Biophysical and functional characterization of N-terminal domain of Human Interferon Regulatory Factor 6.

BACKGROUND: Interferon regulatory factor 6 (IRF6) has a key function in palate fusion during palatogenesis during embryonic development, and mutations in IRF6 cause orofacial clefting disorders. METHODS AND RESULTS: The in silico analysis of IRF6 is done to obtain leads for the domain boundaries and subsequently the sub-cloning of the N-terminal domain of IRF6 into the pGEX-2TK expression vector and successfully optimized the overexpression and purification of recombinant glutathione S-transferase-fused NTD-IRF6 protein under native conditions. After cleavage of the GST tag, NTD-IRF6 was subjected to protein folding studies employing Circular Dichroism and Intrinsic fluorescence spectroscopy at variable pH, temperature, and denaturant. CD studies showed predominantly alpha-helical content and the highest stability of NTD-IRF6 at pH 9.0. A comparison of native and renatured protein depicts loss in the secondary structural content. Intrinsic fluorescence and quenching studies have identified that tryptophan residues are majorly present in the buried areas of the protein and a small fraction was on or near the protein surface. Upon the protein unfolding with a higher concentration of denaturant urea, the peak of fluorescence intensity decreased and red shifted, confirming that tryptophan residues are majorly present in a more polar environment. While regulating IFNß gene expression during viral infection, the N-terminal domain binds to the promoter region of Virus Response Element-Interferon beta (VRE-IFNß). Along with the protein folding analysis, this study also aimed to identify the DNA-binding activity and determine the binding affinities of NTD-IRF6 with the VRE-IFNß promoter region. The protein-DNA interaction is specific as demonstrated by gel retardation assay and the kinetics of molecular interactions as quantified by Biolayer Interferometry showed a strong affinity with an affinity constant (KD) value of 7.96 × 10-10 M. CONCLUSION: NTD-IRF6 consists of a mix of α-helix and ß-sheets that show temperature-dependent cooperative unfolding between 40 °C and 55 °C. Urea-induced unfolding shows moderate tolerance to urea as the mid-transition concentration of urea (Cm) is 3.2 M. The tryptophan residues are majorly buried as depicted by fluorescence quenching studies. NTD-IRF6 has a specific and high affinity toward the promoter region of VRE-IFNß.

Small molecules that disrupt RAD54-BLM interaction hamper tumor proliferation in colon cancer chemoresistance models.

RAD54 and BLM helicase play pivotal roles during homologous recombination repair (HRR) to ensure genome maintenance. BLM amino acids (aa 181-212) interact with RAD54 and enhance its chromatin remodeling activity. Functionally, this interaction heightens HRR, leading to a decrease in residual DNA damage in colon cancer cells. This contributes to chemoresistance in colon cancer cells against cisplatin, camptothecin, and oxaliplatin, eventually promoting tumorigenesis in preclinical colon cancer mouse models. ChIP-Seq analysis and validation revealed increased BLM and RAD54 corecruitment on the MRP2 promoter in camptothecin-resistant colon cancer cells, leading to BLM-dependent enhancement of RAD54-mediated chromatin remodeling. We screened the Prestwick small-molecule library, with the intent to revert camptothecin- and oxaliplatin-induced chemoresistance by disrupting the RAD54-BLM interaction. Three FDA/European Medicines Agency-approved candidates were identified that could disrupt this interaction. These drugs bound to RAD54, altered its conformation, and abrogated RAD54-BLM-dependent chromatin remodeling on G5E4 and MRP2 arrays. Notably, the small molecules also reduced HRR efficiency in resistant lines, diminished anchorage-independent growth, and hampered the proliferation of tumors generated using camptothecin- and oxaliplatin-resistant colon cancer cells in both xenograft and syngeneic mouse models in BLM-dependent manner. Therefore, the 3 identified small molecules can serve as possible viable candidates for adjunct therapy in colon cancer treatment.

Identification of potential novel inhibitors against the SARS-CoV-2 spike protein: targeting RBD and ACE2 interaction.

