Binding Core Domain Research Articles

Notch1 and Notch4 core binding domain peptibodies exhibit distinct ligand-binding and anti-angiogenic properties

The Notch signaling pathway is an important therapeutic target for the treatment of inflammatory diseases and cancer. We previously created ligand-specific inhibitors of Notch signaling comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain, called Notch decoys, which bound ligands, blocked Notch signaling, and showed anti-tumor activity with low toxicity. However, the study of their function depended on virally mediated expression, which precluded dosage control and limited clinical applicability. We have refined the decoy design to create peptibody-based Notch inhibitors comprising the core binding domains, EGF-like repeats 10–14, of either Notch1 or Notch4. These Notch peptibodies showed high secretion properties and production yields that were improved by nearly 100-fold compared to previous Notch decoys. Using surface plasmon resonance spectroscopy coupled with co-immunoprecipitation assays, we observed that Notch1 and Notch4 peptibodies demonstrate strong but distinct binding properties to Notch ligands DLL4 and JAG1. Both Notch1 and Notch4 peptibodies interfere with Notch signaling in endothelial cells and reduce expression of canonical Notch targets after treatment. While prior DLL4 inhibitors cause hyper-sprouting, the Notch1 peptibody reduced angiogenesis in a 3-dimensional in vitro sprouting assay. Administration of Notch1 peptibodies to neonate mice resulted in reduced radial outgrowth of retinal vasculature, confirming anti-angiogenic properties. We conclude that purified Notch peptibodies comprising EGF-like repeats 10–14 bind to both DLL4 and JAG1 ligands and exhibit anti-angiogenic properties. Based on their secretion profile, unique Notch inhibitory activities, and anti-angiogenic properties, Notch peptibodies present new opportunities for therapeutic Notch inhibition.

Mechanism of apoptosis activation by Curcumin rescued mutant p53Y220C in human pancreatic cancer

The mutant p53Y220C (mutp53Y220C) is frequently observed in numerous tumors, including pancreatic cancer. The mutation creates a crevice in the DNA binding core domain and makes p53 a thermally unstable non-functional protein that assists tumor progression and confers resistance to chemotherapeutic drugs. Restoring mutp53 function to its wild type by selectively targeting this crevice with small molecules is a pivotal strategy to promote apoptosis. In this study, we have shown through different biophysical and cell-based studies that curcumin binds and rescues mutp53Y220C to an active wild-type conformation and restores its apoptotic transcription function in BxPC-3-pancreatic cancer cells. In addition, the curcumin-rescued-p53Y220C (CRp53) showed significant hyperphosphorylation at Ser15, Ser20, and acetylation at Lys382 with an 8-fold increase in transcription activity in the BxPC-3 cell lines. We also observed that the active CRp53 escapes Mdm2-mediated proteasomal degradation and the majority of the proteins were localized inside the nucleus with an increased half-life and transcription restoration compared to untreated BxPC-3 cells. By label-free proteomics analysis, we observed that upon curcumin treatment almost 227 proteins were dysregulated with the majority of them being transcriptional targets of p53. Based on our studies, it reflects that apoptosis in pancreatic cancer cells is mediated by curcumin-rescued mutant p53Y220C.

Structural insights into the CP312R protein of the African swine fever virus

African swine fever (ASF) is a lethal hemorrhagic disease that affects domestic pigs and wild boars. There is no medication available for ASF to date. The ability to mount antigen-specific responses to viral vectored CP312R makes it a crucial potential target for designing vaccines or drugs. This study determined the crystal structure of ASFV CP312R at 2.32 Å and found it to be a monomer with a single-stranded DNA binding core domain with a clear five-strands β-barrel OB-fold architecture. Electrophoretic mobility shift assay and size-exclusion chromatography characterization assay further confirmed the single-stranded DNA (ssDNA)-binding property of ASFV CP312R. This study revealed the structure and preliminary ssDNA interaction mechanisms of ASFV CP312R, providing new clues for developing new antiviral strategies.

