Auxiliary Regulators Research Articles

In Silico Identification of Potential Quadruplex Forming Sequences in LncRNAs of Cervical Cancer.

Long non-coding RNAs (lncRNAs) have emerged as auxiliary regulators of gene expression influencing tumor microenvironment, metastasis and radio-resistance in cancer. The presence of lncRNA in extracellular fluids makes them promising diagnostic markers. LncRNAs deploy higher-order structures to facilitate a complex range of functions. Among such structures, G-quadruplexes (G4s) can be detected or targeted by small molecular probes to drive theranostic applications. The in vitro identification of G4 formation in lncRNAs can be a tedious and expensive proposition. Bioinformatics-driven strategies can provide comprehensive and economic alternatives in conjunction with suitable experimental validation. We propose a pipeline to identify G4-forming sequences, protein partners and biological functions associated with dysregulated lncRNAs in cervical cancer. We identified 17 lncRNA clusters which possess transcripts that can fold into a G4 structure. We confirmed in vitro G4 formation in the four biologically active isoforms of SNHG20, MEG3, CRNDE and LINP1 by Circular Dichroism spectroscopy and Thioflavin-T-assisted fluorescence spectroscopy and reverse-transcriptase stop assay. Gene expression data demonstrated that these four lncRNAs can be potential prognostic biomarkers of cervical cancer. Two approaches were employed for identifying G4 specific protein partners for these lncRNAs and FMR2 was a potential interacting partner for all four clusters. We report a detailed investigation of G4 formation in lncRNAs that are dysregulated in cervical cancer. LncRNAs MEG3, CRNDE, LINP1 and SNHG20 are shown to influence cervical cancer progression and we report G4 specific protein partners for these lncRNAs. The protein partners and G4s predicted in lncRNAs can be exploited for theranostic objectives.

Ammonia level influences the assembly of dissimilatory nitrate reduction to ammonia bacterial community in soils under different heavy metal remediation treatments

Heavy metal remediation treatments might influence functional microbial community assembly. Dissimilatory nitrate reduction to ammonia (DNRA) contributes to the nitrogen retention processes in soil ecosystems. We assumed that remediation might reduce heavy metal toxicity and increase some available nutrients for the DNRA microbes, thus balancing the deterministic and stochastic process for DNRA community assembly. Here, we investigated the process of DNRA bacterial community assembly under different heavy metal remediation treatments (including control, biochar, limestone, rice straw, rice straw + limestone, and biochar + limestone) in an Alfisol soil. The abundance of DNRA bacteria diverged across treatments. The α-diversity of the DNRA bacterial community was correlated with pH, available phosphorus (AP), ammonium (NH4+), and extractable Fe (EFe). Metal Cd and Fe significantly affected the abundance of the nrfA gene. The β-diversity was associated with pH, NH4+, and EFe. Deterministic processes dominantly drove the assembly processes of the DNRA bacterial community. NH4+ level played an essential role in the assembly processes than the other soil physicochemical properties and metal availability. High, moderate, and low levels of NH4+ could advocate stochastic process plus selection, heterogeneous selection to stochastic process, and heterogeneous selection, respectively. Network analysis highlighted a predominant role of NH4+ in regulating DNRA bacterial community assembly. However, the relative abundance of modules and some keystone species also were influenced by pH and EFe, respectively. Therefore, the DNRA bacterial community assembly under different heavy metal remediation treatments in this study was dominantly driven by nitrogen availability. pH, phosphorus, and metal availability were auxiliary regulators on DNRA bacterial community.

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

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.

The UzcRS two-component system in Caulobacter crescentus integrates regulatory input from diverse auxiliary regulators.

The UzcRS two-component system in Caulobacter crescentus mediates widespread transcriptional activation in response to the metals U, Zn and Cu. Unexpectedly, a screen for mutations that affected the activity of the UzcR-regulated urcA promoter (PurcA ) revealed four previously uncharacterized proteins whose inactivation led to metal-independent induction of PurcA . Using molecular genetics and functional genomics, we find that these auxiliary regulators control PurcA expression by modulating the activity of UzcRS through distinct mechanisms. An ABC transporter with a periplasmic metallo-aminopeptidase domain forms a sensory complex with UzcRS, antagonizing metal dependent stimulation by virtue of its ATPase and peptidase domains. Two MarR-like transcription factors synergistically regulate UzcRS activity by repressing the expression of the membrane proteins UzcY and UzcZ, which stimulate UzcRS activity and enhance its sensitivity to a more environmentally relevant U/Zn/Cu concentration range. Additionally, the membrane protein UzcX, whose expression is positively regulated by UzcR, provides a mechanism of feedback inhibition within the UzcRS circuit. Collectively, these data suggest that UzcRS functions as the core-signaling unit within a multicomponent signal transduction pathway that includes a diverse set of auxiliary regulators, providing further insight into the complexity of signaling networks.

Ca2+-Activated K+ Channel KCa3.1 as a Therapeutic Target for Immune Disorders.

