Isolation and detection of human IgA using a streptococcal IgA-binding peptide

Bacterial proteins that bind to the Fc part of IgG have found widespread use in immunology. A similar protein suitable for the isolation and detection of human IgA has not been described. Here, we show that a 50-residue synthetic peptide, designated streptococcal IgA-binding peptide (Sap) and derived from a streptococcal M protein, can be used for single-step affinity purification of human IgA. High affinity binding of IgA required the presence in Sap of a C-terminal cysteine residue, not present in the intact M protein. Passage of human serum through a Sap column caused depletion of >99% of the IgA, and elution of the column allowed quantitative recovery of highly purified IgA, for which the proportions of the IgA1 and IgA2 subclasses were the same as in whole serum. Moreover, immobilized Sap could be used for single-step purification of secretory IgA of both subclasses from human saliva, with a recovery of approximately 45%. The Sap peptide could also be used to specifically detect IgA bound to Ag. Together, these data indicate that Sap is a versatile Fc-binding reagent that may open new possibilities for the characterization of human IgA.     

Interferon receptor interaction. Internalization of interferon alpha 2 and modulation of its receptor on human cells

Studies reported earlier [ Joshi et al. (1982) J. Biol. Chem. 257, 13884-13887] have indicated that human interferon-alpha 2 (HuIFN-alpha 2) binds to a specific macromolecular receptor on human cells as identified by cross-linking with bifunctional cross-linking reagents and analysis by polyacrylamide gel electrophoresis. We have carried out experiments to investigate the fate of the interferon-receptor complex on the cell surface under conditions which lead to cellular response. As analyzed by cross-linking and gel electrophoresis, the interferon-receptor complex, formed on incubation with 125I-IFN-alpha 2 at 4 degrees C, persisted at the cell surface for several hours at 4 degrees C; however, if the cells were switched to 37 degrees C, there was a rapid decline in the complex, apparently due to a loss of the interferon receptors from the cell surface. This was associated with an internalization of the 125I-interferon as indicated by the fact that, on incubation at 37 degrees C, an appreciable fraction of the cell-associated interferon (approximately equal to 50%) became resistant to trypsin digestion, or dissociation on incubation in growth medium or low-pH buffer. A large fraction of the trypsin-resistant (internalized) 125I-labeled material migrated as intact interferon in polyacrylamide gels, and it was immunoprecipitated by anti-(HuIFN-alpha)antibodies but not by anti-(HuIFN-beta)antibodies. The bulk of the internalized 125I-interferon was recovered in a particulate fraction and, on cross-linking with disuccinimidyl suberate, a 150000-Mr complex could be detected. The results suggest that interferon may be internalized as a complex with the receptor, which may account for the loss of the interferon-receptors on the cell surface. This modulation of the IFN-alpha/beta receptors was induced by HuIFN-alpha and HuIFN-beta but not by HuIFN-gamma. The recovery of the IFN-alpha/beta receptors, lost upon incubation with HuIFN-alpha, took several hours and required protein synthesis. The significance of the results is discussed.     

The calcium-sensing receptor and its interacting proteins

Seven membrane-spanning, or G protein-coupled receptors were originally thought to act through het-erotrimeric G proteins that in turn activate intracellular enzymes or ion channels, creating relatively simple, linear signalling pathways. Although this basic model remains true in that this family does act via a relatively small number of G proteins, these signalling systems are considerably more complex because the receptors interact with or are located near additional proteins that are often unique to a receptor or subset of receptors. These additional proteins give receptors their unique signalling personalities. The extracellular Ca-sensing receptor (CaR) signals via Galpha(i), Galpha(q) and Galpha(12/13), but its effects in vivo demonstrate that the signalling pathways controlled by these subunits are not sufficient to explain all its biologic effects. Additional structural or signalling proteins that interact with the CaR may explain its behaviour more fully. Although the CaR is less well studied in this respect than other receptors, several CaR-interacting proteins such as filamin, a potential scaffolding protein, receptor activity modifying proteins (RAMPs) and potassium channels may contribute to the unique characteristics of the CaR. The CaR also appears to interact with additional proteins common to other G protein-coupled receptors such as arrestins, G protein receptor kinases, protein kinase C, caveolin and proteins in the ubiquitination pathway. These proteins probably represent a few initial members of CaR-based signalling complex. These and other proteins may not all be associated with the CaR in all tissues, but they form the basis for understanding the complete nature of CaR signalling.     

