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It has previously been documented that Zn[sup 2+] inhibits TrkAmediated effects of NGF. To evaluate the ability of Zn[sup 2+] to attenuate the biological activities of NGF mediated by p75[sup NTR], we characterized the effects of this transition metal cation on both binding and the pro-apoptotic properties of the NGFp75[sup NTR] interaction. Binding of NGF to p75[sup NTR] displayed higher affinity in embryonic chick retinal cells than in PC12 cells. NGF induced apoptosis in dissociated cultures of chick neural retina. The addition of 100 µM Zn[sup 2+] inhibited binding and chemical cross-linking of [sup 125]I-NGF to p75[sup NTR], and also attenuated apoptosis mediated by this ligand-receptor interaction. These studies lead to the conclusion that Zn[sup 2+] antagonizes NGF/p75[sup NTR]-mediated signaling, suggesting that the effect of this transition metal cation can be either pro- or anti-apoptotic depending on the cellular context. [ABSTRACT FROM AUTHOR]

Forty percent of the US population and 1 in 6 individuals worldwide are obese, and the incidence of this disease is surging globally 1,2 . Various dietary interventions, including carbohydrate and fat restriction, and more recently amino acid restriction, have been explored to combat this epidemic 3-6 . We sought to investigate the impact of removing individual amino acids on the weight profiles of mice. Compared to essential amino acid restriction, induction of conditional cysteine restriction resulted in the most dramatic weight loss, amounting to 20% within 3 days and 30% within one week, which was readily reversed. This weight loss occurred despite the presence of substantial cysteine reserves stored in glutathione (GSH) across various tissues 7 . Further analysis demonstrated that the weight reduction primarily stemmed from an increase in the utilization of fat mass, while locomotion, circadian rhythm and histological appearance of multiple other tissues remained largely unaffected. Cysteine deficiency activated the integrated stress response (ISR) and NRF2-mediated oxidative stress response (OSR), which amplify each other, leading to the induction of GDF15 and FGF21, hormones associated with increased lipolysis, energy homeostasis and food aversion 8-10 . We additionally observed rapid tissue coenzyme A (CoA) depletion, resulting in energetically inefficient anaerobic glycolysis and TCA cycle, with sustained urinary excretion of pyruvate, orotate, citrate, α-ketoglutarate, nitrogen rich compounds and amino acids. In summary, our investigation highlights that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism, and stress signaling compared to other amino acid restrictions. These findings may pave the way for innovative strategies for addressing a range of metabolic diseases and the growing obesity crisis., Competing Interests: Conflicts of Interest D.R.L consults for and has equity interest in Vedanta Bioscience, Sonoma Immunotherapeutics, Immunai, IMIDomics, and Pfizer, Inc.

Gene expression in Escherichia coli is controlled by well-established mechanisms that activate or repress transcription. Here, we identify CedA as an unconventional transcription factor specifically associated with the RNA polymerase (RNAP) σ 70 holoenzyme. Structural and biochemical analysis of CedA bound to RNAP reveal that it bridges distant domains of β and σ 70 subunits to stabilize an open-promoter complex. CedA does so without contacting DNA. We further show that cedA is strongly induced in response to amino acid starvation, oxidative stress and aminoglycosides. CedA provides a basal level of tolerance to these clinically relevant antibiotics, as well as to rifampicin and peroxide. Finally, we show that CedA modulates transcription of hundreds of bacterial genes, which explains its pleotropic effect on cell physiology and pathogenesis., (© 2024. The Author(s).)

Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering 1 to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature., (© 2023. The Author(s).)

Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside monophosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull-downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy structures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show specific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states. The weakening of RNAP-RNaseHII interactions compromises RER in vivo. The structure-functional data support a model where RNaseHII scans DNA in one dimension in search for rNMPs while "riding" the RNAP. We further demonstrate that TC-RER accounts for a significant fraction of repair events, thereby establishing RNAP as a surveillance "vehicle" for detecting the most frequently occurring replication errors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glutathione (GSH) is the most abundant cellular antioxidant. As reactive oxygen species (ROS) are widely believed to promote aging and age-related diseases, and antioxidants can neutralize ROS, it follows that GSH and its precursor, N-acetyl cysteine (NAC), are among the most popular dietary supplements. However, the long- term effects of GSH or NAC on healthy animals have not been thoroughly investigated. We employed C. elegans to demonstrate that chronic administration of GSH or NAC to young or aged animals perturbs global gene expression, inhibits skn-1-mediated transcription, and accelerates aging. In contrast, limiting the consumption of dietary thiols, including those naturally derived from the microbiota, extended lifespan. Pharmacological GSH restriction activates the unfolded protein response and increases proteotoxic stress resistance in worms and human cells. It is thus advantageous for healthy individuals to avoid excessive dietary antioxidants and, instead, rely on intrinsic GSH biosynthesis, which is fine-tuned to match the cellular redox status and to promote homeostatic ROS signaling., (© 2021. The Author(s).)

