Low Molecular Weight Effectors Research Articles

Glutamate Positively Regulates Chitinase Activity and the Biocontrol Efficacy of Pseudomonas protegens.

Broad-spectrum biocontrol by Pseudomonas protegens CHA0 and other fluorescent pseudomonads is achieved through the generation of various secondary metabolites with antibiotic activities against not only other microbes but, also, nematodes and insects present in the rhizosphere. A previous metabolomic study demonstrated that intracellular low-molecular weight effectors, such as guanosine tetraphosphate and γ-aminobutyrate, function as important signals in niche adaptation by strain CHA0 to plant roots. We investigated the role of amino acids in the biocontrol trait of P. protegens Cab57 towards Pythium damping off and root rot in cucumber. Among the 11 amino acids tested, only glutamate markedly enhanced the efficacy of biocontrol. An RNA-Seq analysis revealed that glutamate upregulated the expression of a chitinase gene cluster (c21370-c21380, in which the c21370 gene was annotated as a gene encoding the chitin-binding protein cbp and the c21380 gene encoded chitinase chiC) in strain CHA0. Glutamate upregulated the expression of the regulatory small RNA rsmZ but reduced the production levels of other Gac/Rsm-regulated biocontrol factors, such as 2,4-diacetylphloroglucinol and pyoluteorin. The promoter activity of cbp and chitinase activity were characterized in detail; their activities were up-regulated in response to glutamate and their expression was under the control of GacA. Therefore, glutamate appears to be essential for biocontrol activity in which chitinase production is regulated in response to glutamate. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The N-terminal Helix-Turn-Helix Motif of Transcription Factors MarA and Rob Drives DNA Recognition.

DNA-binding proteins play an important role in gene regulation and cellular function. The transcription factors MarA and Rob are two homologous members of the AraC/XylS family that regulate multidrug resistance. They share a common DNA-binding domain, and Rob possesses an additional C-terminal domain that permits binding of low-molecular weight effectors. Both proteins possess two helix-turn-helix (HTH) motifs capable of binding DNA; however, while MarA interacts with its promoter through both HTH-motifs, prior studies indicate that Rob binding to DNA via a single HTH-motif is sufficient for tight binding. In the present work, we perform microsecond time scale all-atom simulations of the binding of both transcription factors to different DNA sequences to understand the determinants of DNA recognition and binding. Our simulations characterize sequence-dependent changes in dynamical behavior upon DNA binding, showcasing the role of Arg40 of the N-terminal HTH-motif in allowing for specific tight binding. Finally, our simulations demonstrate that an acidic C-terminal loop of Rob can control the DNA binding mode, facilitating interconversion between the distinct DNA binding modes observed in MarA and Rob. In doing so, we provide detailed molecular insight into DNA binding and recognition by these proteins, which in turn is an important step toward the efficient design of antivirulence agents that target these proteins.

Abstract 4303: Modulation of protein interaction states through the cell cycle

Abstract Global profiling of protein expression through the cell cycle has revealed subsets of periodically expressed proteins. However, expression levels alone only give a partial view of processes determining cellular events. The cell cycle progression events are to a large extent controlled by the dynamic biochemical interactions of proteins with physiological ligands such as other proteins, metabolites, lipids, nucleic acids or low molecular weight effectors. Using a proteome-wide implementation of the Cellular Thermal Shift Assay (CETSA) to study specific cell cycle phases in K562 cell, we uncover modulations of interaction states for more than 750 proteins along the cell cycle. Notably, many protein complexes are modulated in specific cell cycle phases, reflecting their roles in processes such as DNA replication, chromatin remodeling, transcription, translation, and nuclear membrane decomposition. Surprisingly, only small differences in interaction states were seen between G1 and G2 phases, suggesting similar hardwiring of biochemical processes in these two phases. The present work reveals novel molecular details of the cell cycle. CETSA, therefore emerge as a novel approach to discover cellular modulations during cancer development and therapy. Citation Format: Lingyun Dai, Tianyun Zhao, Xavier Bisteau, Wendi Sun, Nayana Prabhu, Yan Ting Lim, Radoslaw Sobota, Philipp Kaldis, Pär Nordlund. Modulation of protein interaction states through the cell cycle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4303.

Antibiotics and evolution: food for thought.