Reduction of RUNX1 transcription factor activity by a CBFA2T3-mimicking peptide: application to B cell precursor acute lymphoblastic leukemia

BackgroundB Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia.MethodsWe worked with BCP-ALL mononuclear bone marrow patients’ cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models.ResultsWe demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients’ cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located − 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation.ConclusionsAltogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein.

Bisphenol A stabilizes Nrf2 via Ca2+ influx by direct activation of the IP3 receptor.

Bisphenol A (BPA) is an endocrine-disrupting chemical used in polycarbonate and epoxy resins. Previously, we found that BPA stabilized the protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) by inducing Ca2+ efflux into the cytosol, followed by nitric oxide synthase activation, resulting in the enhanced nitrosylation of Keap1, which is a negative regulator of Nrf2. However, the mechanisms behind the stimulation of Ca2+ efflux by BPA remain unknown. In the present study, we found that BPA stimulated Ca2+ efflux into the cytosol from the ER, but not from outside of cells through the plasma membrane in Hep3B cells. Ca2+ efflux and Nrf2 stabilization by BPA were inhibited by an inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor, 2-aminoethoxydiphenylborane, in the endoplasmic reticulum. IP3 is produced by activation of phospholipase C (PLC) from a membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2). The induction of Nrf2 by BPA was not inhibited by a PLC inhibitor, U-73122, suggesting that BPA does not induce the production of IP3 via PLC activation. We found that BPA bound directly to the IP3 binding core domain of the IP3 receptor, and BPA competed with IP3 on this site. In addition, overexpression of this domain of the IP3 receptor in Hep3B cells inhibited the stabilization of Nrf2 by BPA. These results clarified that the IP3 receptor is a new target of BPA, and that BPA induces Ca2+ efflux from the endoplasmic reticulum via activation of the IP3 receptor.

Early-Life Stress Induced Epigenetic Changes of Corticotropin-Releasing Factor Gene in Anorexic Low Body Weight-Selected Chicks.

The expression of neuropeptide Y (NPY) in the arcuate nucleus (ARC) and corticotropin-releasing factor (CRF) in the paraventricular nucleus (PVN) were increased when low body weight–selected (LWS) line chicks, which are predisposed to anorexia, were subjected to a combination of nutritional and thermal stressors at hatch. We hypothesized that such changes resulted from epigenetic modifications. We determined global DNA methylation, DNA methyltransferase (DNMT) activity, and methylation near the promoter regions of NPY and CRF, in the hypothalamus of LWS chicks on day 5 post-hatch. Stress exposure at hatch induced global hypermethylation and increased DNMT activity in the ARC but not PVN. In the PVN of stressed LWS chicks, there was decreased methylation of a CpG site located at the core binding domain of methyl cytosine binding domain protein 2 (MBD2), near the CRF gene promoter. We then demonstrated that this was associated with disrupted binding of MBD2. There was also reduced utilization of yolk reserves and lean and fat masses in chicks that were stress-exposed. These findings provide novel insights on molecular mechanisms through which stressful events induce or intensify anorexia in predisposed individuals and a novel molecular target for further studies.

The scaffolding protein JADE1 physically links the acetyltransferase subunit HBO1 with its histone H3–H4 substrate