The MEK1 kinase plays a critical role in key cellular processes, and as such, its dysfunction is strongly linked to several human diseases, particularly cancer. MEK1 has consequently received considerable attention as a drug target, and a significant number of small-molecule inhibitors of this kinase have been reported. The majority of these inhibitors target an allosteric pocket proximal to the ATP binding site which has proven to be highly druggable, with four allosteric MEK1 inhibitors approved to date. Despite the significant attention that the MEK1 allosteric site has received, chemotypes which have been shown structurally to bind to this site are limited. With the aim of discovering novel allosteric MEK1 inhibitors using a fragment-based approach, we report here a screening method which resulted in the discovery of multiple allosteric MEK1 binders, one series of which was optimized to sub-μM affinity for MEK1 with promising physicochemical and ADMET properties., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

RNA polymerase-binding RNA aptamers (RAPs) are natural RNA elements that control transcription in cis by directly contacting RNA polymerase. Many RAPs inhibit transcription by inducing Rho-dependent termination in Escherichia coli. Here, we studied the role of inhibitory RAPs (iRAPs) in modulation of antisense transcription (AT) using in silico and in vivo approaches. We revisited the antisense transcriptome in cells with impaired AT regulators (Rho, H-NS and RNaseIII) and searched for the presence of RAPs within antisense RNAs. Many of these RAPs were found at key genomic positions where they terminate AT. By exploring the activity of several RAPs both in a reporter system and in their natural genomic context, we confirmed their significant role in AT regulation. RAPs coordinate Rho activity at the antisense strand and terminate antisense transcripts. In some cases, they stimulated sense expression by alleviating ongoing transcriptional interference. Essentially, our data postulate RAPs as key determinants of Rho-mediated AT regulation in E. coli., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter-proximal regions and within the first 20 to 30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20 to 30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an "accelerator" over specific early gene body regions., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Prokaryotes and eukaryotes alike endogenously generate the gaseous molecule hydrogen sulfide (H 2 S). Bacterial H 2 S acts as a cytoprotectant against antibiotics-induced stress and promotes redox homeostasis. In E. coli, endogenous H 2 S production is primarily dependent on 3-mercaptopyruvate sulfurtransferase (3MST), encoded by mstA. Here, we show that cells lacking 3MST acquire a phenotypic suppressor mutation resulting in compensatory H 2 S production and tolerance to antibiotics and oxidative stress. Using whole genome sequencing, we identified a non-synonymous mutation within an uncharacterized LacI-type transcription factor, ycjW. We then mapped regulatory targets of YcjW and discovered it controls the expression of carbohydrate metabolic genes and thiosulfate sulfurtransferase PspE. Induction of pspE expression in the suppressor strain provides an alternative mechanism for H 2 S biosynthesis. Our results reveal a complex interaction between carbohydrate metabolism and H 2 S production in bacteria and the role, a hitherto uncharacterized transcription factor, YcjW, plays in linking the two.

Bacteria naturally form communities of cells known as biofilms. However the physiological roles of biofilms produced by non-pathogenic microbiota remain largely unknown. To assess the impact of a biofilm on host physiology we explored the effect of several non-pathogenic biofilm-forming bacteria on Caenorhabditis elegans. We show that biofilm formation by Bacillus subtilis, Lactobacillus rhamnosus and Pseudomonas fluorescens induces C. elegans stress resistance. Biofilm also protects against pathogenic infection and prolongs lifespan. Total mRNA analysis identified a set of host genes that are upregulated in response to biofilm formation by B. subtilis. We further demonstrate that mtl-1 is responsible for the biofilm-mediated increase in oxidative stress resistance and lifespan extension. Induction of mtl-1 and hsp-70 promotes biofilm-mediated thermotolerance. ilys-2 activity accounts for biofilm-mediated resistance to Pseudomonas aeruginosa killing. These results reveal the importance of non-pathogenic biofilms for host physiology and provide a framework to study commensal biofilms in higher organisms.