The SARS-CoV-2, responsible for the COVID-19 pandemic has wrecked devastation throughout the globe. The SARS-CoV-2 spike (S) glycoprotein plays crucial role in virus attachment, fusion, and entry. This study aims to identify inhibitors targeting the receptor binding domain (RBD) of the S protein using computational and experimental techniques. We carried out virtual screening of four datasets against the S-RBD. Six potential candidate inhibitors were selected for experimental evaluation. Here, we provide experimental evidence that the molecules 9‴-MethyllithosperMate, Epimedin A, Pentagalloylglucose, and Theaflavin-3-gallate have a high binding affinity towards SARS-CoV-2 S-RBD. 9‴-MethyllithosperMate with a KD value of 1.3 nM serves as the best inhibitor, followed by others with KD values in micromolar range. We performed molecular dynamics simulation to assess the binding stability of these inhibitors. Hence, our study reports novel inhibitors against the SARS-CoV-2 S-RBD and their predicted binding mode also suggest the possibility to interfere with the ACE2 binding.Communicated by Ramaswamy H. Sarma.

Insight into the Conformational Transitions of Serine Acetyl Transferase Isoforms in E. histolytica: Implications for Structural and Functional Balance.

Serine acetyl transferase (SAT) is one of the crucial enzymes in the cysteine biosynthetic pathway and an essential enzyme for the survival of Entamoeba histolytica, the causative agent of amoebiasis. E. histolytica expresses three isoforms of SAT, where SAT1 and SAT2 are inhibited by the final product cysteine, while SAT3 is not inhibited. SAT3 has a slightly elongated C-terminus compared to SAT1. To understand the stability and conformational transition between two secondary structures of proteins, we measured the effect of urea, a chemical denaturant, on two isoforms of SAT (SAT1 and SAT3) of E. histolytica. The effect of urea on the structure and stability of SAT1 and SAT3 was determined by measuring changes in their far-UV circular dichroism (CD), Trp fluorescence, and near-UV absorption spectra. The urea-induced normal transition curves suggested that the structural transition is reversible and follows a two-state process. Analysis of the urea-induced transition of all optical properties for the stability parameters ΔG D° (Gibbs free energy change (ΔG D) in the absence of urea), m (dependence of ΔG D on urea concentration), and C m (midpoint of urea transition) suggested that SAT1 is more stable than SAT3. Characterization of the end product of the urea-induced transition of both proteins by the far-UV CD and Trp-fluorescence and near-UV absorbance suggested that urea causes α-helix to ß-sheet transition and burial of Trp residues, respectively. To support the in vitro findings, 100 ns molecular dynamics simulations (in silico study) were performed. Both the spectroscopic and molecular dynamics approaches clearly indicated that SAT1 is more stable than SAT3. SAT3 has evolved to escape the feedback inhibition to keep producing cysteine, but in the process, it compromises its structural stability relative to SAT1.

Structural and functional studies of serine acetyltransferase isoform from Entamoeba histolytica reveals novel role of the C-terminal tail in loss of regulation from feedback inhibition.

Serine acetyltransferase (SAT) catalyzes the acetylation of l-serine in the first step of the two-step pathway to synthesize L-cysteine in bacteria, protozoans and plants. L-cysteine is known to be involved in feedback regulation of SAT. However, in E. histolytica, SAT exists in three isoforms where third isoform SAT3 is nearly insensitive to feedback inhibition. Here, we explored the previously unknown precise mechanism of the insensitivity of EhSAT3 to L-cysteine. The C-terminal deletion mutants of EhSAT3 were inhibited completely by L-cysteine in contrast to the wildtype EhSAT3. The crystal structure of EhSAT3ΔC22 in complex with cysteine revealed that C-terminal region swaps over the neighboring monomer in the trimer. This structure combined with the modeled C-terminal residues suggests that EhSAT3 C-terminal end interacts with the active site and play crucial role in feedback inhibition. The interacting distances between sulfur of cysteine and protein indicate cysteine is in deprotonated (S-) state, thus making stronger interactions than serine. In the full length SAT3, C-terminal tail provides an acidic environment at the active site pocket, so that cysteine can't be deprotonated and bind strongly at the active site. These results conveyed a unique role of the C-terminal region of EhSAT3 in regulating the feedback inhibition.

Unravelling the Biology of EhActo as the First Cofilin From Entamoeba histolytica.