In lymphoid and myeloid cells, membrane hyperpolarization by the opening of K+ channels increases the activity of Ca2+ release-activated Ca2+ (CRAC) channels and transient receptor potential (TRP) Ca2+ channels. The intermediate-conductance Ca2+-activated K+ channel KCa3.1 plays an important role in cell proliferation, differentiation, migration, and cytokine production in innate and adaptive immune systems. KCa3.1 is therefore an attractive therapeutic target for allergic, inflammatory, and autoimmune disorders. In the past several years, studies have provided new insights into 1) KCa3.1 pharmacology and its auxiliary regulators; 2) post-transcriptional and proteasomal regulation of KCa3.1; 3) KCa3.1 as a regulator of immune cell migration, cytokine production, and phenotypic polarization; 4) the role of KCa3.1 in the phosphorylation and nuclear translocation of Smad2/3; and 5) KCa3.1 as a therapeutic target for cancer immunotherapy. In this review, we have assembled a comprehensive overview of current research on the physiological and pathophysiological significance of KCa3.1 in the immune system.

Interaction of the RcsB Response Regulator with Auxiliary Transcription Regulators in Escherichia coli

The Rcs phosphorelay is a two-component signal transduction system that is induced by cell envelope stress. RcsB, the response regulator of this signaling system, is a pleiotropic transcription regulator, which is involved in the control of various stress responses, cell division, motility, and biofilm formation. RcsB regulates transcription either as a homodimer or together with auxiliary regulators, such as RcsA, BglJ, and GadE in Escherichia coli. In this study, we show that RcsB in addition forms heterodimers with MatA (also known as EcpR) and with DctR. Our data suggest that the MatA-dependent transcription regulation is mediated by the MatA-RcsB heterodimer and is independent of RcsB phosphorylation. Furthermore, we analyzed the relevance of amino acid residues of the active quintet of conserved residues, and of surface-exposed residues for activity of RcsB. The data suggest that the activity of the phosphorylation-dependent dimers, such as RcsA-RcsB and RcsB-RcsB, is affected by mutation of residues in the vicinity of the phosphorylation site, suggesting that a phosphorylation-induced structural change modulates their activity. In contrast, the phosphorylation-independent heterodimers BglJ-RcsB and MatA-RcsB are affected by only very few mutations. Heterodimerization of RcsB with various auxiliary regulators and their differential dependence on phosphorylation add an additional level of control to the Rcs system that is operating at the output level.

Transcriptional regulation by BglJ–RcsB, a pleiotropic heteromeric activator in Escherichia coli

The bacterial Rcs phosphorelay signals perturbations of the bacterial cell envelope to its response regulator RcsB, which regulates transcription of multiple loci related to motility, biofilm formation and various stress responses. RcsB is unique, as its set of target loci is modulated by interaction with auxiliary regulators including BglJ. The BglJ–RcsB heteromer is known to activate the HNS repressed leuO and bgl loci independent of RcsB phosphorylation. Here, we show that BglJ–RcsB activates the promoters of 10 additional loci (chiA, molR, sfsB, yecT, yqhG, ygiZ, yidL, ykiA, ynbA and ynjI). Furthermore, we mapped the BglJ–RcsB binding site at seven loci and propose a consensus sequence motif. The data suggest that activation by BglJ–RcsB is DNA phasing dependent at some loci, a feature reminiscent of canonical transcriptional activators, while at other loci BglJ–RcsB activation may be indirect by inhibition of HNS-mediated repression. In addition, we show that BglJ–RcsB activates transcription of bgl synergistically with CRP where it shifts the transcription start by 20 bp from a position typical for class I CRP-dependent promoters to a position typical for class II CRP-dependent promoters. Thus, BglJ–RcsB is a pleiotropic transcriptional activator that coordinates regulation with global regulators including CRP, LeuO and HNS.

Three (and more) component regulatory systems – auxiliary regulators of bacterial histidine kinases

Two-component signal transduction (TCST) is the most prevalent mechanism employed by microbes to sense and respond to environmental changes. It is characterized by the signal-induced transfer of phosphate from a sensor histidine kinase (HK) to a response regulator (RR), resulting in a cellular response. An emerging theme in the field of TCST signalling is the discovery of auxiliary factors, distinct from the HK and RR, which are capable of influencing phosphotransfer. One group of TCST auxiliary proteins accomplishes this task by acting on HKs. Auxiliary regulators of HKs are widespread and have been identified in all cellular compartments, where they can influence HK activity through interactions with the sensing, transmembrane or enzymatic domains of the HK. The effects of an auxiliary regulator are controlled by its regulated expression, modification and/or through ligand binding. Ultimately, auxiliary regulators can connect a given TCST system to other regulatory networks in the cell or result in regulation of the TCST system in response to an expanded range of stimuli. The studies highlighted in this review draw attention to an emerging view of bacterial TCST systems as core signalling units upon which auxiliary factors act.

