Direct Interaction of the NifL Regulatory Protein with the GlnK Signal Transducer Enables theAzotobacter vinelandiiNifL-NifA Regulatory System to Respond to Conditions Replete for Nitrogen

ABSTRACT The BipA protein of Escherichia coli has intriguing similarities to the elongation factor subfamily of GTPases, including EF-Tu, EF-G, and LepA. In addition, phenotypes of a bipA deletion mutant suggest that BipA is involved in regulation of a variety of pathways. These two points have led to speculation that BipA may be a novel regulatory protein that affects efficient translation of target genes through direct interaction with the ribosome. We isolated and characterized suppressors of the cold-sensitive growth phenotype exhibited by ΔbipA strains and identified insertion mutations in rluC. The rluC gene encodes a pseudouridine synthase responsible for pseudouridine modification of 23S rRNA at three sites, all located near the peptidyl transferase center. Deletion of rluC not only suppressed cold sensitivity but also alleviated the decrease in capsule synthesis exhibited by bipA mutants, suggesting that the phenotypic effects of BipA are manifested through an effect on the ribosome. The suppressor effect is specific to rluC, as deletion of other rlu genes did not relieve cold sensitivity, and further, more than a single pseudouridine residue is involved, as alteration of single residues did not produce suppressors. These results are consistent with a role for BipA in either the structure or the function of the ribosome and imply that wild-type ribosomes are dependent on BipA for efficient expression of target mRNAs and that the lack of pseudouridylation at these three sites renders the ribosomes BipA independent.

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RocA Binds CsrS To Modulate CsrRS-Mediated Gene Regulation in Group A Streptococcus

mBio ◽

10.1128/mbio.01495-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 4

Author(s):

Nicola N. Lynskey ◽

Jorge J. Velarde ◽

Meredith B. Finn ◽

Simon L. Dove ◽

Michael R. Wessels

Keyword(s):

Gene Regulation ◽

Target Genes ◽

Response Regulator ◽

Critical Role ◽

Regulatory Protein ◽

Regulatory System ◽

Fine Tuning ◽

Human Pathogen ◽

Two Component ◽

Group A

ABSTRACT The orphan regulator RocA plays a critical role in the colonization and pathogenesis of the obligate human pathogen group A Streptococcus. Despite multiple lines of evidence supporting a role for RocA as an auxiliary regulator of the control of virulence two-component regulatory system CsrRS (or CovRS), the mechanism of action of RocA remains unknown. Using a combination of in vitro and in vivo techniques, we now find that RocA interacts with CsrS in the streptococcal membrane via its N-terminal region, which contains seven transmembrane domains. This interaction is essential for RocA-mediated regulation of CsrRS function. Furthermore, we demonstrate that RocA forms homodimers via its cytoplasmic domain. The serotype-specific RocA truncation in M3 isolates alters this homotypic interaction, resulting in protein aggregation and impairment of RocA-mediated regulation. Taken together, our findings provide insight into the molecular requirements for functional interaction of RocA with CsrS to modulate CsrRS-mediated gene regulation. IMPORTANCE Bacterial two-component regulatory systems, comprising a membrane-bound sensor kinase and cytosolic response regulator, are critical in coordinating the bacterial response to changing environmental conditions. More recently, auxiliary regulators which act to modulate the activity of two-component systems, allowing integration of multiple signals and fine-tuning of bacterial responses, have been identified. RocA is a regulatory protein encoded by all serotypes of the important human pathogen group A Streptococcus. Although RocA is known to exert its regulatory activity via the streptococcal two-component regulatory system CsrRS, the mechanism by which it functions was unknown. Based on new experimental evidence, we propose a model whereby RocA interacts with CsrS in the streptococcal cell membrane to enhance CsrS autokinase activity and subsequent phosphotransfer to the response regulator CsrR, which mediates transcriptional repression of target genes.

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Salicylic acid-mediated plasmodesmal closure via Remorin-dependent lipid organization

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1911892116 ◽

2019 ◽

Vol 116 (42) ◽

pp. 21274-21284 ◽

Cited By ~ 21

Author(s):

Dingquan Huang ◽

Yanbiao Sun ◽

Zhiming Ma ◽

Meiyu Ke ◽

Yong Cui ◽

...