Actin-depolymerising factors (ADF) are a known family of proteins that regulate actin dynamics. Actin regulation is critical for primitive eukaryotes since it drives their key cellular processes. Entamoeba histolytica, a protist human pathogen harbours eleven proteins within this family, however, with no actin depolymerising protein reported to date. We present here the NMR model of EhActo, the first Cofilin from E. histolytica that severs actin filaments and also participates in cellular events like phagocytosis and pseudopod formation. The model typically represents the ADF-homology domain compared to other cofilins. Uniquely, EhActo lacks the critical Serine3 residue present in all known actophorins mediating its phospho-regulation. The second mode of regulation that cofilin's are subjected to is via their interaction with 14-3-3 proteins through the phosphorylated Serine residue and a consensus binding motif. We found a unique interaction between EhActo and 14-3-3 without the presence of the consensus motif or the phosphorylated Serine. These interesting results present unexplored newer mechanisms functional in this pathogen to regulate actophorin. Through our structural and biochemical studies we have deciphered the mechanism of action of EhActo, implicating its role in amoebic biology.

Cdc42/Rac Interactive Binding Containing Effector Proteins in Unicellular Protozoans With Reference to Human Host: Locks of the Rho Signaling.

Small GTPases are the key to actin cytoskeleton signaling, which opens the lock of effector proteins to forward the signal downstream in several cellular pathways. Actin cytoskeleton assembly is associated with cell polarity, adhesion, movement and other functions in eukaryotic cells. Rho proteins, specifically Cdc42 and Rac, are the primary regulators of actin cytoskeleton dynamics in higher and lower eukaryotes. Effector proteins, present in an inactive state gets activated after binding to the GTP bound Cdc42/Rac to relay a signal downstream. Cdc42/Rac interactive binding (CRIB) motif is an essential conserved sequence found in effector proteins to interact with Cdc42 or Rac. A diverse range of Cdc42/Rac and their effector proteins have evolved from lower to higher eukaryotes. The present study has identified and further classified CRIB containing effector proteins in lower eukaryotes, focusing on parasitic protozoans causing neglected tropical diseases and taking human proteins as a reference point to the highest evolved organism in the evolutionary trait. Lower eukaryotes' CRIB containing proteins fall into conventional effector molecules, PAKs (p21 activated kinase), Wiskoit-Aldrich Syndrome proteins family, and some have unique domain combinations unlike any known proteins. We also highlight the correlation between the effector protein isoforms and their selective specificity for Cdc42 or Rac proteins during evolution. Here, we report CRIB containing effector proteins; ten in Dictyostelium and Entamoeba, fourteen in Acanthamoeba, one in Trypanosoma and Giardia. CRIB containing effector proteins that have been studied so far in humans are potential candidates for drug targets in cancer, neurological disorders, and others. Conventional CRIB containing proteins from protozoan parasites remain largely elusive and our data provides their identification and classification for further in-depth functional validations. The tropical diseases caused by protozoan parasites lack combinatorial drug targets as effective paradigms. Targeting signaling mechanisms operative in these pathogens can provide greater molecules in combatting their infections.

A unique aromatic cluster near the active site of H. pylori CPA is essential for catalytic function.

Polyamines are essential for cell growth and proliferation. In plants and many bacteria, including Helicobacter pylori, the parent polyamine putrescine is only produced through the metabolism of N-carbamoylputrescine by N-carbamoylputrescine amidase (CPA). Thus, CPA is a crucial intermediate enzyme. Moreover, the absence of CPA in humans makes its presence in H. pylori a potential target for the development of new therapeutics against this pathogen. Despite this enzyme's presence in plants and bacteria, its function is not completely explored. Using structure-guided biochemical and biophysical studies on H. pylori CPA, we discovered an aromatic cluster containing four conserved tryptophans near the catalytic site and elucidated its role. Mutational studies revealed that they are individually vital to enzyme function. Unlike wild-type, which forms a hexamer, the Trp to Ala mutants only formed dimers. Interestingly, two other conserved residues, Gln155 and Asp278, interact with the tryptophan cluster and perform similar roles. Our results indicate that aromatic-aromatic and H-bonding contacts between the residues (Trp156-Trp273, Trp196-Gln155, and Trp153-Asp278) play a crucial role in stimulating activity through hexamer formation. Additionally, Trp156 is essential to generating a catalytically efficient hexamer. These results suggest dual roles for the tryptophans; in hexamer formation and in generating its functionally active form, thereby providing a mechanistic understanding into the role of the cluster. We also elucidated the catalytic roles of Glu43, Lys115, and Cys152, which are present at the active site. Our findings highlight, for the first time, the importance of a tryptophan cluster in H. pylori CPA that can be exploited to design therapeutic inhibitors.

Functional implications of residues of the B' motif of geminivirus replication initiator protein in its helicase activity.