The time course of miniature endplate currents and its modification by receptor blockade and ethanol

Miniature endplate currents (MEPCs) recorded from mouse diaphragms with a point voltage clamp, without inhibition of acetylcholinesterase (AChE) and in the absence of any drug, showed in their decay phase consistent deviations from an exponential time course, consisting of (a) "curvature," a progressive increase of decay rate during most of the decay phase, followed by (b) "late" tails. Both phenomena persisted when MEPCs (and channel lifetime) were prolonged by ethanol. Curvature was increased by muscle fiber depolarization and decreased by hyperpolarization. Receptor blockade by (+)-tubocurarine, alpha-bungarotoxin, hexamethonium, or myasthenic IgG accelerated the decay of the main part of MEPCs and eliminated curvature; the time constant of MEPCs became close to the channel time constant. We conclude that curvature arises from repeated action of ACh with cooperativity in ACh-receptor interaction; the voltage sensitivity of curvature follows from the voltage sensitivity of channel closing. Ethanol, in addition to its effect to prolong channel lifetime, enhances the tendency of ACh to act more than once to open channels before being lost to the system. Analysis of the rising phase of the MEPC, in terms of driving functions, also indicated that ethanol promotes channel opening by ACh; this action can account for a substantial increase of MEPC height by ethanol when MEPCs are made small by receptor blockade. Driving functions were also voltage sensitive, in a manner indicating acceleration of channel opening, but reduction of channel conductance, with hyperpolarization. Poisoning or inhibition of AChE prolonged MEPCs without altering the duration of ionic channels. Since ethanol caused further prolongation of MEPCs after poisoning of AChE, with little change in MEPC height, we conclude that the extension of mean channel lifetime by ethanol is accompanied by a similar extension of ACh binding to receptors. After poisoning of AChE, MEPCs became very variable in time course and the decay rate (tau-1) was correlated with MEPC height with a slope of log tau vs. log height of 0.77 for MEPCs of greater than 60% mean size. This slope is larger than expected from cooperativity in ACh-receptor interaction. Correlation of tau and height of MEPCs also exists when AChE is intact; the slope of log tau vs. log height was 0.12 with or without prolongation of MEPCs by ethanol.     

The interaction of Fc alpha RI with IgA and its implications for ligand binding by immunoreceptors of the leukocyte receptor cluster

This study defines the molecular basis of the FcalphaRI (CD89):IgA interaction, which is distinct from that of the other leukocyte Fc receptors and their Ig ligands. A comprehensive analysis using both cell-free (biosensor) and cell-based assays was used to define and characterize the IgA binding region of FcalphaRI. Biosensor analysis of mutant FcalphaRI proteins showed that residues Y35, Y81, and R82 were essential for IgA binding, and R52 also contributed. The role of the essential residues (Y35 and R82) was confirmed by analysis of mutant receptors expressed on the surface of mammalian cells. These receptors failed to bind IgA, but were detected by the mAb MY43, which blocks IgA binding to FcalphaRI, indicating that its epitope does not coincide with these IgA binding residues. A homology model of the ectodomains of FcalphaRI was generated based on the structures of killer Ig-like receptors, which share 30-34% identity with FcalphaRI. Key structural features of killer Ig-like receptors are appropriately reproduced in the model, including the structural conservation of the interdomain linker and hydrophobic core (residues V17, V97, and W183). In this FcalphaRI model the residues forming the IgA binding site identified by mutagenesis form a single face near the N-terminus of the receptor, distinct from other leukocyte Fc receptors where ligand binding is in the second domain. This taken together with major differences in kinetics and affinity for IgA:FcalphaRI interaction that were observed depending on whether FcalphaRI was immobilized or in solution suggest a mode of interaction unique among the leukocyte receptors.     

