Gotowa bibliografia na temat „Intramolecular signal transduction”

Dynamics and functions of the peroxisome proliferator-activated receptor (PPAR)-α are modulated by the types of ligands that bind to the orthosteric sites. While several X-ray crystal structures of PPAR-α have been determined in their agonist-bound forms, detailed structural information in their apo and antagonist-bound states are still lacking. To address these limitations, we apply unbiased molecular dynamics simulations to three different PPAR-α systems to determine their modulatory mechanisms. Herein, we performed hydrogen bond and essential dynamics analyses to identify the important residues involved in polar interactions and conformational structural variations, respectively. Furthermore, betweenness centrality network analysis was carried out to identify key residues for intramolecular signaling. The differences observed in the intramolecular signal flow between apo, agonist- and antagonist-bound forms of PPAR-α will be useful for calculating maps of information flow and identifying key residues crucial for signal transductions. The predictions derived from our analysis will be of great help to medicinal chemists in the design of effective PPAR-α modulators and additionally in understanding their regulation and signal transductions.

Heat shock proteins (HSP) are essential for intracellular protein folding during stress and protect cells from denaturation and aggregation cascades that can lead to cell death. HSP genes are regulated at the transcriptional level by heat shock transcription factor 1 (HSF1) that is activated by stress and binds to heat shock elements in HSP genes. The activation of HSF1 during heat shock involves conversion from an inert monomer to a DNA binding trimer through a series of intramolecular folding rearrangements. However, the trigger for HSF1 at the molecular level is unclear and hypotheses for this process include reversal of feedback inhibition of HSF1 by molecular chaperones and heat-induced binding to large non-coding RNAs. Heat shock also causes a profound modulation in cell signaling pathways that lead to protein kinase activation and phosphorylation of HSF1 at a number of regulatory serine residues. HSP genes themselves exist in an accessible chromatin conformation already bound to RNA polymerase II. The RNA polymerase II is paused on HSP promoters after transcribing a short RNA sequence proximal to the promoter. Activation by heat shock involves HSF1 binding to the promoter and release of the paused RNA polymerase II followed by further rounds of transcriptional initiation and elongation. HSF1 is thus involved in both initiation and elongation of HSP RNA transcripts. Recent studies indicate important roles for histone modifications on HSP genes during heat shock. Histone modification occurs rapidly after stress and may be involved in promoting nucleosome remodeling on HSP promoters and in the open reading frames of HSP genes. Understanding these processes may be key to evaluating mechanisms of deregulated HSP expression that plays a key role in neurodegeneration and cancer.

Five ferrocene alkymethylimidazolium cations 1a–1d and 2 with different alkyl spacer lengths were reinvestigated using voltammetry and density functional theory (DFT) calculations. The voltammetric responses of ligand 2 toward various anions are described in detail. An interesting and unprecedented finding from both experimental and theoretical studies is that coupled electron and intramolecular anion (F−) transfer may be present in these molecules. In addition, it was also observed that, in these studied molecules, the electrostatic attraction interaction toward F− would effectively vanish beyond 1 nm, which was previously reported only for cations.

Objective: The molecular mechanisms of TSH receptor (TSHR) activation and intramolecular signal transduction are largely unknown. Deletion of the extracellular domain (ECD) of the TSHR results in increased constitutive activity, which suggests a self-inhibitory interaction between the ECD and the extracellular loops (ECLs) or the transmembrane domains (TMDs). To investigate these potential interactions and to pursue the idea that mutations in the ECD affect the constitutive activity of mutants in the ECLs or TMDs we generated double mutants between position 281 in the ECD and mutants in all three ECLs as well as the 6th TMD. Design: We combined mutation S281D, characterized by an impaired TSH-stimulated cAMP response, with the constitutively activating in vivo mutations I486F (1st ECL), I568T (2nd ECL), V656F (3rd ECL) and D633F (6th TMD). Further, we constructed double mutants containing the constitutively activating mutation S281N and one of the inactivating mutations D474E, T477I (1st ECL) and D633K (6th TMD). Results: The cAMP level of the double mutants with S281N and the inactive mutants in the 1st ECL was decreased below the level of the inactive single mutants, demonstrating that a constitutively activating mutation in the ECD cannot bypass disruption of signal transduction in the serpentine domain. In double mutants with S281D, basal and TSH-induced cAMP and inositol phosphate production of constitutively active mutants was reduced to the level of S281D. Conclusion: The dominance of S281D and the dependence of constitutively activating mutations in the ECLs on the functionally intact ECD strongly suggest that interactions between these receptor domains are required for TSHR activation and intramolecular signal transduction.