Keyword(s):

Salicylic Acid ◽

Lipid Raft ◽

Plant Pathogens ◽

Regulatory Protein ◽

Cell Communication ◽

Direct Interaction ◽

Membrane Lipid ◽

Dependent Manner ◽

Transmission Electron ◽

Lipid Organization

Plasmodesmata (PD) are plant-specific membrane-lined channels that create cytoplasmic and membrane continuities between adjacent cells, thereby facilitating cell–cell communication and virus movement. Plant cells have evolved diverse mechanisms to regulate PD plasticity in response to numerous environmental stimuli. In particular, during defense against plant pathogens, the defense hormone, salicylic acid (SA), plays a crucial role in the regulation of PD permeability in a callose-dependent manner. Here, we uncover a mechanism by which plants restrict the spreading of virus and PD cargoes using SA signaling by increasing lipid order and closure of PD. We showed that exogenous SA application triggered the compartmentalization of lipid raft nanodomains through a modulation of the lipid raft-regulatory protein, Remorin (REM). Genetic studies, superresolution imaging, and transmission electron microscopy observation together demonstrated that Arabidopsis REM1.2 and REM1.3 are crucial for plasma membrane nanodomain assembly to control PD aperture and functionality. In addition, we also found that a 14-3-3 epsilon protein modulates REM clustering and membrane nanodomain compartmentalization through its direct interaction with REM proteins. This study unveils a molecular mechanism by which the key plant defense hormone, SA, triggers membrane lipid nanodomain reorganization, thereby regulating PD closure to impede virus spreading.

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Agonist-induced association of tropomyosin with protein kinase Cα in colonic smooth muscle

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00330.2004 ◽

2005 ◽

Vol 288 (2) ◽

pp. G268-G276 ◽

Cited By ~ 19

Author(s):

Sita Somara ◽

Haiyan Pang ◽

Khalil N. Bitar

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Thin Filament ◽

Regulatory Protein ◽

Direct Interaction ◽

Muscle Cells ◽

Smooth Muscle Contraction ◽

Particulate Fraction ◽

Calcium Dependent

Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKCα in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKCα in the particulate fraction may be due to acetylcholine-induced translocation of PKCα to the particulate fraction. To investigate whether the association of PKCα with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKCα indicated direct interaction of tropomyosin with PKCα. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKCα with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction.

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BldG and SCO3548 Interact Antagonistically To Control Key Developmental Processes in Streptomyces coelicolor

Journal of Bacteriology ◽

10.1128/jb.01695-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2541-2550 ◽

Cited By ~ 13

Author(s):

Archana Parashar ◽

Kimberley R. Colvin ◽

Dawn R. D. Bignell ◽

Brenda K. Leskiw

Keyword(s):

Sigma Factor ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Affinity Purification ◽

Regulatory System ◽

Direct Interaction ◽

Morphological Differentiation ◽

Sigma Factors ◽

Developmental Processes ◽

Two Hybrid

ABSTRACT The similarity of BldG and the downstream coexpressed protein SCO3548 to anti-anti-sigma and anti-sigma factors, respectively, together with the phenotype of a bldG mutant, suggests that BldG and SCO3548 interact as part of a regulatory system to control both antibiotic production and morphological differentiation in Streptomyces coelicolor. A combination of bacterial two-hybrid, affinity purification, and far-Western analyses demonstrated that there was self-interaction of both BldG and SCO3548, as well as a direct interaction between the two proteins. Furthermore, a genetic complementation experiment demonstrated that SCO3548 antagonizes the function of BldG, similar to other anti-anti-sigma/anti-sigma factor pairs. It is therefore proposed that BldG and SCO3548 form a partner-switching pair that regulates the function of one or more sigma factors in S. coelicolor. The conservation of bldG and sco3548 in other streptomycetes demonstrates that this system is likely a key regulatory switch controlling developmental processes throughout the genus Streptomyces.

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Transferrin receptor hyperexpression in primary erythroblasts is lost on transformation by avian erythroblastosis virus

Blood ◽

10.1182/blood.v100.1.289 ◽

2002 ◽

Vol 100 (1) ◽

pp. 289-298 ◽

Cited By ~ 5

Author(s):

Lioba Lobmayr ◽

Thomas Sauer ◽

Iris Killisch ◽

Matthias Schranzhofer ◽

Robert B. Wilson ◽

...

Keyword(s):

Plasma Membrane ◽

Transferrin Receptor ◽

Messenger Rna ◽

Large Fraction ◽

Regulatory Protein ◽

Regulatory System ◽

Cell Types ◽

Binding Activity ◽

Chicken Cell ◽

Avian Erythroblastosis Virus

Abstract In primary chicken erythroblasts (stem cell factor [SCF] erythroblasts), transferrin receptor (TfR) messenger RNA (mRNA) and protein were hyperexpressed as compared to nonerythroid chicken cell types. This erythroid-specific hyperexpression was abolished in transformed erythroblasts (HD3E22 cells) expressing the v-ErbA and v-ErbB oncogenes of avian erythroblastosis virus. TfR expression in HD3E22 cells could be modulated by changes in exogenous iron supply, whereas expression in SCF erythroblasts was not subject to iron regulation. Measurements of TfR mRNA half-life indicated that hyperexpression in SCF erythroblasts was due to a massive stabilization of transcripts even in the presence of high iron levels. Changes in mRNA binding activity of iron regulatory protein 1 (IRP1), the primary regulator of TfR mRNA stability in these cells, correlated well with TfR mRNA expression; IRP1 activity in HD3E22 cells and other nonerythroid cell types tested was iron dependent, whereas IRP1 activity in primary SCF erythroblasts could not be modulated by iron administration. Analysis of avian erythroblasts expressing v-ErbA alone indicated that v-ErbA was responsible for these transformation-specific alterations in the regulation of iron metabolism. In SCF erythroblasts high amounts of TfR were detected on the plasma membrane, but a large fraction was also located in early and late endosomal compartments, potentially concealing temporary iron stores from the IRP regulatory system. In contrast, TfR was almost exclusively located to the plasma membrane in HD3E22 cells. In summary, stabilization of TfR mRNA and redistribution of Fe-Tf/TfR complexes to late endosomal compartments may contribute to TfR hyperexpression in primary erythroblasts, effects that are lost on leukemic transformation.

