DNA Metabolic Enzymes Research Articles

Degron-Controlled Protein Degradation in Escherichia coli: New Approaches and Parameters.

Protein degron tags have proven to be uniquely useful for the characterization of gene function. Degrons can mediate quick depletion, usually within minutes, of a protein of interest, allowing researchers to characterize cellular responses to the loss of function. To develop a general-purpose degron tool in Escherichia coli, we sought to build upon a previously characterized system of SspB-dependent inducible protein degradation. For this, we created a family of expression vectors containing a destabilized allele of SspB, capable of a rapid and nearly perfect "off-to-on" induction response. Using this system, we demonstrated excellent control over several DNA metabolism enzymes. However, other substrates did not respond to degron tagging in such an ideal manner, indicating the apparent limitations of SspB-dependent systems. Several degron-tagged proteins were degraded too slowly to be completely depleted during active growth, whereas others appeared to be completely refractory to degron-promoted degradation. Thus, only a minority of our, admittedly biased, selection of degron substrates proved to be amenable to efficient SspB-catalyzed degradation. We also uncovered an apparent stalling and/or disengagement of ClpXP from a degron-tagged allele of beta-galactosidase (beta-gal). While a degron-containing fusion peptide attached to the carboxy-terminus of beta-gal was degraded quantitatively, no reductions in beta-gal activity or concentration were detected, demonstrating an apparently novel mechanism of protease resistance. We conclude that substrate-dependent effects of the SspB system present a continued challenge to the widespread adoption of this degron system. For substrates that prove to be degradable, we provide a series of titratable SspB-expression vehicles.

Unlocking the Antibacterial Potential of Xanthone from Calophyllum Species: Inhibition of Nucleic Acid Synthesis

AbstractPlants are valuable resources for the development of novel pharmaceutical products. The increasing threat to global health caused by antibiotic resistance remains a serious concern, driven a need to discover and evaluate novel anti‐bacterial agents. Calophyllum species are known for having excellent biological activity due to its secondary metabolites, such as xanthone. Numerous xanthones have been found to possess anti‐bacterial properties that are effective against plant pathogens, hence can be applied to fight human pathogens. Topoisomerase enzymes (DNA gyrase and topoisomerase IV) are DNA metabolism enzymes that possess distinct roles as unlinking enzymes during DNA replication. Nucleic acid synthesis inhibition reduces bacteria proliferation through the inhibition of topoisomerase enzymes that are essential for bacterial growth. The xanthone isolated from Calophyllum and its anti‐bacterial were discussed in this review. Besides, molecular docking simulations were applied to explore the potential binding mode of xanthones to DNA metabolism enzymes. The docking study displayed that biscaloxanthone is a good topoisomerase enzymes inhibitor compared to their co‐cystalize ligand, novobiocin and BDBM50198240. The complied information and molecular docking simulations suggested that xanthone isolated possesses potential anti‐bacterial agents inhibiting nucleic acid synthesis. Besides, it suggested that the anti‐microbial activity of xanthone contributes from the topoisomerase enzyme‘s inhibition.

