrane or endosome, and can be regarded as the sensor of extracellular nucleic acid. The research aim is to study the structure and the function of the mammalian cytoplasm protein LSm14A. According to provious results, hLSm14A firstly detects virus DNA or RNA. Then P-bodies will interact with RIG-1/VISA/MITA proteins, initiate the releasing of IFNs and then launch the immune responses, such as immunity, and inflammatory responses. Though the 3D structures of N-terminal LSm domain of hLSm14A has been discovered, the structure of C-terminal (including DFDF domain and FFD/TFG/RGG domain) remained unknown. Thus, little is know about how foreign DNA or RNA of virus interact with hLSm14A protein. To obtain more information about the interaction between hLSm14A and virus DNA or RNA, we have constructed the hLSm14A clone by total gene synthesis and expressed the hLSm14A(261-463) protein fragment. Currently, wa have obtained the hLSm14A(261-463) crystal. But due to the poor crystal quality, we were unable to collect good diffraction data to determine its structure. From the gel-filtration data, hLSm14A(261-463) was found to exist as a mixture of tetramer and monomer in solution, which may lead to the imperfect protein crystals. It may be necessary to study hLSm14A protein in different lengh to get a better crystal quality to determine its structure.