The role of secondary metabolites in effecting and modulating reactions during early biochemical evolution has been largely unappreciated. It is possible that low molecular weight effectors were gradually replaced by polypeptides as polymerizing reactions became more complex, but retained some ability to interact with original receptor sites. Indeed, by reviewing the era of antibiotics in this light we can begin to reconcile the ancient and contemporary activities of these molecules. The corollary being that secondary metabolites participate in a vast array of interactions in nature and investigating their intended receptors will be revealing in both pharmacological and evolutionary terms.

Low molecular weight regulators of the intracellular insulin signal transduction as a correction method of the insulin resistance in the treatment of type 2 diabetes

Insulin resistance is the characteristic feature of type 2 diabetes. This condition is manifested in the reduction of peripheral tissues sensitivity to the biological action of insulin and is expressed in the inhibition of cellular glucose absorption and metabolism in response to hormonal stimulation. At the cellular level, disorders which are realized both at the receptor and the postreceptor levels can serve a prerequisite to the formation of insulin resistance and are associated with a change in the amount or dysfunction of major molecular signaling cascade. Thus, the insulin receptor, as well as the other related signaling molecules can be considered as ideal therapeutic targets for the correction of insulin resistance and thus low molecular weight effectors which act on the individual links of insulin signaling cascade may be positioned as a new generation of anti-diabetic agents. This report provides information on the regulators of insulin receptor cascade, main advantages and disadvantages of their impact on biological targets and prospects for their therapeutic use as anti-diabetic drugs.

Low molecular weight regulators of the intracellular insulin signal transduction as the method of correction of insulin resistance in the treatment of type 2 diabetes mellitus

Insulin resistance is the characteristic feature of type 2 diabetes mellitus. This condition is manifested in decreased sensitivity of peripheral tissues to the biological action of insulin and is expressed in the inhibition of cell glucose uptake and metabolism in response to the hormonal stimulation. At the cellular level, impairments realized both at the receptor and the postreceptor levels and associated with changes in the content or dysfunction of the main molecules of the signal cascade can serve a molecular prerequisite to the formation of insulin resistance. Thus, the insulin receptor, as well as the other related signaling molecules can be considered as ideal therapeutic targets for the correction of insulin resistance and low molecular weight effectors, which act on the individual links of the insulin signaling cascade, may be positioned as a new generation of anti-diabetic agents. This review summarizes current knowledge on regulators of the insulin receptor cascade, main advantages and disadvantages of their effects on biological targets and prospects for their therapeutic use as anti-diabetic agents.

CcpA forms complexes with CodY and RpoA in Bacillus subtilis

The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator that is controlled by interactions with the phosphoproteins histidine-containing protein (HPr)Ser46P and the catabolite responsive HPr (Crh)Ser46P and with low molecular weight effectors, depending on the availability of preferred carbon sources such as glucose. Distinct point mutations in CcpA abolish the regulation of some but not all target genes, suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and MS were applied to identify CcpA complexes active in repression and activation. To compensate for an excess of promoters only repressed by CcpA, this experiment was accomplished with cells using multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase subunits and the global regulator transcriptional pleiotropic repressor (CodY) were observed. Bacterial two-hybrid assays combining each RNA polymerase subunit with CcpA localized CcpA binding at the α-subunit of the RNA polymerase (RpoA). In vivo crosslinking combined with immunoblot analyses revealed CcpA-RpoA complexes in cultures with or without glucose, whereas CcpA-HPr and CcpA-CodY complexes occurred only or predominantly in cultures with glucose. Surface plasmon resonance analyses confirmed the binding of CcpA to the N-terminal domain (αNTD) and C-terminal domain (αCTD) of RpoA, as well as to CodY. Furthermore, interactions of CodY with the αNTD and the αCTD were detected by surface plasmon resonance. The K(D) values of complexes of CcpA or CodY with the αNTD or the αCTD are in the range 5-8 μm. CcpA and CodY form a loose complex with a K(D) of 60 μm. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.

Study of low molecular weight effectors on the binding between cell membrane receptor IGF-1R and its substrate protein IRS-1 by SPR biosensor