Geminivirus replication initiator protein (Rep) is a multifunctional viral protein required for replication. During the process of viral replication, Rep acts as a site- and strand-specific endonuclease, ligase, ATPase, and helicase. B' motif and ß-hairpin loop of the geminivirus Rep are conserved and important for Rep-mediated helicase activity required for viral replication. To dissect the roles of various amino acid residues of the B' motif and ß-hairpin loop of the geminivirus Rep helicase in its process of unwinding DNA, we investigated eight conserved residues near the ATP active site or the ssDNA contact channel. Our strategy was to mutate these residues to alanines and investigate the effects of these mutations on various biochemical activities associated with DNA unwinding. We looked into the ATP binding, ATP hydrolysis, DNA binding, and DNA unwinding activities of the wild-type and mutant Rep proteins. These investigations showed four residues (Arg279, Asp280, Tyr287, and Pro290) affecting the DNA unwinding activity. A structural model analysis confirmed the B' loop and ssDNA binding loop to be connected through a ß-hairpin structure, suggesting that changes on one loop might affect the other and that these residues function by acting in concert. Viral genomes containing Rep proteins having these mutations in the B' motif did not replicate in planta. Taken together, these results indicated all four residues to be implicated in helicase activity mediated by Rep and demonstrated the significance, for viral replication, of the B' motif and ß-hairpin loop of the C-terminal region of the Rep protein.

Inhibiting Pyridoxal Kinase of Entamoeba histolytica Is Lethal for This Pathogen.

Pyridoxal 5'-phosphate (PLP) functions as a cofactor for hundreds of different enzymes that are crucial to the survival of microorganisms. PLP-dependent enzymes have been extensively characterized and proposed as drug targets in Entamoeba histolytica. This pathogen is unable to synthesize vitamin B6via de-novo pathway and relies on the uptake of vitamin B6 vitamers from the host which are then phosphorylated by the enzyme pyridoxal kinase to produce PLP, the active form of vitamin B6. Previous studies from our lab shows that EhPLK is essential for the survival and growth of this protozoan parasite and its active site differs significantly with respect to its human homologue making it a potential drug target. In-silico screening of EhPLK against small molecule libraries were performed and top five ranked molecules were shortlisted on the basis of docking scores. These compounds dock into the PLP binding site of the enzyme such that binding of these compounds hinders the binding of substrate. Of these five compounds, two compounds showed inhibitory activity with IC50 values between 100-250 µM when tested in-vitro. The effect of these compounds proved to be extremely lethal for Entamoeba trophozoites in cultured cells as the growth was hampered by 91.5% and 89.5% when grown in the presence of these compounds over the period of 72 hours.

Redox homeostasis in Mycobacterium tuberculosis is modulated by a novel actinomycete-specific transcription factor.

Mycobacterium tuberculosis (Mtb) has evolved diverse cellular processes in response to the multiple stresses it encounters within the infected host. We explored available TnSeq datasets to identify transcription factors (TFs) that are essential for Mtb survival inside the host. The analysis identified a single TF, Rv1332 (AosR), conserved across actinomycetes with a so-far uncharacterized function. AosR mitigates phagocyte-derived oxidative and nitrosative stress, thus promoting mycobacterial growth in the murine lungs and spleen. Oxidative stress induces formation of a single intrasubunit disulphide bond in AosR, which in turn facilitates AosR interaction with an extracytoplasmic-function sigma factor, SigH. This leads to the specific upregulation of the CysM-dependent non-canonical cysteine biosynthesis pathway through an auxiliary intragenic stress-responsive promoter, an axis critical in detoxifying host-derived oxidative and nitrosative radicals. Failure to upregulate AosR-dependent cysteine biosynthesis during the redox stress causes differential expression of 6% of Mtb genes. Our study shows that the AosR-SigH pathway is critical for detoxifying host-derived oxidative and nitrosative radicals to enhance Mtb survival in the hostile intracellular environment.

Structure-based inhibitor screening of natural products against NSP15 of SARS-CoV-2 revealed thymopentin and oleuropein as potent inhibitors.