Abstract 2733: TYMS as a novel target for AAV-based therapy

Abstract The purpose of this study is to develop a strategy for treating pancreatic neuroendocrine tumor (PanNET) through blocking Thymidylate Synthase (TYMS or TS), a DNA metabolic enzyme. Elevated TYMS levels are frequently observed in human PanNET and are associated with worse prognosis and resistance to cytotoxic agents such as 5FU. Elevated TYMS levels directly promote tumorigenesis in vitro and ectopic expression of human TS (hTS) in transgenic mice promotes adenoma development in the endocrine pancreas in vivo. Pancreatic islet tumor formation in hTS transgenic mice occurred with a long latency period, suggesting that additional somatic events are required for PanNET formation and progression. Men1 was shown to be a frequent target for somatic mutation in PanNETs (44% have MEN1 mutations). Therefore, we established a new mouse model designated hTS/Men1-/-, where hTS is overexpressed in pancreatic islet cells carrying conditional Men1 null alleles. We observed that ectopic hTS cooperates with Men1 inactivation to significantly accelerate PanNET progression and shorten survival. Since traditional TYMS inhibitors, such as 5-FU, consistently induce tumor resistance and cannot cure advanced disease, we have now tested the ability of TS-shRNA delivered using adeno-associated viral (AAV) vectors as a pharmacological safe and effective anti-tumor strategy. We packaged TS shRNA under the mouse insulin promoter (mIP) for islet-cell specific expression using capsid optimized Y to F double mutant AAV (AAV8-2M) to generate viral particles of scAAV8-2M-mIP-GFP-hTS-shRNA. We treated hTS/Men1-/- mice with intraperitoneal (IP) injection (1x1011 viral genomes/mice). Treated and control mice were analyzed for PanNET progression, TYMS expression and survival. In addition, we tested a human PanNET tumor cell line (BON) that was isogenic with or without hTS expression for tumor formation in xenograft model. We confirmed sustained reduction of TYMS levels 3 weeks after IP delivery of AA8-2M-mIP-GFP-hTS-shRNA into PanNET tissues of hTS/Men1-/- mice. In addition, AA8-2M-mIP-GFP-hTS-shRNA delivery blocked PanNET tumor progression and enhanced the survival of treated mice as compared to vector controls. We also confirmed that TYMS knock-down in BON cells by hTS-shRNA reduced xenograft tumor growth as compared to non-specific shRNA controls. In conclusion, the ability of AAV-mediated TS shRNA to block PanNET tumor progression directly demonstrates the importance of elevated TYMS in tumorigenesis and offers a novel anti-cancer strategy. Citation Format: Vinod Vijayakurup, Benjamin Meyer, Akbar Nawab, Frederic J. Kaye, Maria V. Guijarro, Maria Zajac-Kaye. TYMS as a novel target for AAV-based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2733.

Maintenance of genome integrity by the late-acting cytoplasmic iron-sulfur assembly (CIA) complex.

Iron-sulfur (Fe-S) clusters are unique, redox-active co-factors ubiquitous throughout cellular metabolism. Fe-S cluster synthesis, trafficking, and coordination result from highly coordinated, evolutionarily conserved biosynthetic processes. The initial Fe-S cluster synthesis occurs within the mitochondria; however, the maturation of Fe-S clusters culminating in their ultimate insertion into appropriate cytosolic/nuclear proteins is coordinated by a late-acting cytosolic iron-sulfur assembly (CIA) complex in the cytosol. Several nuclear proteins involved in DNA replication and repair interact with the CIA complex and contain Fe-S clusters necessary for proper enzymatic activity. Moreover, it is currently hypothesized that the late-acting CIA complex regulates the maintenance of genome integrity and is an integral feature of DNA metabolism. This review describes the late-acting CIA complex and several [4Fe-4S] DNA metabolic enzymes associated with maintaining genome stability.

Multistep loading of a DNA sliding clamp onto DNA by replication factor C.

The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3' end (3' ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3' ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.

The Clustered Regularly Interspaced Short Palindromic Repeats-Associated System and Its Relationship With Mobile Genetic Elements in Klebsiella.

Microorganisms have developed many strategies in the process of long-term defense against external attacks, one of which is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) bacterial immunological system. In this study, the whole genome of 300 strains of Klebsiella was collected, the CRISPR-Cas system in the strains was statistically analyzed, and the types and structures of CRISPR system in Klebsiella were explored, as well as the correlation between CRISPR and mobile genetic elements (MGEs). Through principal component analysis (PCA), we found that Cas gene, plasmids, integron, IS1, IS609, and enzymes of DNA metabolism were closely related to CRISPR-Cas. Compared the structural characteristics of plasmids, the DinG family helicases, Cas6, Csf2, and IS5 were observed near the CRISPR loci in plasmid, which is also confirmed by the results of PCA that they may be important factors affecting the plasmid with CRISPR.