The human enzyme histone acetyltransferase binding to ORC1 (HBO1) regulates DNA replication, cell proliferation, and development. HBO1 is part of a multiprotein histone acetyltransferase (HAT) complex that also contains inhibitor of growth family member (ING) 4/5, MYST/Esa1-associated factor (MEAF) 6, and the scaffolding proteins Jade family PHD finger (JADE) 1/2/3 or bromodomain and PHD finger-containing protein (BRPF) 2/3 to acetylate histone H4 H4K5/8/12 or H3K14, respectively. Within this four-protein complex, JADE1 determines histone H4 substrate specificity of the HBO1-HAT complex. However, the mechanism by which JADE1 controls the H4-specific acetyltransferase activity of HBO1 is unknown. Here we used recombinant proteins in vitro to dissect the specific regions and activities of HBO1 and JADE1 that mediate histone H3-H4 acetylation via the HBO1-HAT domain. We found that JADE1 increases the catalytic efficiency of HBO1 acetylation of an H3-H4 substrate by about 5-fold through an N-terminal, 21-residue HBO1- and histone-binding domain and a nearby second histone core-binding domain. We also demonstrate that HBO1 contains an N-terminal histone-binding domain (HBD) that makes additional contacts with H3-H4 independent of JADE1 interactions with histones and that the HBO1 HBD does not significantly contribute to HBO1's overall HAT activity. Experiments with JADE1 deletions in vivo recapitulated these in vitro interactions and their roles in HBO1 histone acetylation activity. Together, these results indicate that the N-terminal region of JADE1 functions as a platform that brings together the catalytic HBO1 subunit with its cognate H3-H4 substrate for histone acetylation.

A minimalist mitochondrial threonyl-tRNA synthetase exhibits tRNA-isoacceptor specificity during proofreading

Yeast mitochondria contain a minimalist threonyl-tRNA synthetase (ThrRS) composed only of the catalytic core and tRNA binding domain but lacking the entire editing domain. Besides the usual tRNAThr2, some budding yeasts, such as Saccharomyces cerevisiae, also contain a non-canonical tRNAThr1 with an enlarged 8-nucleotide anticodon loop, reprograming the usual leucine CUN codons to threonine. This raises interesting questions about the aminoacylation fidelity of such ThrRSs and the possible contribution of the two tRNAThrs during editing. Here, we found that, despite the absence of the editing domain, S. cerevisiae mitochondrial ThrRS (ScmtThrRS) harbors a tRNA-dependent pre-transfer editing activity. Remarkably, only the usual tRNAThr2 stimulated pre-transfer editing, thus, establishing the first example of a synthetase exhibiting tRNA-isoacceptor specificity during pre-transfer editing. We also showed that the failure of tRNAThr1 to stimulate tRNA-dependent pre-transfer editing was due to the lack of an editing domain. Using assays of the complementation of a ScmtThrRS gene knockout strain, we showed that the catalytic core and tRNA binding domain of ScmtThrRS co-evolved to recognize the unusual tRNAThr1. In combination, the results provide insights into the tRNA-dependent editing process and suggest that tRNA-dependent pre-transfer editing takes place in the aminoacylation catalytic core.

Molecular dynamic studies on the impact of mutations on the structure, stability, and N-terminal orientation of annexin A1: implications for membrane aggregation.

Multiple MD simulations were performed for the full-length wild-type A1, the full length A1 mutations S27E and S27A, as well as the N-terminal peptide (AMVSEFLKQAWFIDNEEQEYIKTVKGS²⁷KGGPGSAVSPYPTFN) of wild-type A1 and mutations S27E and S27A. The MD simulation trajectories of about 350 ns were generated and analyzed to examine the changes of core domain calcium binding affinity, core domain and N-terminal domain structures, and N-terminal domain orientation. Our results indicated that S27A and S27E mutations caused little changes on the calcium-binding affinity of the core domain of A1. However, the S27A mutation made the N-terminal domain of A1 less helical, and made the N-terminal domain migrate faster toward the core domain; these impacts on A1 are beneficial to the membrane aggregation process. On the contrary, the S27E mutation made the N-terminal domain of A1 more stable, and hindered the migration to the core domain; these changes on A1 are antagonistic for the membrane aggregation process. Our results using MD simulations provide an atomistic explanation for experimental observations that the S27E mutant showed a higher calcium concentration requirement and lower maximal extent of aggregation, while the wild-type and two mutants S27E and S27A required identical calcium concentrations for liposome binding.