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The DNA Architectural Protein HMGB1 Displays Two Distinct Modes of Action That Promote Enhanceosome Assembly

Molecular and Cellular Biology ◽

10.1128/mcb.22.12.4390-4401.2002 ◽

2002 ◽

Vol 22 (12) ◽

pp. 4390-4401 ◽

Cited By ~ 55

Author(s):

Katherine Mitsouras ◽

Ben Wong ◽

Charina Arayata ◽

Reid C. Johnson ◽

Michael Carey

Keyword(s):

Dna Binding ◽

Mutational Analysis ◽

Regulatory Protein ◽

Specific Interaction ◽

Regulatory Region ◽

Direct Interaction ◽

Single Amino Acid ◽

Target Sequence ◽

Mobility Shift ◽

Epstein Barr

ABSTRACT HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites. Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA. Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1. HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer. DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex. Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence. The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU. These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence. The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA. We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts.

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Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

Microbiology ◽

10.1099/mic.0.28903-0 ◽

2006 ◽

Vol 152 (7) ◽

pp. 2075-2089 ◽

Cited By ~ 28

Author(s):

Yaoping Zhang ◽

David M. Wolfe ◽

Edward L. Pohlmann ◽

Mary C. Conrad ◽

Gary P. Roberts

Keyword(s):

Translational Regulation ◽

Rhodospirillum Rubrum ◽

Nitrogenase Activity ◽

Regulatory Protein ◽

Regulatory System ◽

Poor Response ◽

Photosynthetic Bacterium ◽

Nitrogen Acquisition ◽

Consistent Model ◽

Energy Deprivation

The AmtB protein transports uncharged NH3 into the cell, but it also interacts with the nitrogen regulatory protein PII, which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three PII homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB 1 and amtB 2, have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB1 are defective in their response to both addition and darkness, while mutants lacking AmtB2 show little effect on the regulation of nitrogenase activity. These responses to darkness and appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB1 is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB1-PII regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by PII is proposed. Other results demonstrate a degree of specificity in interaction of AmtB1 with the different PII homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which PII proteins regulate processes involved in nitrogen acquisition and utilization.

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Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity

FEBS Journal ◽

10.1111/febs.12618 ◽

2013 ◽

Vol 281 (3) ◽

pp. 724-738 ◽

Cited By ~ 9

Author(s):

Marta Menegazzi ◽

Sofia Mariotto ◽

Martina Dal Bosco ◽

Elena Darra ◽

Nadia Vaiana ◽

...

Keyword(s):

Direct Interaction ◽

Signal Transducer ◽

Natural And Synthetic

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GTP and Na+ modulate receptor-adenyl cyclase coupling and receptor-mediated function

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1984.247.1.e59 ◽

1984 ◽

Vol 247 (1) ◽

pp. E59-E68 ◽

Cited By ~ 2

Author(s):

L. E. Limbird

Keyword(s):

Adenylate Cyclase ◽

Binding Protein ◽

Regulatory Protein ◽

Direct Interaction ◽

Receptor Occupancy ◽

Gtp Binding Protein ◽

Protein Activation ◽

Gtp Binding ◽

Molecular Events ◽

The Relationship

Activation and inhibition of adenylate cyclase activity is mediated via two distinct GTP-binding proteins. In both stimulatory and inhibitory systems, receptor occupancy by a hormone or agonist drug stabilizes receptor interactions with its functionally associated GTP-binding protein and, as a consequence, facilitates GTP occupancy of the regulatory protein. Activation of cyclase proceeds until GTP is hydrolyzed to GDP. Although hormonal inhibition of cyclase is elicited by a sequence of molecular events seemingly parallel to those for activation of cyclase, it is not known whether hormonal inhibition of cyclase involves the direct interaction of the GTP-occupied inhibitory regulatory protein with the catalytic subunit or results indirectly from destabilization of the interaction of the stimulatory GTP-binding protein with the catalytic moiety. Sodium ion also modulates receptor-mediated inhibition of adenylate cyclase, although apparently via a component distinct from the GTP-binding subunit of the inhibitory GTP-binding protein. Sodium also influences the physiological functions elicited by receptors that mediate inhibition of cyclase, at least for alpha 2-adrenergic-promoted platelet secretion. The relationship between sodium effects on hormonal attenuation of cAMP accumulation and elicited physiological effects is discussed.

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