Iron-Sulfur Cluster Biogenesis as a Critical Target in Cancer.

Cancer cells preferentially accumulate iron (Fe) relative to non-malignant cells; however, the underlying rationale remains elusive. Iron–sulfur (Fe–S) clusters are critical cofactors that aid in a wide variety of cellular functions (e.g., DNA metabolism and electron transport). In this article, we theorize that a differential need for Fe–S biogenesis in tumor versus non-malignant cells underlies the Fe-dependent cell growth demand of cancer cells to promote cell division and survival by promoting genomic stability via Fe–S containing DNA metabolic enzymes. In this review, we outline the complex Fe–S biogenesis process and its potential upregulation in cancer. We also discuss three therapeutic strategies to target Fe–S biogenesis: (i) redox manipulation, (ii) Fe chelation, and (iii) Fe mimicry.

Composition of Transcription Machinery and Its Crosstalk with Nucleoid-Associated Proteins and Global Transcription Factors.

The coordination of bacterial genomic transcription involves an intricate network of interdependent genes encoding nucleoid-associated proteins (NAPs), DNA topoisomerases, RNA polymerase subunits and modulators of transcription machinery. The central element of this homeostatic regulatory system, integrating the information on cellular physiological state and producing a corresponding transcriptional response, is the multi-subunit RNA polymerase (RNAP) holoenzyme. In this review article, we argue that recent observations revealing DNA topoisomerases and metabolic enzymes associated with RNAP supramolecular complex support the notion of structural coupling between transcription machinery, DNA topology and cellular metabolism as a fundamental device coordinating the spatiotemporal genomic transcription. We analyse the impacts of various combinations of RNAP holoenzymes and global transcriptional regulators such as abundant NAPs, on genomic transcription from this viewpoint, monitoring the spatiotemporal patterns of couplons—overlapping subsets of the regulons of NAPs and RNAP sigma factors. We show that the temporal expression of regulons is by and large, correlated with that of cognate regulatory genes, whereas both the spatial organization and temporal expression of couplons is distinctly impacted by the regulons of NAPs and sigma factors. We propose that the coordination of the growth phase-dependent concentration gradients of global regulators with chromosome configurational dynamics determines the spatiotemporal patterns of genomic expression.

Generation of Fluorescent Versions of Saccharomyces cerevisiae RPA to Study the Conformational Dynamics of Its ssDNA-Binding Domains.

Replication protein A (RPA) is an essential single-stranded DNA (ssDNA)-binding protein that sequesters ssDNA and protects it from nucleolytic degradation. The RPA-ssDNA nucleoprotein acts as a hub to recruit over two dozen DNA metabolic enzymes onto ssDNA to coordinate DNA replication, repair, and recombination. RPA functions as a heterotrimer composed of RPA70, RPA32, and RPA14 subunits and has multiple DNA-binding and protein-interaction domains. Several of these domains are connected by disordered linkers allowing RPA to adopt a wide variety of conformations on ssDNA. Here we describe a fluorescence-based tool to monitor the dynamics of selectDNA-binding domains of RPA. Noncanonical amino acids are utilized to site-specifically engineer fluorescent probes in Saccharomyces cerevisiae RPA heterologously expressed in BL21 (DE3) and its derivatives. A procedure to synthesize 4-azido-L-phenylalanine (4AZP), a noncanonical amino acid, is also described. Sites for fluorophore positioning that produce a measurable change in fluorescence upon binding to ssDNA are detailed. This fluorescence enhancement through noncanonical amino acid (FEncAA) approach can also be applied to other DNA-binding proteins to investigate the dynamics of protein-nucleic acid interactions.

Canonical non-homologous end-joining promotes genome mutagenesis and translocations induced by transcription-associated DNA topoisomerase 2 activity.