Engineering receptor-mediated cytotoxicity into human ribonucleases by steric blockade of inhibitor interaction

Several nonmammalian members of the RNase A superfamily exhibit anticancer activity that appears to correlate with resistance to the cytosolic ribonuclease inhibitor (RI). We mutated two human ribonucleases-pancreatic RNase (hRNAse) and eosinophil-derived neurotoxin (EDN)-to incorporate cysteine residues at putative sites of close contact to RI, but distant from the catalytic sites. Coupling of Cys89 of RNase and Cys87 of EDN to proteins at these sites via a thioether bond produced enzymatically active conjugates that were resistant to RI. To elicit cellular targeting as well as to block RI binding, transferrin was conjugated to a mutant human RNase, rhRNase(Gly89)-->Cys) and a mutant EDN (Thr87-->Cys). The transferrin-rhRNase(Gly89-->Cys) thioether conjugate was 5000-fold more toxic to U251 cells than recombinant wild-type hRNase. In addition, transferrin-targeted EDN exhibited tumor cell toxicities similar to those of hRNase. Thus, we endowed two human RI-sensitive RNases with greater cytotoxicity by increasing their resistance to RI. This strategy has the potential to generate a novel set of recombinant human proteins useful for targeted therapy of cancer.     

The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

Interleukin‐8 (CXCL8, IL‐8) is a proinflammatory chemokine important for the regulation of inflammatory and immune responses via its interaction with G‐protein coupled receptors, including CXC receptor 1 (CXCR1). CXCL8 exists as both a monomer and as a dimer at physiological concentrations, yet the molecular basis of CXCL8 interaction with its receptor as well as the importance of CXCL8 dimer formation remain poorly characterized. Although several biological studies have indicated that both the CXCL8 monomer and dimer are active, biophysical studies have reported conflicting results regarding the binding of CXCL8 to CXCR1. To clarify this problem, we expressed and purified a peptide (hCXCR1pep) corresponding to the N‐terminal region of human CXCR1 (hCXCR1) and utilized nuclear magnetic resonance (NMR) spectroscopy to interrogate the binding of wild‐type CXCL8 and a previously reported mutant (CXCL8M) that stabilizes the monomeric form. Our data reveal that the CXCL8 monomer engages hCXCR1pep with a slightly higher affinity than the CXCL8 dimer, but that the CXCL8 dimer does not dissociate upon binding hCXCR1pep. These investigations also showed that CXCL8 is dynamic on multiple timescales, which may help explain the versatility in this interleukin for engaging its target receptors.     

The detection of streptococcal cell-wall proteins that form complexes with human macroglobulins]

The composition of the extracts of the cultures of individual streptococcal strains, studied by immunoblotting techniques, has been shown to contain proteins with a molecular weight of 70-80 KD. These proteins have pronounced affinity to human macroglobulins: alpha-macroglobulin, alpha-glycoprotein associated with pregnancy and protein A. The significance of this phenomenon on the cellular and somatic levels is discussed.     

Quantitative characterization of the interaction between purified human estrogen receptor alpha and DNA using fluorescence anisotropy