Glutamate dehydrogenase (GDH) is a ubiquitous enzyme that catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate. It acts as an important branch-point enzyme between carbon and nitrogen metabolisms. Due to the multifaceted roles of GDH in cancer, hyperinsulinism/hyperammonemia, and central nervous system development and pathologies, tight control of its activity is necessitated. To date, several GDH structures have been solved in its closed form; however, intrinsic structural information in its open and apo forms are still deficient. Moreover, the allosteric communications and conformational changes taking place in the three different GDH states are not well studied. To mitigate these drawbacks, we applied unbiased molecular dynamic simulations (MD) and network analysis to three different GDH states i.e., apo, active, and inactive forms, for investigating their modulatory mechanisms. In this paper, based on MD and network analysis, crucial residues important for signal transduction, conformational changes, and maps of information flow among the different GDH states were elucidated. Moreover, with the recent findings of allosteric modulators, an allosteric wiring illustration of GDH intramolecular signal transductions would be of paramount importance to obtain the process of this enzyme regulation. The structural insights gained from this study will pave way for large-scale screening of GDH regulators and could support researchers in the design and development of new and potent GDH ligands.

Protein homeostasis in all organisms is a complex process involving regulatory mechanisms that govern protein synthesis, post-translational events and degradation. The protein degradation mechanism serves multiple functions ranging from maturation of cellular proteins, protein recycling and intracellular signal transduction. This mechanism, therefore, has a major role in diverse cellular and developmental contexts. In prokaryotes, the protein degradation mechanism has been shown to influence the cellular response to environmental stimuli and thus pathogenesis and virulence. The prokaryotic protein degradation machinery involves both regulated and processive proteases. Of these, the regulated proteases are particularly important as controlled and specific proteolysis are often key events in a signal transduction cascade triggered by intracellular or extracellular stimuli. The studies described in this thesis were designed to examine a specific aspect of regulated proteases wherein proteolytic activity could be either triggered or inhibited by a regulatory domain. These studies involve three regulatory proteases belonging to the High temperature requirement A (HtrA) family in Mycobacterium tuberculosis. These M. tuberculosis HtrA paralogues share common structural features with the well-characterized regulated protease E. coli DegS. HtrA proteases, in general, have a serine protease domain that is flexibly tethered to one or more PDZ (Post synaptic density protein-95; Drosophila disc-large tumor suppressor; Zonula occludens-1) domains at the C-terminus. HtrA proteases are membrane tethered. The membrane localization has been suggested to be significant from a functional perspective. The common features noted from multiple HtrA homologous characterized thus far include changes in quaternary structure and typically two distinct conformational states of catalytic triad (referred interchangeably as the inactive or tense state and the active or relaxed state). Tightly regulated proteolytic activity is thus achieved by an enzyme that is primarily in an inactive conformation and adopts an active conformation upon a specific trigger. Characterized triggers for HtrA activity are environmental triggers (such as high temperature), peptides or other effector ligands or substrate proteins. The modular organization of HtrA enzymes relies on the interactions between the sensory (tethered PDZ) domain with an effector ligand. Information from the PDZ domain is conveyed by concerted conformational changes to activate the catalytic domain. The intramolecular signal transduction paths thus play a key role in dictating the regulatory mechanism in HtrA enzymes. This thesis describes the structural features of M. tuberculosis HtrA and characterization of its biochemical features and regulatory mechanism(s). The first chapter of this thesis provides a brief introduction to regulated proteolytic mechanisms. Similarities and broad differences between the eukaryotic and prokaryotic mechanisms are discussed to phrase the research problem in the context of reported studies. The extensive studies performed on this enzyme, especially E. coli DegS, are summarized to place the known features and observations in the context of M. tuberculosis homologues. The characterized features of the M. tuberculosis HtrA paralogues are summarized to describe the scope of the studies reported in this thesis. A brief introduction to the diverse techniques employed in the course of these studies is also provided to place the methodology in context. The detailed methodology is included in different chapters alongside experimental