DNA topoisomerase II (TOP2) is a major DNA metabolic enzyme, with important roles in replication, transcription, chromosome segregation and spatial organisation of the genome. TOP2 is the target of a class of anticancer drugs that poison the DNA-TOP2 transient complex to generate TOP2-linked DNA double-strand breaks (DSBs). The accumulation of DSBs kills tumour cells but can also result in genome instability. The way in which topoisomerase activity contributes to transcription remains unclear. In this work we have investigated how transcription contributes to TOP2-dependent DSB formation, genome instability and cell death. Our results demonstrate that gene transcription is an important source of abortive TOP2 activity. However, transcription does not contribute significantly to apoptosis or cell death promoted by TOP2-induced DSBs. On the contrary: transcription-dependent breaks greatly contribute to deleterious mutations and translocations, and can promote oncogenic rearrangements. Importantly, we show that TOP2-induced genome instability is mediated by mutagenic canonical non-homologous end joining whereas homologous recombination protects cells against these insults. Collectively, these results uncover mechanisms behind deleterious effects of TOP2 abortive activity during transcription, with relevant implications for chemotherapy.

Synthesis of water soluble tetra-substituted phthalocyanines: Investigation of DNA cleavage, cytotoxic effects and metabolic enzymes inhibition

In this study, we have reported the synthesis and characterization of 4-(sodium 2-mercaptoethane sulfonate) phthalonitrile (1) as ligand and its water-soluble phthalocyanine compounds [M = 2H (2), Zn(II) (3), Ga(III)Cl (4), In(III)Cl (5)] containing sodium 2-mercaptoethanesulfonate substituents at the peripheral positions. All compounds were characterized by UV–Vis, FT-IR and NMR spectroscopies, and MALDI-TOF mass spectra. Besides, the DNA cleavage effects of the phthalocyanine compounds were investigated using agarose gel electrophoresis and cytotoxic effects of them were determined on BT-20, DU-145, A549, SNU-398 and HFC cell lines using MTT assay. The compounds cleaved supercoiled pBR322 plasmid DNA upon irradiation (white, 17.5 mW/cm2, 60 min) in the presence of H2O2. The IC50 values of (2) were determined as 145.76, 93.75, 124.73, 128.81 and 136 μM on BT-20, DU-145, A549, SNU-398 and HFC cell lines, respectively. The IC50 values of others were above 200 μM on five cell lines. These results showed that the compounds had low cytotoxic effects against breast, prostate, lung and liver cell lines. These water-soluble phthalocyanine compounds [M = 2H (2), Zn(II) (3), Ga(III)Cl (4), In(III)Cl (5)] were found to be good inhibitor against the α-glycosidase, human carbonic anhydrase I and II isoforms, and both cholinesterase enzymes with Ki values in the range of 84.02 ± 16.95–166.47 ± 14.87 μM for α-glycosidase, 7.54 ± 1.02–31.65 ± 3.08 μM for hCA I, 5.97 ± 0.97–36.13 ± 6.97 μM for hCA II, 1.02 ± 0.14–3.42 ± 0.86 μM for BChE, and 2.01 ± 0.32–3.98 ± 0.12 μM for AChE, respectively.

Super-resolution imaging reveals changes in Escherichia coli SSB localization in response to DNA damage.

The E.coli single-stranded DNA-binding protein (SSB) is essential to viability. It plays key roles in DNA metabolism where it binds to nascent single strands of DNA and to target proteins known as the SSB interactome. There are >2,000 tetramers of SSB per cell with 100-150 associated with the genome at any one time, either at DNA replication forks or at sites of repair. The remaining 1,900 tetramers could constantly diffuse throughout the cytosol or be associated with the inner membrane as observed for other DNA metabolic enzymes. To visualize SSB localization and to ascertain potential spatiotemporal changes in response to DNA damage, SSB-GFP chimeras were visualized using a novel, super-resolution microscope optimized for the study of prokaryotic cells. In the absence of DNA damage, SSB localizes to a small number of foci and the excess protein is associated with the inner membrane where it binds to the major phospholipids. Within five minutes following DNA damage, the vast majority of SSB disengages from the membrane and is found almost exclusively in the cell interior. Here, it is observed in a large number of foci, in discreet structures or, in diffuse form spread over the genome, thereby enabling repair events.

Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA.

In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during de novo HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B.IMPORTANCE Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both de novo synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.

Activation of interferon-α signaling by resveratrol, genistein and quercetin

Resveratrol, genistein and quercetin from the group of polyphenols from secondary plant metabolites reveal cancer preventive and antivirus effects realized via their pleiotropic influence on the different macromolecules in cells. These compounds can interact with DNA without the formation of covalent bonds. This process is usually followed by changes in spatial, physical-chemical and structural DNA characteristics that can result in disfunction of DNA metabolism enzymes and chromatin destabilization. Similar effects were described for anticancer drug Curaxine CBL0137 in association with activation of interferon-α signaling. We demonstrated dose-dependent stimulating effects of resveratrol, genistein and quercetin on interferon-α signaling using HeLa cells expressed mCherry protein with interferon-stimulated response elements (ISRE) in promoter. Furthermore, it was shown by live-cell fluorescent microscopy in HT1080 cells with mCherry-labeled histone H1.5 that described polyphenols induced the redistribution of this linker histone in cell nuclei. The data obtained suggest an existence of DNA-dependent mechanism of anticancer effects of plant polyphenols and a need for further study of crosslinks between the polyphenols’ influence on chromatin structure and the changes in genome function, in particular, induction of interferon- interferon-α signaling.

In-silico identification and validation of miRNAs in pearl millet [Pennisetum glaucum L.

Pearl millet [Pennisetum glaucum (L.) R. Br.] is an important C4 crop in the arid and semi-arid regions of Asian and African countries which is under cultivation for grain, forage and stover. Here, we report identification of the conserved miRNAs in pearl millet by mining publicly available data sets including expressed sequence tag (EST) sequences, genome survey sequences (GSS), and nucleotide sequences. Homology search of 7240 known mature miRNAs of plant species against 6014 Unigene sequences of pearl millet resulted in identification of 14 potential miRNAs belonging to 11 different families. The length of predicated precursor and mature miRNAs ranged from 60 to 109 nucleotides and 18–23 nucleotides, respectively. The negative minimal folding free energy of precursor miRNAs varied significantly between −13 to −41.73 kcal/mol. Five out of 14 predicted miRNAs were validated in root and leaf/shoot tissues of seedlings of two pearl millet genotypes (J2290 and 7042S) using quantitative real time polymerase chain reaction (qRT-PCR). The psRNATarget server predicted 33 potential target genes and their probable functions. Most of the pearl millet miRNA target genes were involved in stress response, transcriptional regulation, signal transduction, DNA replication proteins, male sterility, metabolic enzymes, plant growth and development. The results of the present study will help in the understanding of mechanism of miRNA functioning in pearl millet.

Novel ATP-cone-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit.

Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, with allosteric mechanisms controlling substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leeuwenhoekiella blandensis radical-generating subunit regulates activity via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzymes. The tetramer forms by interactions between ATP-cones, shown by a 2.45 Å crystal structure. We also present evidence for an MnIIIMnIV metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by transfer of the domain to the radical-generating subunit. To our knowledge, this represents the first observation of transfer of an allosteric domain between components of the same enzyme complex.

The Phage Nucleus and Tubulin Spindle Are Conserved among Large Pseudomonas Phages

We recently demonstrated that the large Pseudomonas chlororaphis bacteriophage 201φ2-1 assembles a nucleus-like structure that encloses phage DNA and segregates proteins according to function, with DNA processing proteins inside and metabolic enzymes and ribosomes outside the nucleus. Here,we investigate the replication pathway of the Pseudomonas aeruginosa bacteriophages φKZ and φPA3. Bacteriophages φKZ and φPA3 encode a proteinaceous shell that assembles a nucleus-like structure that compartmentalizes proteins and DNA during viral infection. We show that the tubulin-like protein PhuZ encoded by each phage assembles abipolar spindle that displays dynamic instability and positions the nucleus at midcell. Our results suggest that the phage spindle and nucleus play the same functional role in all three phages, 201φ2-1, φKZ, and φPA3, demonstrating that these key structures are conserved among large Pseudomonas phages.