In an effort to better define the molecular mechanisms of the functional specificity of human estrogen receptor α, we have carried out equilibrium binding assays to study the interaction of the receptor with a palindromic estrogen response element derived from the vitellogenin ERE. These assays are based on the observation of the fluorescence anisotropy of a fluorescein moiety covalently bound to the target oligonucleotide. The low anisotropy value due to the fast tumbling of the free oligonucleotide in solution increases substantially upon binding the receptor to the labeled ERE. The quality of our data are sufficient to ascertain that the binding is clearly cooperative in nature, ruling out a simple monomer interaction and implicating a dimerization energetically coupled to DNA binding in the nanomolar range. The salt concentration dependence of the affinity reveals formation of high stoichiometry, low specificity complexes at low salt concentration. Increasing the KCl concentration above 200 mM leads to specific binding of ER dimer. We interpret the lack of temperature dependence of the apparent affinity as indicative of an entropy driven interaction. Finally, binding assays using fluorescent target EREs bearing mutations of each of the base pairs in the palindromic ERE half-site indicate that the energy of interaction between ER and its target is relatively evenly distributed throughout the site.     

Episodes of natural selection shaped the interactions of IgA-Fc with FcalphaRI and bacterial decoy proteins

FcalphaRI, a receptor for IgA-Fc, recruits myeloid cells to attack IgA-coated pathogens. By competing with FcalphaRI for IgA, bacterial decoys, like SSL7 of Staphylococcus aureus, subvert this defense. We examined how pathogen selection has driven the diversification and coevolution of IgA and FcalphaRI. In higher primates, the IgA binding site of FcalphaRI diversified under positive selection, a strong episode occurring in hominoid ancestors about the time of the IgA gene duplication. The differential binding of SSL7 to IgA-Fc of different species correlates with substitution at seven positions in IgA-Fc, two of which were positively selected in higher primates. Two others, which reduce SSL7 binding, emerged during episodes of positive selection in the rabbit and rodent lineages. The FcalphaRI-IgA interaction evolves episodically under two types of positive selection: pressure from pathogen decoys selects for IgA escape variants which, in turn, selects for FcalphaRI variants to keep up with the novel IgA. When FcalphaRI cannot keep up, its function is lost and the gene becomes susceptible to elimination, as occurred in the mouse genome, either by chance or selection on one of the many linked, variable immune system genes. A cluster of positively selected residues presents a putative binding site for unknown IgA-binding factors.     

Molecular analysis of the interaction of LCMV with its cellular receptor [alpha]-dystroglycan

alpha-Dystroglycan (DG) has been identified as the cellular receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LFV). This subunit of DG is a highly versatile cell surface molecule that provides a molecular link between the extracellular matrix (ECM) and a beta-DG transmembrane component, which interacts with the actin-based cytoskeleton. In addition, DG exhibits a complex pattern of interaction with a wide variety of ECM and cellular proteins. In the present study, we characterized the binding of LCMV to alpha-DG and addressed the role of alpha-DG-associated host-derived proteins in virus infection. We found that the COOH-terminal region of alpha-DG's first globular domain and the NH2-terminal region of the mucin-related structures of alpha-DG together form the binding site for LCMV. The virus-alpha-DG binding unlike ECM alpha-DG interactions was not dependent on divalent cations. Despite such differences in binding, LCMV and laminin-1 use, in part, an overlapping binding site on alpha-DG, and the ability of an LCMV isolate to compete with laminin-1 for receptor binding is determined by its binding affinity to alpha-DG. This competition of the virus with ECM molecules for receptor binding likely explains the recently found correlation between the affinity of LCMV binding to alpha-DG, tissue tropism, and pathological potential. LCMV strains and variants with high binding affinity to alpha-DG but not low affinity binders are able to infect CD11c+ dendritic cells, which express alpha-DG at their surface. Infection followed by dysfunction of these antigen-presenting cells contributes to immunosuppression and persistent viral infection in vivo.     