Surface plasmon resonance (SPR) biosensor technique was used to study the effects of Mg 2+, Mn 2+, ATP, genistein, and quercetin on the binding kinetics between insulin-like growth factor-1 receptor (IGF-1R) and its intracellular substrate protein insulin receptor substrate-1 (IRS-1). IGF-1R was captured from cell lysates using anti-IGF-1R (α-subunit) monoclonal antibodies immobilized on biosensor chip surface, which retained the binding capability with its intracellular substrate. With IGF-1 stimulation, the association rate of IRS-1binding to IGF-1R increased and the affinity constant (1.37 × 10 9 M) is about 10 times higher than that without IGF-1 stimulation (1.27 × 10 8 M). The association and dissociation rates of IRS-1 binding to phosphorylated IGF-1R in the presence of either or both of Mg 2+ and Mn 2+, and in the absence or presence of ATP, genistein, and quercetin, were determined from the real-time binding and dissociation curves and the results indicate that ATP, genistein, and quercetin reduced the affinity constants of IRS-1 and IGF-1R by 4.7-, 6.0-, and 6.6-folds, respectively, in the presence of Mg 2+ and Mn 2+. Mn 2+ slightly increased the affinity mainly due to the increased association rate, while Mg 2+ and Mn 2+ together showed no effect on the affinity. In addition, ATP alone did not affect the binding affinity. The results demonstrate that SPR biosensor could be used as a quantitative technique for studying effects of small molecules on the binding kinetics and affinity of receptor–cellular substrate. The method developed in the study may also be used in other biosensor techniques for studying the mechanism of molecule interactions.

Inducible DNA-loop formation blocks transcriptional activation by an SV40 enhancer

It is well established that gene expression in eukaryotes is controlled by sequence-dependent binding of trans-acting proteins to regulatory elements like promoters, enhancers or silencers. A less well understood level of gene regulation is governed by the various structural and functional states of chromatin, which have been ascribed to changes in covalent modification of core histone proteins. And, much on how topological domains in the genome take part in establishing and maintaining distinct gene expression patterns is still unknown. Here we present a set of regulatory proteins that allow to reversibly alter the DNA structure in vivo and in vitro by adding low molecular weight effectors that control their oligomerization and DNA binding. Using this approach, we completely regulate the activity of an SV40 enhancer in HeLa cells by reversible loop formation to topologically separate it from the promoter. This result establishes a new mechanism for DNA-structure-dependent gene regulation in vivo and provides evidence supporting the structural model of insulator function.

Analysis of Transcription Factor Interactions at Sedimentation Equilibrium

Publisher Summary This chapter explores the large numbers of proteins participating in the assemblies that regulate and catalyze transcription. Among methods available for characterizing their interactions, sedimentation equilibrium (SE) ultracentrifugation stands out as a direct and rigorous means of determining molecular masses, interaction stoichiometries, association constants, and the influences of low molecular weight effectors, ions, and crowding on the stabilities of protein complexes. The chapter also reviews the availability of modern instrumentation and the development of improved analysis methods that have resulted in an upsurge of interest in SE during the past decade. This chapter describes the application of SE techniques to the characterization of transcription factors and their interactions. It focuses on three situations that are encountered most frequently in studies of transcription factors: self-association, heteroassociation, and the presence of inactive components. Analysis of such interactions provides crucial information on the role of protein–protein interactions in the assembly of transcription and transcription–regulatory complexes.

Thermodynamic properties of nucleotide-free EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors of its GTPase activity

The thermal transition of elongation factor EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors was studied by differential scanning calorimetry. The effectors of GTPase activity used were the antibiotic kirromycin and the cations Li +, Na +, K + and NH 4 + in the chloride form. The temperature of thermal denaturation and the cooperativity of the transition of nucleotide-free EF-Tu (EF-Tu f) in the presence of kirromycin are comparable with those of the EF-Tu·guanosine-5′-[β,γ-imido]triphosphate (GppNHp) form, indicating similar conformational states. Increased concentrations of Na + and K + stabilized EF-Tu f in a manner similar to GppNHp. NH 4 + decreased the transition temperature of EF-Tu f and Li + decreased both the temperature and the calorimetric enthalpy of the thermal transition of EF-Tu f. In the presence of salts, binding of kirromycin had a stabilizing effect on EF-Tu f. Correlation between the GTPase activity and thermodynamic characteristics of EF-Tu f induced by kirromycin in the absence or presence of the cations is discussed.

Analysis of interaction of regulatory protein TyrR with DNA

Publisher Summary This chapter describes the methodology used to characterize the linkage between the binding of ligands to TyrR and the interaction of TyrR with DNA. The experimental strategies, based primarily on the analysis of sedimentation behavior, provide a quantitative model for the role of tyrosine as a low molecular weight effector of the TyrR regulon. TyrR is a DNA-binding protein that regulates several genes involved in aromatic amino acid biosynthesis in Escherichia coli . Regulation occurs via the repression or activation of several unlinked operons that comprise the TyrR regulon. It discusses that repression varies considerably in magnitude between the different operons and usually involves the coeffectors ATP and tyrosine whereas activation is ATP independent and requires any of the three aromatic amino acids. Strong boxes bind TyrR strongly in vitro in the absence of coeffectors whereas weak boxes have less identity with the consensus sequence and do not bind TyrR unless there is an adjacent strong box and both ATP and tyrosine are present. In most operons repressed by tyrosine, overlap of the promoter by the operator is predominantly via the weak box.