A high-sugar diet has been associated with reduced lifespan in organisms ranging from worms to mammals. However, the mechanisms underlying the harmful effects of glucose are poorly understood. Here we establish a causative relationship between endogenous glucose storage in the form of glycogen, resistance to oxidative stress and organismal aging in Caenorhabditis elegans. We find that glycogen accumulated on high dietary glucose limits C. elegans longevity. Glucose released from glycogen and used for NADPH/glutathione reduction renders nematodes and human hepatocytes more resistant against oxidative stress. Exposure to low levels of oxidants or genetic inhibition of glycogen synthase depletes glycogen stores and extends the lifespan of animals fed a high glucose diet in an AMPK-dependent manner. Moreover, glycogen interferes with low insulin signalling and accelerates aging of long-lived daf-2 worms fed a high glucose diet. Considering its extensive evolutionary conservation, our results suggest that glycogen metabolism might also have a role in mammalian aging.

Bacterial small RNAs (sRNAs) have been implicated in various aspects of post-transcriptional gene regulation. Here, we demonstrate that sRNAs also act at the level of transcription termination. We use the rpoS gene, which encodes a general stress sigma factor σ(S), as a model system, and show that sRNAs DsrA, ArcZ, and RprA bind the rpoS 5'UTR to suppress premature Rho-dependent transcription termination, both in vitro and in vivo. sRNA-mediated antitermination markedly stimulates transcription of rpoS during the transition to the stationary phase of growth, thereby facilitating a rapid adjustment of bacteria to global metabolic changes. Next generation RNA sequencing and bioinformatic analysis indicate that Rho functions as a global "attenuator" of transcription, acting at the 5'UTR of hundreds of bacterial genes, and that its suppression by sRNAs is a widespread mode of bacterial gene regulation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing, may be skewed by mutational ‘hot’ or ‘cold’ spots. Both approaches are affected by numerous caveats. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 10(4)-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcription–replication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.

Although the anti-inflammatory role of the A2a receptor is well established, controversy remains with regard to the therapeutic value for A2a agonists in treatment of inflammatory lung diseases, also as a result of unwanted A2a-mediated cardiovascular effects. In this paper, we describe the discovery and characterization of a new, potent and selective A2a agonist (compound 2) with prolonged lung retention and limited systemic exposure following local administration. To support the lead optimization chemistry program with compound selection and profiling, multiple in vitro and in vivo assays were used, characterizing compound properties, pharmacodynamics (PD), and drug concentrations. Particularly, pharmacokinetic-PD modeling was applied to quantify the effects on the cardiovascular system, and an investigative toxicology study in rats was performed to explore potential myocardial toxicities. Compound 2, in comparison to a reference A2a agonist, UK-432,097, demonstrated higher solubility, lower lipophilicity, lower plasma protein binding, high rat lung retention (28% remaining after 24 h), and was efficacious in a lung inflammatory rat model following intratracheal dosing. Despite these properties, compound 2 did not provide a sufficient therapeutic index, that is, separation of local anti-inflammatory efficacy in the lung from systemic side effects in the cardiovascular system. The plasma concentration that resulted in induction of hypotension (half maximal effective concentration; EC50 0.5 nmol/L) correlated to the in vitro A2a potency (rIC50 0.6 nmol/L). Histopathological lesions in the heart were observed at a dose level which is threefold above the efficacious dose level in the inflammatory rat lung model. In conclusion, compound 2 is a highly potent and selective A2a agonist with significant lung retention after intratracheal administration. Despite its local anti-inflammatory efficacy in rat lung, small margins to the cardiovascular effects suggested limited therapeutic value of this compound for treatment of inflammatory lung disease by the inhaled route.