Regulation of chemoattractant receptor interaction with transducing proteins by organizational control in the plasma membrane of human neutrophils

Isolated purified plasma membrane domains from unstimulated human neutrophils were photoaffinity labeled with F-Met-Leu-Phe-N epsilon-(2- (p-azido-[125I]salicylamido)ethyl- 1,3'-dithiopropionyl)-Lys also referred to as FMLPL-SASD[125I]. Most of the photoaffinity-labeled N- formyl peptide receptors were found in light plasma membrane fraction (PM-L) which has been previously shown to be enriched in guanyl nucleotide binding proteins and the plasma membrane marker alkaline phosphatase (Jesaitis, A. J., G. M. Bokoch, J. O. Tolley, and R. A. Allen. 1988. J. Cell Biol. 107:921-928). Furthermore, the heavy plasma membrane fraction (PM-H), which is enriched in actin and fodrin, was depleted in receptors. Solubilization of PM-L and PM-H in divalent cation-free buffer containing octylglucoside and subsequent sedimentation at 180,000 g in detergent-containing sucrose gradients revealed two receptor forms. The major population, found in PM-L sedimented as a globular protein with an apparent sedimentation coefficient of 6-7S, while a minor fraction found in the PM-H fraction sedimented as a 4S particle. In addition, the 6-7S form could be converted to the 4S form by inclusion of guanosine 5'-O-(3- thiotriphosphate) (GTP gamma S) in the extraction buffer (ED50 = 10-30 nM). ATP was not effective at doses of up to 10 microM. In contrast, isolation and solubilization of receptors from desensitized cells (photoaffinity labeled after a 15 degrees C incubation with FMLPL- SASD[125I]) revealed that the majority of receptors (greater than 60- 90%), which are found in PM-H, sedimented as 4S particles. A minor fraction of receptors found in the PM-L sedimented as 6-7S species. The receptors in the PM-H fraction, however, were still capable of interacting with G-proteins, since addition of unlabeled PM-L membrane fraction as a G-protein source reconstituted a more rapidly sedimenting form showing sensitivity to GTP gamma S. These results suggest that receptors in unstimulated human neutrophils have a higher probability of interacting with G-proteins because they are in the light plasma membrane domain. The results also suggest that receptors that have been translocated to the heavy plasma membrane domain during the process of desensitization or response termination have a lower probability of interacting with G-protein. Since the latter receptors are still capable of forming G protein associations, then their lateral segregation would represent a mechanism of controlling of receptor G- protein interactions. This reorganization of the plasma membrane, therefore, may form the molecular basis for response termination or homologous desensitization in human neutrophils.     

Estrogen regulation of trefoil factor 1 expression by estrogen receptor alpha and Sp proteins

Estrogen-responsive genes in human breast cancer cells often have an estrogen response element (ERE) positioned next to an Sp1 binding site. In chromatin immunoprecipitation (ChIP) assays, we investigated the binding of estrogen receptor alpha (ER), Sp1, and Sp3 to the episomal and native estrogen-responsive trefoil factor 1 (TFF1; formerly pS2) promoter in MCF-7 breast cancer cells. Mutation of the Sp site upstream of the ERE reduced estrogen responsiveness and prevented binding of Sp1 and Sp3, but not ER to the episomal promoter. In the absence of estradiol (E2), Sp1, Sp3, histone deacetylase 1 (HDAC), and HDAC2, and low levels of acetylated H3 and H4 are associated with the native promoter, with the histones being engaged in dynamic reversible acetylation. Following E2 addition, levels of ER and acetylated H3 and H4 bound to the native promoter increases. There is clearance of Sp1, but not of Sp3, from the promoter while HDAC1 and HDAC2 remain bound. These data are consistent with a model in which Sp1 or Sp3 aid in recruitment of HDACs and histone acetyltransferases (HATs) to mediate dynamic acetylation of histones associated with the TFF1 promoter, which is in a state of readiness to respond to events occurring following the addition of estrogen.     