Human foamy virus Bel 1 transactivator/estrogen receptor fusion proteins allow inducible transactivation of both human foamy virus promoters.

Recombinant plasmids that express human foamy virus (HFV) Bel 1 transactivator and human estrogen receptor (ER) fusion proteins were constructed. The HFV bel 1 gene was inserted up- and downstream of the ER gene. Recombinant Bel 1-ER and ER-Bel 1 fusion proteins were expressed in eukaryotic cells. In the absence of estrogen, the ER moiety of the fusion proteins suppressed Bel 1-mediated transactivation as measured in CAT reporter gene-based transactivation assays. However, transactivation of the HFV LTR and the HFV internal promoter by Bel 1-ER and ER-Bel 1 fusion proteins was recovered in the presence of estrogen. Thus, the transactivation function of the Bel 1 moiety of the chimeric Bel 1-ER fusion proteins can be efficiently, specifically, and intentionally activated and inactivated by simply adding the low-molecular weight effector estrogen.

Cyclic AMP and low molecular weight effector(s) present in yeast extract are involved in pectin lyase production by Penicillium griseoroseum cultured on sucrose.

Pectin lyase (PL) induction by organic and inorganic components of yeast extract (YE) was evaluated in Penicillium griseoroseum, cultured in a mineral medium containing sucrose, by determining PL activity (A235) and mycelial growth (mycelial dry weight). The lowest YE concentration that promoted significant PL induction without acting as a carbon source for the fungus corresponded to 0.0075%. Neither calcined YE nor a nutrient solution containing micronutrients induced PL production, indicating that the inducer was an organic compound. Vitamins, phospholipid components, amino acids, and nitrogenous bases were tested in place of YE and promoted no significant PL induction. A PL inducer compound was found to be soluble in the nucleotide fraction obtained during extraction of YE. The inducer was shown to be a thermostable polar substance dialyzable at 2000 Daltons, hydrolyzable by HCl, and activated by boiling for up to 60 min. Cyclic AMP (cAMP) exogenously added to the culture medium at 5 and 10 mM was capable of inducing PL in P. griseoroseum grown on sucrose, suggesting that at least one compound may be present in YE acting in a cooperative fashion for the maintenance of high levels of cAMP into the cell. PL activity and the level of cAMP inside the fungal cells increased after the addition of YE to the culture medium, suggesting the participation of this messenger in this enzyme's synthesis.

The effect of pH on biological activity of plant cytotoxin cauloside C

The effect of plant carboxyl-containing glycoside cauloside C upon eucaryotic cells has been studied. The glycoside interacts with cells as a pH-dependent cytotoxin and increases K + leakage and Ca 2+ uptake with strong action in acidic media. Cell viability after glycoside action at acidic pH may be recovered by the shift of medium pH from 5.6 to 7.4. Directed transport of low molecular weight effectors such as cAMP and Ca 2+ to human embryo fibroblasts under the action of cauloside C has been demonstrated. Calcium uptake is accompanied by about a twofold stimulation of fibroblast proliferation in serum-free medium. The manifestation of the effect depends on the strictly determined time of the'open' state of the membrane permeability (2 min) and upon concentration of glycoside in the medium (1 ng/ml). Cauloside C-stimulated Ca-transport is not blocked by Ca-channel blockers such as verapamil, diltiasem, and nitrendipine (all at a concentration of 1×10 −6 M) but these blockers inhibit cauloside C-stimulated proliferation of fibroblasts. We conclude that stimulation of fibroblast proliferation is caused by activation of membrane associated Ca-channels at the expense of calcium, incorporated into cells with cauloside C. The use of cauloside C as a new biochemical tool for cell permeabilisation is suggested.