Label-Free Detection of the P53-DNA Complex

The tumour suppressor p53 plays a crucial role in cell cycle regulation in response to cellular stress such as DNA damage. p53 has been found to be mutated in 50% of all human cancers, the majority of which occur in the DNA binding core domain of the protein. These mutations result in loss of sequence specific binding abilities that activate the transcription of genes to regulate the cell cycle and prevent cell damage proliferation. It is, therefore, crucial to understand the p53-DNA binding interaction to further our ability to detect and combat any breakdown in wild type function.In this research we investigate the interaction of p53 and sequence specific DNA with label-free techniques. The solid-state nanopore platform is a label-free biosensor that utilises ion transport to drive analytes through the nanopore aperture. The result is a current-time signal that is characteristic to the charge, shape and size of the analyte of interest (see figure). This method allows for detection and differentiation between analytes without a need for labels.The use of this platform to study the p53-DNA specific binding interaction is combined with AFM analysis to examine binding statistics.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Bacteriophage T5 Encodes a Homolog of the Eukaryotic Transcription Coactivator PC4 Implicated in Recombination-Dependent DNA Replication

The RNA polymerase II cofactor PC4 globally regulates transcription of protein-encoding genes through interactions with unwinding DNA, the basal transcription machinery and transcription activators. Here, we report the surprising identification of PC4 homologs in all sequenced representatives of the T5 family of bacteriophages, as well as in an archaeon and seven phyla of eubacteria. We have solved the crystal structure of the full-length T5 protein at 1.9Å, revealing a striking resemblance to the characteristic single-stranded DNA (ssDNA)-binding core domain of PC4. Intriguing novel structural features include a potential regulatory region at the N-terminus and a C-terminal extension of the homodimerisation interface. The genome organisation of T5-related bacteriophages points at involvement of the PC4 homolog in recombination-dependent DNA replication, strongly suggesting that the protein corresponds to the hitherto elusive replicative ssDNA-binding protein of the T5 family. Our findings imply that PC4-like factors intervene in multiple unwinding-related processes by acting as versatile modifiers of nucleic acid conformation and raise the possibility that the eukaryotic transcription coactivator derives from ancestral DNA replication, recombination and repair factors.

P73 – constitutively open for business

p73 – constitutively open for business

The L–VP35 and L–L interaction domains reside in the amino terminus of the Ebola virus L protein and are potential targets for antivirals

The Ebola virus (EBOV) RNA-dependent RNA polymerase (RdRp) complex consists of the catalytic subunit of the polymerase, L, and its cofactor VP35. Using immunofluorescence analysis and coimmunoprecipitation assays, we mapped the VP35 binding site on L. A core binding domain spanning amino acids 280–370 of L was sufficient to mediate weak interaction with VP35, while the entire N-terminus up to amino acid 380 was required for strong VP35–L binding. Interestingly, the VP35 binding site overlaps with an N-terminal L homo-oligomerization domain in a non-competitive manner. N-terminal L deletion mutants containing the VP35 binding site were able to efficiently block EBOV replication and transcription in a minigenome system suggesting the VP35 binding site on L as a potential target for the development of antivirals.