Expression of retinoic acid-related orphan receptor alpha and its responsive genes in human endometrium regulated by cholesterol sulfate

Cholesterol sulfate (CS) is a major sterol sulfate in human plasma that is detected in the uterine endometrium. CS plays a role in steroidogenesis, cellular membrane stabilization, and regulation of the skin barrier. We previously reported that CS increased in rabbit endometrium during the implantation period. Recently, CS has been reported to be a ligand of retinoic acid receptor-related orphan receptor alpha (RORA). NR1D1 is one of the genes regulated by RORA. In the present study, we investigated the regulation of RORA and NR1D1 by CS in human endometrium. We determined the association-dissociation curves for the interaction of CS with RORA and the kinetic rates by surface plasmon resonance. Immunohistochemical staining and in situ hybridization revealed that RORA and NR1D1 were expressed in human endometrial stromal and epithelial cells. CS treatment significantly induced the mRNA expression of RORA and NR1D1 mRNA in ESCs. The results of a luciferase assay showed that RORA significantly activated the human NR1D1 promoter regardless of CS. Our results suggest that CS regulates the expression of RORA responsive genes in human endometrial cells but not as a ligand for RORA.     

生物功能化色谱技术研究胰岛素与其受体间的相互作用

生物功能化色谱技术是通过模拟细胞膜表面环境,在色谱系统内实现研究生物分子间相互作用过程的新技术。利用生物功能化色谱方法研究了胰岛素与其受体间的相互作用。将提取出的胰岛素受体混合物固定化在人工膜(Immobilized artificial membrane, IAM)的表面,并确定了固定化的最佳pH值为7.2,最佳离子强度为20 mmol,最佳有机溶剂浓度为2% (v/v)。通过区带洗脱法确定了胰岛素受体的存在及其与胰岛素间存在相互作用;通过前沿分析法确定了胰岛素受体与胰岛素间相互作用的结合常数大小约为0.701 nmol/L。     

Analysis of the interaction between the bacterial superantigen streptococcal pyrogenic exotoxin A (SpeA) and the human T-cell receptor

Streptococcus pyogenes that produces the bacterial superantigen streptococcal pyrogenic exotoxin A (SpeA) is associated with outbreaks of streptococcal toxic shock syndrome (STSS) in the United States and Europe. SpeA stimulates Vβ2.1, 12.2, 14.1, and 15.1-positive T cells, and the lymphokine production from the activated T cells is believed to result in the symptoms associated with STSS. The T-cell receptor (TCR)–SpeA interaction is crucial for superantigenic activity, and studies were undertaken to determine regions of both SpeA and the TCR involved in the formation of MHC/SpeA/TCR complexes. Previously, recombinant toxins encoded by speA alleles 1, 2, and 3 as well as toxins resulting from 19 distinct point mutations in speA1 were generated. Here, these 22 toxin forms were incubated with human peripheral blood mono- nuclear cells (PBMCs), and the percentages of T-cell blasts bearing Vβ chains 2.1, 12.2, and 14.1 were quantified by flow cytometry. The analysis indicates that the residues of SpeA needed for a productive TCR interaction differ for each Vβ chain examined. An amino acid substitution at only one site significantly affected the toxin’s ability to stimulate Vβ2.1-expressing T cells, three individual amino acid substitutions resulted in significant loss of ability to stimulate Vβ12.2-expressing T cells, and substitution at 13 individual sites significantly affected the ability to stimulate Vβ14.1-expressing T cells. To elucidate the regions of the Vβ chains that interacted with SpeA, synthetic peptides representative of the human Vβ12.2 complementary-determining regions (CDRs) 1, 2, and 4 were used to block the SpeA-mediated proliferation of human PBMCs. The CDR1, CDR2 and CDR4 peptides were each able to block proliferation, with the activity of CDR1 > CDR2 > CDR4. Combinations of CDR1 peptide with CDR2 or CDR4 peptides allosterically enhanced the ability of each to block proliferation, suggesting SpeA has distinct binding sites for the CDR loops.     