Coronaviruses are enveloped, non-segmented positive-sense RNA viruses with the largest genome among RNA viruses. Their genome contains a large replicase ORF which encodes nonstructural proteins (NSPs), structural, and accessory genes. NSP15 is a nidoviral RNA uridylate-specific endoribonuclease (NendoU) with C-terminal catalytic domain. The endoribonuclease activity of NSP15 interferes with the innate immune response of the host. Here, we screened Selleckchem Natural product database of the compounds against NSP15, and we found that thymopentin and oleuropein displayed highest binding energies. The binding of these molecules was further validated by molecular dynamic simulations that revealed them as very stable complexes. These drugs might serve as effective counter molecules in the reduction of virulence of this virus; may be more effective if treated in combination with replicase inhibitors. Future validation of both these inhibitors is worth the consideration for patients being treated for COVID-19. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42485-021-00059-w.

Queuine Is a Nutritional Regulator of Entamoeba histolytica Response to Oxidative Stress and a Virulence Attenuator.

Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His, and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here, we describe the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in [Formula: see text] Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in the OS response, such as heat shock protein 70 (Hsp70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in [Formula: see text], parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.IMPORTANCEEntamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.

Structural analysis of EhPSP in complex with 3-phosphoglyceric acid from Entamoeba histolytica reveals a basis for its lack of phosphoglycerate mutase activity.

Entamoeba histolytica phosphoserine phosphatase (EhPSP), a regulatory enzyme in the serine biosynthetic pathway, is also a structural homolog of cofactor-dependent phosphoglycerate mutase (dPGM). However, despite sharing many of its catalytic residues with dPGM, EhPSP displays no significant mutase activity. In the current work, we determined a crystal structure of EhPSP in complex with 3-PGA to 2.5 Å resolution and observed striking differences between the orientation of 3-PGA bound to EhPSP and that to its other homologous structures. We also performed computational modeling and simulations of the intermediate 2,3-bisphosphoglyceric acid into the active site of EhPSP to better understand its mechanistic details. Based on these results and those of a similar study with the dPGMs from E. coli and B. pseudomallei, the affinity of EhPSP for 2,3-BPG was concluded to be lower than those of the other proteins. Moreover, a different set of 2,3-BPG interacting residues was observed in EhPSP compared to dPGMs, with all of the crucial interacting residues of dPGMs either missing or substituted with weakly interacting residues. This study has expanded our understanding, at the structural level, of the inability of EhPSP to catalyze the mutase reaction and has strengthened earlier conclusions indicating it to be a true phosphatase.

Phosphoserine Aminotransferase has Conserved Active Site from Microbes to Higher Eukaryotes with Minor Deviations.

Serine is ubiquitously synthesized in all living organisms from the glycolysis intermediate 3-phosphoglycerate (PGA) by phosphoserine biosynthetic pathway, consisting of three different enzymes, namely: 3-phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Any functional defect or mutation in these enzymes may cause deliberating conditions, such as colon cancer progression and chemoresistance in humans. Phosphoserine aminotransferase (PSAT) is the second enzyme in this pathway that converts phosphohydroxypyruvate (PHP) to O-phospho-L-serine (OPLS). Humans encode two isoforms of this enzyme: PSAT1 and PSAT2. PSAT1 exists as a functional dimer, where each protomer has a large and a small domain; each large domain contains a Lys residue that covalently binds PLP. The PLP-binding site of human PSAT1 and most of its active site residues are highly conserved in all known PSAT structures except for Cys-80. Interestingly, Two PSAT structures from different organisms show halide binding near their active site. While the human PSAT1 shows a water molecule at this site with different interacting residues, suggesting the inability of halide binding in the human enzyme. Analysis of the human PSAT1 structure showed a big patch of positive charge around the active site, in contrast to the bacterial PSATs. Compared to human PSAT1, the PSAT2 isoform lacks 46 residues at its C-terminal tail. This tail region is present at the opening of the active site as observed in the other PSAT structures. Further structural work on human PSAT2 may reveal the functional importance of these 46 residues.

An evolutionary non-conserved motif in Helicobacter pylori arginase mediates positioning of the loop containing the catalytic residue for catalysis.

The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (-153ESEEKAWQKLCSL165-) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122-Trp159-Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more significant, indicating they are crucial to function. Our data suggest that the Lys57-Glu155-Ser152 interaction influences the positioning of the loop containing the catalytic His133 so that this His can participate in catalysis, thereby providing a mechanistic understanding into the role of this motif in catalytic function. Lys57 was also found only in the arginases of other Helicobacter gastric pathogens. Based on the non-conserved motif, we found a new molecule, which specifically inhibits this enzyme. Thus, the present study not only provides a molecular basis into the role of this motif in function, but also offers an opportunity for the design of inhibitors with greater efficacy.