information. Chapter two describes the crystal structure of M. tuberculosis HtrA at 1.83 Å resolution. This membrane-associated protease is essential for the survival of M. tuberculosis. The crystal structure revealed that the catalytic triad in HtrA is in an inactive conformation. This finding is consistent with the proposed role of M. tuberculosis HtrA as a regulatory protease that is conditionally activated upon appropriate environmental triggers. This structure provided a basis for directed studies to evaluate the role of this essential protein and the intracellular signal transduction mechanism that governs the activity of this protease. The intramolecular signal transduction process that is essential for regulated enzyme activity in HtrA proteases is described in chapter three. The first part of the study involves computational analysis and molecular dynamics (MD) simulations. This work is based on several ‘seed structures’ including those of E. coli DegS in peptide-bound and free forms, M. tuberculosis HtrA2 (PepD) in peptide-bound and apo forms and M. tuberculosis HtrA (HtrA1). These simulations provided multiple insights. The first was that of conformational sampling wherein the switch between the inactive (tense) arrangement of catalytic triad and the active (relaxed) arrangement of catalytic triad could be visualized. This finding, in effect, suggested that concerted conformational changes between the site in the PDZ domain that binds the effector ligand (peptide or any substrate) and the active site were essential for regulated proteolysis. While this aspect has long been discussed, the paths for intramolecular signal transduction remain poorly understood. In this study, we adopted a methodology involving difference energy calculations between the peptide bound and free forms of HtrA homologous. The emphasis in this context was on electrostatic interactions. Difference energy calculations, in turn, provided sites on the enzyme that could serve as nodes in the intramolecular signal transduction pathways. This analysis suggested multiple paths between the PDZ and the protease domain that could potentially be adopted for signal transduction. Three representative intramolecular signal transduction paths were experimentally validated by mutational analysis. Together, the computational studies and experimental observations provided a framework to understand intramolecular signal transduction paths that serve to transmit regulatory information. Chaperone activity in HtrA enzymes was suggested to be associated with the ability of these enzymes to adopt large oligomeric assemblies. The oligomeric assemblies of M. tuberculosis HtrA paralogues were evaluated in the presence of generic substrates such as β-casein or denatured lysozyme. These studies are described in chapter four of this thesis. Size exclusion chromatography suggested that M. tuberculosis HtrA (also referred to as HtrA1) and PepD (also referred to as HtrA2) existed in hexameric, trimeric and monomeric states. PepA (HtrA3) was prominently a trimer in solution. An important experimental insight on the differences between these paralogues came from an assay to evaluate the ability of these enzymes to inhibit protein aggregation. These assays suggested that PepD (HtrA2) was perhaps the best suited amongst three paralogues to serve this function. Small angle X-ray scattering methods provided information on the oligomeric assembly. While these were not conclusive due to experimental limitations, these studies suggested that higher order oligomers (such as dodecamer or di-dodecamer in E. coli DegP and DegQ) were not likely in the case of the M. tuberculosis paralogues. We note that conformational heterogeneity is positively correlated with chaperone activity as both HtrA and PepD adopt multiple oligomeric states. PepA (HtrA3), on the other hand, is the most homogeneous (trimer) and also the least effective in inhibiting protein aggregation. A broad summary of the findings from studies on regulated proteases is described in chapter five. The observations on the allosteric pathways also provide an insight into the mechanism of intramolecular signal transduction. These findings are likely to be useful from an enzyme engineering perspective wherein catalytic activity can be specifically triggered by effector ligand binding to the PDZ domain. Future studies based on the work reported in this thesis include identification of cognate triggers for the three M. tuberculosis HtrA paralogues. Together, the finding that the human pathogen M. tuberculosis has three paralogues of HtrA with diverse functional roles thus exemplifies how a functional diversity is embedded within a conserved structural scaffold. This thesis has four annexures. Annexure-I summarizes the experimental details and strategies that could not be incorporated in the main text of this thesis. Annexure-II enlisted the pairs of residues having difference electrostatic energy. Annexure-III describes a collaborative study performed on the M. tuberculosis σ factor σJ. Annexure-IV describes a collaborative project on the E. coli Arginine transporter ArgO.