STKE Focus Issue on GPCRs: The Evolution of G Protein-Coupled Receptor Signaling

G protein-coupled receptors (GPCRs) are one of the largest families of signaling proteins and are characterized by the presence of seven transmembrane domains and their ability to activate the heterotrimeric guanosine triphosphate (GTP) binding proteins (G proteins). Through the coupling to G proteins, GPCRs can mediate many different cellular effects and regulate processes as different as mitogenesis, cell movement, and cellular excitability. The original dogma about GPCR signaling was a fairly simple straightforward model in which each individual GPCR activated a single class of G protein. Furthermore, the G proteins had a high intrinsic GTPase activity and were initially believed not to utilize auxiliary regulators like those identified for the small GTPases. However, over time, the GPCR signaling field has gained in complexity, with GPCRs interacting with one another and other plasma membrane receptors, individual receptors coupling to more than one type of G protein, and the G proteins being regulated by GTPase activating proteins (GAPs). The STKE has information relevant to many aspects of G protein signaling and includes Reviews, Perspectives, Protocols, Connections Maps, relevant meetings in the Events section of the site, and other Web sites in the ST on the Web section of the site. If you are looking for others interested in G protein signaling, there are many people and labs in the Directory that list GPCRs as a focus of their research. Three new articles this issue highlight some of the changing views in the GPCR field. The ability and consequences of GPCRs coupling to more than one G protein is the subject of the review by Xiao ( ). GPCR multimerization is the topic of the perspective by Thelen and Baggiolini ([http://stke.sciencemag.org/cgi/content/full/sigtrans;2001/104/pe3][1]). The role of regulators of G protein signaling (RGS) in connecting GPCRs to other receptors and membrane proteins is the topic of the review by Druey ( ). Xiao focuses on the coupling of β-adrenergic receptors in the heart and the consequences of the dual coupling of the β2 subtype to both Gi and Gs pathways in terms of signal compartmentalization and cell survival signaling. These effects have important physiological ramifications when considering the underlying pathologies associated with cardiac heart failure and therapeutic approaches for treatment. GPCRs can oligomerize with other GPCR or with other types of receptors, such as ligand-gated ion channels. The direct interaction between GPCRs and ligand-gated ion channels is the topic of a perspective from the STKE archive by Brandon and Moss ( ). In this issue, Thelen and Bagglioni critically evaluate whether chemokine receptors heterodimerize or homodimerize and whether the multimerization is physiologically important. Druey focuses on the G proteins themselves. RGS proteins enhance the rate of GTPase activity of the Gα subunit. RGS proteins have additional protein-protein interaction motifs through which they couple G protein signaling to other membrane proteins, such as N-type calcium channels, receptor tyrosine kinases, and ephrin B proteins. For those interested in understanding the basics of GPCR signaling, the STKE has three Connections Maps by Pathway Authority R. Iyengar and members of his laboratory. These are Canonical Pathways that describe the signaling cascades initiated by Gs ([http://stke.sciencemag.org/cgi/cm/CMP_6634][2]), Gq ([http://stke.sciencemag.org/cgi/cm/CMP_6680][2]), and Gi ([http://stke.sciencemag.org/cgi/cm/CMP_7430][2]). The pathway highlighted in this issue, the Gαi Pathway ([http://stke.sciencemag.org/cgi/cm/CMP_7430][2]), describes the signaling from both the activated α subunits and the βγ subunits. Featured in This Focus Issue on GPCRs Related Resources at STKE [1]: http://stke.sciencemag.org/cgi/content/full/sigtrans;2001/104/pe4 [2]: pending:yes

The control of asymmetric gene expression during Caulobacter cell differentiation.

The dimorphic bacterium Caulobacter crescentus provides a simple model for cellular differentiation. Each cell division produces two distinct cell types: a swarmer cell and a stalked cell. These cells possess distinct functional morphologies and differential programs of transcription and DNA replication. The synthesis of a single polar flagellum is restricted to the swarmer pole of the predivisional cell by a genetic hierarchy comprising at least 50 genes whose transcription is regulated by novel and ubiquitous promoters, cognate sigma factors, and auxiliary transcriptional regulators. Chromosome replication is restricted to the stalked cell by a unique chromosome origin of replication that may be regulated by a novel cell-specific transcriptional control system. Phosphorylation signals, DNA methylation, differential chromosome structures, protein targeting, and selective protein degradation are also involved in establishing and maintaining cellular asymmetry. The molecular details of these universal cellular processes in C. crescentus will provide paradigms applicable to many general aspects of cellular differentiation.

Chiral and conformal anomalies in lattice gauge theory

The continuum limit of the chiral and conformal (Weyl) Ward-Takahashi identities in the lattice Wilson action is studied. The Wilson term works for the chiral anomaly, but it gives rise to-15 times the conventional conformal anomaly for a smallr-parameter and a very sensitiver-dependence of the Λ-parameter. This shows that the strong symmetry breaking by the Wilson term by itself does not necessarily generate correct anomalies. In the lattice regularization the functional Jacobian factors becomec-numbers and do not contribute to anomalies, corresponding to the cut-off of short distance components; the naive continuum limit of lattice WT identities can thus behave differently from continuum ones. To reconstruct conventional identities from lattice relations, the lattice composite operators should be rewritten in terms of relevant continuum operators. In general, this identification of relevant operators is facilitated either by the procedure corresponding to Zimmermann's normal product algorithm or simply by the use of auxiliary regulators such as the dimensional regulator.