Nuclear receptor SHP dampens transcription function and abrogates mitotic chromatin association of PXR and ERα via intermolecular interactions.

Mitosis is a cellular process that produces two identical progenies. Genome-wide transcription is believed to be silenced during mitosis. However, some transcription factors have been reported to associate with the mitotic chromatin to uphold a role in 'gene-bookmarking'. Here, we investigated the dynamic role of nuclear receptor SHP during cell cycle, and observed intermolecular interactions with PXR and ERα. This was reflected in altered subcellular localization, transcription function and mitotic chromatin behavior of these receptors. Subsequently, by in silico and live cell imaging approaches we identified the minimal domain(s) and crucial amino-acid residues required for such receptor-receptor interactions. It was apparent that both PXR/ERα interact with SHP to translocate cytoplasmic RFP-tagged SHP into the nucleus. In addition, during mitosis SHP interacted with some of the key nuclear receptors, altering partners, as well as, its own relationship with mitotic chromatin. SHP displaced a major fraction of PXR and ERα from the mitotic chromatin while promoted its own weak association reflected in its binding. Since SHP lacks DBD this association is attributed to receptor-receptor interactions rather than SHP-DNA interactions. The abrogation of PXR and ERα from the mitotic chromatin by SHP implies potential implications in regulation of gene bookmarking events in cellular development. Overall, it is concluded that intermolecular interactions between SHP and partner PXR/ERα result in attenuation of target promoter activities. It is proposed that SHP may act as an indirect physiological regulator and functions in a hog-tie manner by displacing the interacting transcription factor from gene regulatory sites.

Uncovering the Cyclic AMP Signaling Pathway of the Protozoan Parasite Entamoeba histolytica and Understanding Its Role in Phagocytosis.

Second messenger signaling controls a surprisingly diverse range of processes in several eukaryotic pathogens. Molecular machinery and pathways involving these messengers thus hold tremendous opportunities for therapeutic interventions. Relative to Ca2+ signaling, the knowledge of a crucial second messenger cyclic AMP (cAMP) and its signaling pathway is very scant in the intestinal parasite Entamoeba histolytica. In the current study, mining the available genomic resources, we have for the first time identified the cAMP signal transduction pathway of E. histolytica. Three heptahelical proteins with variable G-protein-coupled receptor domains, heterotrimeric G-proteins (Gα, Gß, and Gγ subunits), soluble adenylyl cyclase, cyclase-associated protein, and enzyme carbonic anhydrase were identified in its genome. We could also identify several putative candidate genes for cAMP downstream effectors such as protein kinase A, A-kinase anchoring proteins, and exchange protein directly activated by the cAMP pathway. Using specific inhibitors against key identified targets, we could observe changes in the intracellular cAMP levels as well as defect in the rate of phagocytosis of red blood cells by the parasite E. histolytica. We thus strongly believe that characterization of some of these unexplored crucial signaling determinants will provide a paradigm shift in understanding the pathogenicity of this organism.

Characterization and functional insights into the Entamoeba histolytica pyridoxal kinase, an enzyme essential for its survival.

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6 and a cofactor for more than 140 enzymes. This coenzyme plays a pivotal role in catalysis of various enzymatic reactions that are critical for the survival of organisms. Entamoeba histolytica depends on the uptake of pyridoxal (PL), a B6 vitamer from the external environment which is then phosphorylated by pyridoxal kinase (EhPLK) to form PLP via the salvage pathway. E. histolytica cannot synthesise vitamin B6de-novo, and also lacks pyridoxine 5'-phosphate oxidase, a salvage pathway enzyme required to produce PLP from pyridoxine phosphate (PNP) and pyridoxamine phosphate (PMP). Analysing the importance of PLK in E. histolytica, we have determined the high-resolution crystal structures of the dimeric pyridoxal kinase in apo, ADP-bound, and PLP-bound states. These structures provided a snapshot of the transition state and help in understanding the reaction mechanism in greater detail. The EhPLK structure significantly differed from the human homologue at its PLP binding site, and the phylogenetic study also revealed its divergence from human PLK. Further, gene regulation of EhPLK using sense and antisense RNA showed that any change in optimal level is harmful to the pathogen. Biochemical and in vivo studies unveiled EhPLK to be essential for this pathogen, while the molecular differences with human PLK structure can be exploited for the structure-guided design of EhPLK inhibitors.