In vitro selection of RNA lectins: using combinatorial chemistry to interpret ribozyme evolution

Background: It has been hypothesized that the fact that both ribosomal RNA and the group I intron can bind to aminoglycoside antibiotics implies that these RNAs are evolutionarily related. This hypothesis requires the assumption that there are relatively few ways for RNA molecules to form aminoglycoside-binding sites. Results: We have used in vitro selection to determine the diversity of aminoglycoside-binding sites that can be formed by RNA molecules. We have generated RNA ‘lectins’ that can bind aminoglycosides tightly and specifically. Sequence analysis indicates that there are many different ways to form tight and specific aminoglycoside binding sites. These artificially selected binding sites are functionally similar to those that have arisen from natural selection. Conclusions: Our results suggest that the presence of aminoglycoside-binding sites on RNA molecules may not be a useful trait for determining evolutionary relatedness. Instead, the fact that RNA molecules can bind these ‘low molecular-weight effectors’ may indicate that natural products such as aminoglycosides have evolved to exploit sequence- and structure-specific recognition of nucleic acids, in much the same way that lexitropsins have been designed by chemists to recognise specific nucleic acid sequences.

Antibiotic inhibition of group I ribozyme function

The discovery of catalytically active RNA has provided the basis for the evolutionary concept of an RNA world. It has been proposed that during evolution the functions of ancient catalytic RNA were modulated by low molecular weight effectors, related to antibiotics, present in the primordial soup. Antibiotics and RNA may have coevolved in the formation of the modern ribosome. Here we report that a set of aminoglycoside antibiotics, which are known to interact with the decoding region of the 16S ribosomal RNA of Escherichia coli, inhibit the second step of splicing of the T4 phage-derived td intron. Thus catalytic RNA seems to interact not only with a mononucleotide and an amino acid, but also with another class of biomolecules, the sugars. Splicing of other group I introns but not group II introns was inhibited. The similarity in affinity and specificity of these antibiotics for group I introns and rRNAs may result from recognition of evolutionarily conserved structures.

What are antibiotics? Archaic functions for modern activities

Secondary metabolites are proposed to have played important roles in the evolution of the reactions of living forms on earth, in effecting and modulating reactions during biochemical evolution by chemical and structural interaction with 'receptor' sites in primitive macromolecular templates. For example, in the evolution of the translation system, as the polymerizing reactions became more complex and proteins became involved, the low molecular-weight effectors were functionally replaced by polypeptides, but retained their ability to interact with receptor sites in nucleic acids and proteins. Many of these low molecular-weight effectors now play a different role, that of antagonists, by interacting with the original receptor sites in macromolecular structures; this explains their contemporary activity as antibiotics.

Mutants of the methylotrophic yeast Pichia pinus defective in C2 metabolism

AbstractA collection of mutants of Pichia pinus which are unable to grow on ethanol but retain the ability to grow on glucose and methanol, was obtained. Genetic and biochemical analysis of these strains revealed mutations in seven nuclear genes affecting activities of isocitrate lyase (icl1), malate synthase (mls1), phosphoenolpyruvate carboxykinase (pck1), ‘malic’ enzyme (mdd1) and acetyl‐CoA synthetase (acs1, acs2 and acs3). All mutations except acs1‐acs3 have no effect on the activities of other enzymes involved in C2 metabolism. Mutations acs1, acs2 and acs3 have a pleiotropic action, leading to partial reduction in activities of isocitrate lyase and malate synthase. Ethanol‐induced repression of the synthesis of the methanol oxidative enzymes, alcohol oxidase and catalase, is not impaired in these seven mutant classes. On the other hand, C2 compound‐induced inactivation of alcohol oxidase and catalase is impaired in mutants acs1, acs2, acs3 and icl1. It was suggested that glyoxylate and acetate (or acetate precursors) act as low molecular weight effectors, ‘switching on’ inactivation and repression, respectively, of alcohol oxidase and catalase in the medium containing ethanol or acetate.

Effects of Insulin on Gene Transcription

for these changes in mRNA concentration. The steady-state concentration of a messenger RNA is determined by a sequence of events that includes initiation of transcription by RNA polymerase II, elongation of the trans cr ipt , termina­ tion of transcription, mRNA capping, polyadenylation, splicing, transport to the cytoplasm, and eventual degradation. Recent progress in the study of eukaryotic gene expression demonstrates that the first step in this process, initiation of transcription, can be regulated by the binding of nuclear proteins to cis-actin g DNA sequences (26). For example, DNA-binding proteins are known to mediate transcription al effects of steroid hormones, cAMP, and various low-molecu lar-weight effectors. This is not a well established mech­ anism of action for peptide hormones. Whether the effects of insulin on enzyme concentration result from this type of transcriptional regulation is the