Dissecting the Dynamics of HIV-1 Protein Sequence Diversity

The rapid mutation of human immunodeficiency virus-type 1 (HIV-1) and the limited characterization of the composition and incidence of the variant population are major obstacles to the development of an effective HIV-1 vaccine. This issue was addressed by a comprehensive analysis of over 58,000 clade B HIV-1 protein sequences reported over at least 26 years. The sequences were aligned and the 2,874 overlapping nonamer amino acid positions of the viral proteome, each a possible core binding domain for human leukocyte antigen molecules and T-cell receptors, were quantitatively analyzed for four patterns of sequence motifs: (1) “index”, the most prevalent sequence; (2) “major” variant, the most common variant sequence; (3) “minor” variants, multiple different sequences, each with an incidence less than that of the major variant; and (4) “unique” variants, each observed only once in the alignment. The collective incidence of the major, minor, and unique variants at each nonamer position represented the total variant population for the position. Positions with more than 50% total variants contained correspondingly reduced incidences of index and major variant sequences and increased minor and unique variants. Highly diverse positions, with 80 to 98% variant nonamer sequences, were present in each protein, including 5% of Gag, and 27% of Env and Nef, each. The multitude of different variant nonamer sequences (i.e. nonatypes; up to 68%) at the highly diverse positions, represented by the major, multiple minor, and multiple unique variants likely supported variants function both in immune escape and as altered peptide ligands with deleterious T-cell responses. The patterns of mutational change were consistent with the sequences of individual HXB2 and C1P viruses and can be considered applicable to all HIV-1 viruses. This characterization of HIV-1 protein mutation provides a foundation for the design of peptide-based vaccines and therapeutics.

Structure based energy calculation to determine the regulation of G protein signalling by RGS and RGS-G protein interaction specificity

The RGS proteins act as GTPase activating proteins and therefore regulate the lifespan of the active G alpha-GTP by accelerating the GTP hydrolysis. Modulatory residues in the RGS protein are present at the periphery of the RGS domain-G protein interface which is essential to fine-tune the G protein recognition and interaction. The docking energies of the mutant complex and the native complex were compared to see the effects of the mutations in the Modulatory regions. Mutations of Modulatory residues in high-activity RGS proteins lead to loss of function, whereas multiple mutations in the low-activity RGS proteins in critical Modulatory positions lead to complete gain of function. In the RGS proteins the Significant and Conserved core residues with peripheral Modulatory residues selectively optimize G protein recognition and inactivation. The flexibility of the structures of the mutant complexes were seen to be higher and the accessible surface area for the complexes increased after the mutations in the Modulatory residues. Through this approach we analyzed the interaction specificity among the RGS and the G alpha protein, the approach can also be applied to other protein families to find the residues which along with the core binding domain, fine tune the protein recognition and are crucial in the loss or gain of function.

Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

Congenital defects in the Na/H exchanger regulatory factor-1 (NHERF1) are linked to disordered phosphate homeostasis and skeletal abnormalities in humans. In the kidney, these mutations interrupt parathyroid hormone (PTH)-responsive sequestration of the renal phosphate transporter, Npt2a, with ensuing urinary phosphate wasting. We now report that NHERF1, a modular PDZ domain scaffolding protein, coordinates the assembly of an obligate ternary complex with Npt2a and the PKA-anchoring protein ezrin to facilitate PTH-responsive cAMP signaling events. Activation of ezrin-anchored PKA initiates NHERF1 phosphorylation to disassemble the ternary complex, release Npt2a, and thereby inhibit phosphate transport. Loss-of-function mutations stabilize an inactive NHERF1 conformation that we show is refractory to PKA phosphorylation and impairs assembly of the ternary complex. Compensatory mutations introduced in mutant NHERF1 re-establish the integrity of the ternary complex to permit phosphorylation of NHERF1 and rescue PTH action. These findings offer new insights into a novel macromolecular mechanism for the physiological action of a critical ternary complex, where anchored PKA coordinates the assembly and turnover of the Npt2a-NHERF1-ezrin complex.