Comparative proteomics of oxidative stress response of Lactobacillus acidophilus NCFM reveals effects on DNA repair and cysteine de novo synthesis

Probiotic cultures encounter oxidative conditions during manufacturing, yet protein abundance changes induced by such stress have not been characterized for some of the most common probiotics and starters. This comparative proteomics investigation focuses on the response by Lactobacillus acidophilus NCFM to H2 O2, simulating an oxidative environment. Bacterial growth was monitored by BioScreen and batch cultures were harvested at exponential phase for protein profiling of stress responses by 2D gel based comparative proteomics. Proteins identified in 19 of 21 spots changing in abundance due to H2 O2 were typically related to carbohydrate and energy metabolism, cysteine biosynthesis, and stress. In particular, increased cysteine synthase activity may accumulate a cysteine pool relevant for protein stability, enzyme catalysis, and the disulfide-reducing pathway. The stress response further included elevated abundance of biomolecules reducing damage such as enzymes from DNA repair pathways and metabolic enzymes with active site cysteine residues. By contrast, a protein-refolding chaperone showed reduced abundance, possibly reflecting severe oxidative protein destruction that was not overcome by refolding. The proteome analysis provides novel insight into resistance mechanisms in lactic acid bacteria against reactive oxygen species and constitutes a valuable starting point for improving industrial processes, food design, or strain engineering preserving microorganism viability.

Stress signaling between organs in metazoa.

Many organisms have developed a robust ability to adapt and survive in the face of environmental perturbations that threaten the integrity of their genome, proteome, or metabolome. Studies in multiple model organisms have shown that, in general, when exposed to stress, cells activate a complex prosurvival signaling network that includes immune and DNA damage response genes, chaperones, antioxidant enzymes, structural proteins, metabolic enzymes, and noncoding RNAs. The manner of activation runs the gamut from transcriptional induction of genes to increased stability of transcripts to posttranslational modification of important biosynthetic proteins within the stressed tissue. Superimposed on these largely autonomous effects are nonautonomous responses in which the stressed tissue secretes peptides and other factors that stimulate tissues in different organs to embark on processes that ultimately help the organism as a whole cope with stress. This review focuses on the mechanisms by which tissues in one organ adapt to environmental challenges by regulating stress responses in tissues of different organs.

5-O-Acyl plumbagins inhibit DNA polymerase activity and suppress the inflammatory response

We previously found that vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase (pol) γ. In this study, we focused on plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), and chemically synthesized novel plumbagins conjugated with C2:0 to C22:6 fatty acids (5-O-acyl plumbagins). These chemically modified plumbagins displayed enhanced mammalian pol inhibition, with plumbagin conjugated to docosahexaenoic acid (C22:6-acyl plumbagin) exhibiting the strongest inhibition of pol λ among the ten 5-O-acyl plumbagins synthesized. C22:6-acyl plumbagin selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols or DNA metabolic enzymes tested. The inhibition of pol λ, a DNA repair/recombination pol, by these compounds was significantly correlated with both their suppression of lipopolysaccharide (LPS) induced tumor necrosis factor-α (TNF-α) production by mouse RAW264.7 macrophages and the reduction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in the mouse ear. These data indicate that 5-O-acyl plumbagins act as anti-inflammatory agents by inhibiting mammalian pol λ. These results further suggest that C22:6-acyl plumbagin is a promising anti-inflammatory candidate and that acylation could be an effective chemical modification to improve the anti-inflammatory activity of vitamin K3 derivatives, such as plumbagin.