Steroid hormone-dependent interaction of human progesterone receptor with its target enhancer element

We investigated the requirement of steroid hormone for the specific binding of progesterone receptor to its cognate progesterone responsive element (PRE) in cell-free experiments. We prepared unfractionated nuclear extracts from human breast cancer (T47D) cells which are rich in progesterone receptors and used a gel retardation assay to monitor receptor-DNA complex formation. Exposure of receptor to either progesterone, R5020, or the antiprogestin RU38 486 in vivo or in vitro led to the formation of two protein-DNA complexes (1 and 2) which were not detected in nuclear extracts unexposed to hormone. Similar treatment with cortisol or estradiol failed to induce the formation of these complexes. The complexes were specific for PRE, since they could be competed efficiently in binding competition experiments by oligonucleotides containing PRE. A monoclonal antibody which recognizes both A and B forms of human progesterone receptor, interacted with both complexes 1 and 2 and shifted them to slower migrating forms. Another antibody which only recognizes the B form interacted with only complex 1 but not with complex 2, establishing that the complexes 1 and 2 were indeed formed by progesterone receptor forms B and A, respectively. We conclude from the above studies that in vivo or in vitro treatment of nuclear progesterone receptor with either progesterone or R5020 or RU38 486 alone can lead to detection of high affinity complexes formed between the PRE and the receptor present in unpurified nuclear extracts.     

Molecular determinants for the interaction of human neutrophil alpha defensin 1 with its propeptide

Human neutrophil α-defensins (HNPs) are cationic antimicrobial peptides that are synthesized in vivo as inactive precursors (proHNPs). Activation requires proteolytic excision of their anionic N-terminal inhibitory pro peptide. The pro peptide of proHNP1 also interacts specifically with and inhibits the antimicrobial activity of HNP1 inter-molecularly. In the light of the opposite net charges segregated in proHNP1, functional inhibition of the C-terminal defensin domain by its propeptide is generally thought to be of electrostatic nature. Using a battery of analogs of the propeptide and of proHNP1, we identified residues in the propeptide region important for HNP1 binding and inhibition. Only three anionic residues in the propeptide, Glu¹⁵, Asp²⁰ and Glu²³, were modestly important for interactions with HNP1. By contrast, the hydrophobic residues in the central part of the propeptide, and the conserved hydrophobic motif Val²⁴Val²⁵Val²⁶Leu²⁸ in particular, were critical for HNP1 binding and inhibition. Neutralization of all negative charges in the propeptide only partially activated the bactericidal activity of proHNP1. Our data indicate that hydrophobic forces have a dominant role in mediating the interactions between HNP1 and its propeptide — a finding largely contrasting the commonly held view that the interactions are of an electrostatic nature.     

The DNA binding domain of estrogen receptor alpha is required for high-affinity nuclear interaction induced by estradiol

Estrogen receptor alpha (ER) is a member of the nuclear hormone receptor family, which upon binding estrogen shows increased apparent affinity for nuclear components (tight nuclear binding). The nuclear components that mediate this tight nuclear binding have been proposed to include both ER-DNA interactions and ER-protein interactions. In this paper, we demonstrate that tight nuclear binding of ER upon estrogen occupation requires ER-DNA interactions. Hormone-bound ER can be extracted from the nucleus in low-salt buffer using various polyanions, which mimic the phosphate backbone of DNA. The importance of specific ER-DNA interactions in mediating tight nuclear binding is also supported by the 380-fold lower concentration of the ERE oligonucleotide necessary to extract estrogen-occupied ER from the nucleus compared to the polyanions. We also demonstrate that estrogen-induced tight nuclear binding requires both the nuclear localization domain and the DNA binding domain of ER. Finally, enzymatic degradation of nuclear DNA allows us to recover 45% of tight nuclear-bound ER. We further demonstrate that ER-AIB1 interaction is not required for estrogen-induced tight nuclear binding. Taken together, we propose a model in which tight nuclear binding of the estrogen-occupied ER is predominantly mediated by ER-DNA interactions. The effects of estrogen binding on altering DNA binding in whole cells are proposed to occur through estrogen-induced changes in ER-chaperone protein interactions, which alter the DNA accessibility of ER but do not directly change the affinity of the ER for DNA, which is similar for both unoccupied and occupied ER.