Bazedoxifene

Bazedoxifene (Conbriza®, Viviant®) is the first third-generation selective estrogen receptor modulator (SERM) and it is approved for the treatment of postmenopausal osteoporosis in the EU and Japan. Bazedoxifene contains an indole-based core binding domain that binds with high affinity to estrogen receptors and exhibits favourable effects on bone and lipid profiles, with no clinically relevant endometrial or breast stimulation. Oral bazedoxifene once daily reduced the incidence of new vertebral fractures in patients with postmenopausal osteoporosis in a large, well designed trial of 3 years' duration; both bazedoxifene and raloxifene were significantly more effective than placebo. Neither bazedoxifene nor raloxifene reduced the incidence of nonvertebral fractures in the overall study population; however, bazedoxifene, but not raloxifene, reduced the rate of nonvertebral fractures in high-risk patients. Moreover, data from patients who continued to receive the drug during a 2-year extension phase of this trial indicate that bazedoxifene continues to provide protection against new vertebral fractures for up to 5 years. Bazedoxifene also increases bone mineral density and reduces the levels of bone turnover markers. Bazedoxifene was generally well tolerated and did not detrimentally affect the reproductive tract or breast tissue in clinical trials, thereby demonstrating a favourable risk-benefit profile. A pharmacoeconomic analysis conducted from an EU perspective predicted bazedoxifene to be cost effective in some EU countries. Therefore, bazedoxifene presents another useful option for the treatment of postmenopausal osteoporosis, especially in those at high risk for osteoporotic fracture.

Common allosteric mechanisms between ryanodine and inositol-1,4,5-trisphosphate receptors

Ryanodine receptors (RyRs) are calcium release channels found in the membrane of the endoplasmic reticulum (ER). We recently described the crystal structure of the RyR1 N-terminal disease hot spot. It is built up by three domains that show clear structural homology with the inositol-1,4,5-triphosphate (IP3) binding core and suppressor domain of IP3 receptors (IP3Rs) . Here we analyze the structural features of the domains in both calcium release channels, and propose a model for the closed state of the IP3R N-terminal region. This model explains the effect of the suppressor domain on the affinity for IP3 and is supported by mutational studies performed previously. We propose a mechanism whereby opening of both RyR and IP3R is allosterically coupled to a displacement of the N-terminal domain from the following two domains. This displacement can be affected by disease mutations, glutathionylation of a highly reactive cysteine residue, or ligand binding.

A G‐Quadruplex Aptamer Inhibits the Phosphatase Activity of Oncogenic Protein Shp2 in vitro

Shp2 is a member of the protein tyrosine phosphatase (PTP) family, which regulates a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. Using a recombinant Shp2-GST protein as the target and GST as a counter target, we have identified two classes of single-stranded DNA aptamers that selectively bind to Shp2 with a K(d) in the nanomolar range. Structural studies of the most abundant sequence in the enriched library, HJ24, revealed a parallel G-quadruplex as the core binding domain. Furthermore, this aptamer was found to be an effective inhibitor of Shp2 phosphatase, an effect which was readily reversed by using the cDNA of HJ24. In view of these characteristics, this aptamer has the potential to be used for further development of Shp2 assays and therapeutics for the treatment of Shp2-dependent cancers and other diseases.

Conserved Catalytic and C-Terminal Regulatory Domains of the C-Terminal Binding Protein Corepressor Fine-Tune the Transcriptional Response in Development

Transcriptional corepressors play complex roles in developmental gene regulation. These proteins control transcription by recruiting diverse chromatin-modifying enzymes, but it is not known whether corepressor activities are finely regulated in different developmental settings or whether their basic activities are identical in most contexts. The evolutionarily conserved C-terminal binding protein (CtBP) is recruited by a variety of transcription factors that play crucial roles in development and disease. CtBP contains a central NAD(H) binding core domain that is homologous to D2 hydroxy acid dehydrogenase enzymes, as well as an unstructured C-terminal domain. NAD(H) binding is important for CtBP function, but the significance of its intrinsic dehydrogenase activity, as well as that of the unstructured C terminus, is poorly understood. To clarify the biological relevance of these features, we established genetic rescue assays to determine how different forms of CtBP function in the context of Drosophila melanogaster development. The mutant phenotypes and specific gene regulatory effects indicate that both the catalytic site of CtBP and the C-terminal extension play important, if nonessential roles in development. Our results indicate that the structural and enzymatic features of CtBP, previously thought to be dispensable for overall transcriptional control, are critical for modulating this protein's activity in diverse developmental settings.