Nitric oxide (NO) is an important signaling molecule in multicellular organisms. Most animals produce NO from L-arginine via a family of dedicated enzymes known as NO synthases (NOSes). A rare exception is the roundworm Caenorhabditis elegans, which lacks its own NOS. However, in its natural environment, C. elegans feeds on Bacilli that possess functional NOS. Here, we demonstrate that bacterially derived NO enhances C. elegans longevity and stress resistance via a defined group of genes that function under the dual control of HSF-1 and DAF-16 transcription factors. Our work provides an example of interspecies signaling by a small molecule and illustrates the lifelong value of commensal bacteria to their host., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The Legionella pneumophila Dot/Icm type IV secretion system is essential for the biogenesis of a phagosome that supports bacterial multiplication, most likely via the functions of its protein substrates. Recent studies indicate that fundamental cellular processes, such as vesicle trafficking, stress response, autophagy and cell death, are modulated by these effectors. However, how each translocated protein contributes to the modulation of these pathways is largely unknown. In a screen to search substrates of the Dot/Icm transporter that can cause host cell death, we identified a gene whose product is lethal to yeast and mammalian cells. We demonstrate that this protein, called SidI, is a substrate of the Dot/Icm type IV protein transporter that targets the host protein translation process. Our results indicate that SidI specifically interacts with eEF1A and eEF1Bgamma, two components of the eukaryotic protein translation elongation machinery and such interactions leads to inhibition of host protein synthesis. Furthermore, we have isolated two SidI substitution mutants that retain the target binding activity but have lost toxicity to eukaryotic cells, suggesting potential biochemical effect of SidI on eEF1A and eEF1Bgamma. We also show that infection by L. pneumophila leads to eEF1A-mediated activation of the heat shock regulatory protein HSF1 in a virulence-dependent manner and deletion of sidI affects such activation. Moreover, similar response occurred in cells transiently transfected to express SidI. Thus, inhibition of host protein synthesis by specific effectors contributes to the induction of stress response in L. pneumophila-infected cells.

In this issue of Molecular Cell, Mariner et al. (2008) demonstrate that Alu RNA from a human SINE represses RNA polymerase II transcription during heat shock. This noncoding RNA is the first example of a "protein-like" transcription factor with a distinct modular architecture.

The heat-shock transcription factor 1 (HSF1) has an important role in the heat-shock response in vertebrates by inducing the expression of heat-shock proteins (HSPs) and other cytoprotective proteins. HSF1 is present in unstressed cells in an inactive monomeric form and becomes activated by heat and other stress stimuli. HSF1 activation involves trimerization and acquisition of a site-specific DNA-binding activity, which is negatively regulated by interaction with certain HSPs. Here we show that HSF1 activation by heat shock is an active process that is mediated by a ribonucleoprotein complex containing translation elongation factor eEF1A and a previously unknown non-coding RNA that we term HSR1 (heat shock RNA-1). HSR1 is constitutively expressed in human and rodent cells and its homologues are functionally interchangeable. Both HSR1 and eEF1A are required for HSF1 activation in vitro; antisense oligonucleotides or short interfering (si)RNA against HSR1 impair the heat-shock response in vivo, rendering cells thermosensitive. The central role of HSR1 during heat shock implies that targeting this RNA could serve as a new therapeutic model for cancer, inflammation and other conditions associated with HSF1 deregulation.

Neurotrophins are a family of proteins with pleiotropic effects mediated by two distinct receptor types, namely the Trk family, and the common neurotrophin receptor p75NTR. Binding of four mammalian neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), to p75NTR is studied by molecular modeling based on X-ray structures of the neurotrophins and the extracellular domain of p55TNFR, a homologue of p75NTR. The model of neurotrophin/receptor interactions suggests that the receptor binding domains of neurotrophins (loops I and IV) are geometrically and electrostatically complementary to a putative binding site of p75NTR, formed by the second and part of the third cysteine-rich domains. Geometric match of neurotrophin/receptor binding domains in the complexes, as characterized by shape complementarity statistic Sc, is comparable to known protein/protein complexes. All charged residues within the loops I and IV of the neurotrophins, previously determined as being critical for p75NTR binding, directly participate in receptor binding in the framework of the model. Principal residues of the binding site of p75NTR include Asp47, Lys56, Asp75, Asp76, Asp88, and Glu89. The additional involvement of Arg80 and Glu53 is specific for NGF and BDNF, respectively, and Glu73 participates in binding with NT-3 and NT-4/5. Neurotrophins are likely to induce similar, but not identical, conformational changes within the p75NTR binding site.

A role for Zn2+ in a variety of neurological conditions such as stroke, epilepsy and Alzheimer's disease has been postulated. In many instances, susceptible neurons are located in regions rich in Zn2+ where nerve growth factor (NGF) levels rise as a result of insult. Although the interaction of Zn2+ with this neurotrophin has previously been suggested, the direct actions of the ion on NGF function have not been explored. Molecular modeling studies predict that Zn2+ binding to NGF will induce structural changes within domains of this neurotrophin that participate in the recognition of TrkA and p75NTR. We demonstrate here that Zn2+ alters the conformation of NGF, rendering it unable to bind to p75NTR or TrkA receptors or to activate signal transduction pathways and biological outcomes normally induced by this protein. Similar actions of Zn2+ are also observed with other members of the NGF family, suggesting a modulatory role for this metal ion in neurotrophin function.