Different SP1 binding dynamics at individual genomic loci in human cells

Using a tamoxifen-inducible time-course ChIP-sequencing (ChIP-seq) approach, we show that the ubiquitous transcription factor SP1 has different binding dynamics at its target sites in the human genome. SP1 very rapidly reaches maximal binding levels at some sites, but binding kinetics at other sites is biphasic, with rapid half-maximal binding followed by a considerably slower increase to maximal binding. While ∼70% of SP1 binding sites are located at promoter regions, loci with slow SP1 binding kinetics are enriched in enhancer and Polycomb-repressed regions. Unexpectedly, SP1 sites with fast binding kinetics tend to have higher quality and more copies of the SP1 sequence motif. Different cobinding factors associate near SP1 binding sites depending on their binding kinetics and on their location at promoters or enhancers. For example, NFY and FOS are preferentially associated near promoter-bound SP1 sites with fast binding kinetics, whereas DNA motifs of ETS and homeodomain proteins are preferentially observed at sites with slow binding kinetics. At promoters but not enhancers, proteins involved in sumoylation and PML bodies associate more strongly with slow SP1 binding sites than with the fast binding sites. The speed of SP1 binding is not associated with nucleosome occupancy, and it is not necessarily coupled to higher transcriptional activity. These results with SP1 are in contrast to those of human TBP, indicating that there is no common mechanism affecting transcription factor binding kinetics. The biphasic kinetics at some SP1 target sites suggest the existence of distinct chromatin states at these loci in different cells within the overall population.

Endothelial Protein C Receptor Is Expressed on Procoagulant Platelets and Contributes to Factor VIIa Binding and Activity

Recombinant factor VIIa (rFVIIa) is routinely used as an effective bypassing agent to promote hemostasis in hemophilia patients with inhibitory antibodies that compromise factor replacement. In addition, rFVIIa is extensively used off-label as a hemostatic agent in cardiovascular surgery, trauma, and intracranial hemorrhage. Attempts to improve the treatment for these patients have included the production of rFVIIa analogs such as V158D/E296V/M298Q-FVIIa (FVIIa-DVQ). Previous studies have shown that FVIIa-DVQ exhibits enhanced in vitro procoagulant and antifibrinolytic activity, greater factor Xa (FXa) and thrombin generation on activated platelets, and improved hemostatic efficacy relative to wild-type FVIIa in a mouse model of hemophilia. Surprisingly, while FVIIa-DVQ and rFVIIa bind similarly to phospholipid vesicles, FVIIa-DVQ exhibits greater binding to platelets than rFVIIa. It has been established that only a fraction of activated platelets are able to bind high levels of coagulation factors. These highly procoagulant platelets, often called COATed platelets, also preferentially bind rFVIIa and FVIIa-DVQ in a Gla-domain dependent manner. However, the exact mechanism of this interaction remains largely unknown. As previous studies have shown that the endothelial cell protein C receptor (EPCR) also functions as the endothelial cell receptor for FVIIa, the purpose of the current study was to determine whether an interaction with EPCR might be responsible for the increased platelet binding and hemostatic efficacy of FVIIa-DVQ. Following their isolation from anticoagulated whole blood, human platelets were activated with a combination of thrombin plus the collagen receptor agonist convulxin, in order to generate highly procoagulant platelets. We then examined the binding of both rFVIIa and FVIIa-DVQ by flow cytometry in the presence and absence of excess protein C (PC) to determine its ability to compete for platelet binding. As previously reported, maximal binding of FVIIa-DVQ was significantly higher than the maximal binding of rFVIIa in the absence of PC. Interestingly, the addition of PC inhibited the binding of FVIIa-DVQ to a greater extent than the binding of rFVIIa, thereby eliminating the difference in binding seen in the absence of PC. Using both FXa and thrombin generation assays we found that this competition for platelet binding resulted in a corresponding decrease in the procoagulant activity of FVIIa-DVQ. Similar experiments were also performed to evaluate rFVIIa and FVIIa-DVQ binding to platelets in the presence and absence of a rabbit anti-EPCR antibody. Equivalent decreases in the platelet binding and activity of both molecules were seen in the presence of this antibody, thereby confirming that these results are indeed due to interactions with EPCR. However, while mRNA encoding EPCR has been reported in genome-wide sequencing of the human platelet transcriptome, EPCR protein expression has not previously been shown in platelets. We therefore conducted flow cytometric analyses of unactivated, thrombin activated, and thrombin plus convulxin activated platelets to evaluate their expression of EPCR. We found that EPCR is not expressed on either unactivated or thrombin-activated platelets. Conversely, the highly procoagulant platelets do express EPCR. Dual-labeling studies confirmed that those platelets which express EPCR also bind the most rFVIIa. To confirm platelet EPCR expression we first confirmed the presence of EPCR pre-mRNA in unactivated platelets by RT-PCR. We then determined the presence of EPCR protein by immunoprecipitation from unactivated platelet lysates followed by western blotting and mass spectrometric analyses. These data unambiguously demonstrate that EPCR is present in unactivated platelets, and is specifically expressed by the highly procoagulant platelet subpopulation. This work represents the first demonstration that human platelets are capable of expressing EPCR, and suggests that EPCR plays a role in the efficacy of rFVIIa as a therapeutic agent by contributing to platelet-FVIIa interactions. A better understanding of the mechanism by which rFVIIa binds to the activated platelet will facilitate the development of new therapeutic agents to improve the treatment and quality of life for patients requiring emergency hemostasis. Disclosures Hoffman: CSL-Behring: Consultancy, Research Funding; Boehringer Ingelheim: Research Funding; Novo Nordisk: Honoraria, Research Funding.

Functions of the DRY motif and intracellular loop 2 of human melanocortin 3 receptor

The melanocortin 3 receptor (MC3R) regulates several physiological functions, including feed efficiency, nutrient partitioning, fasting response, natriuresis, and immune reactions. Naturally occurring mutations in the MC3R gene have been shown to be associated with increased adiposity and lung diseases such as tuberculosis and cystic fibrosis. The DRY motif at the cytoplasmic end of transmembrane domain 3 (TM3) and the second intracellular loop 2 (ICL2) are known to be important for receptor function in several G protein-coupled receptors (GPCRs). To gain a better understanding of the functions of this domain in MC3R, we performed alanine-scanning mutagenesis on 18 residues. We showed that alanine mutation of 11 residues reduced the maximal binding and maximal cAMP production stimulated by agonists. Mutation of two residues did not change maximal binding but resulted in impaired signaling in the Gs–cAMP pathway. Mutation of five residues impaired signaling in the ERK1/2 pathway. We have also shown that alanine mutants of seven residues that were defective in the cAMP pathway were not defective in the ERK1/2 pathway, demonstrating biased signaling. In summary, we demonstrated that the cytoplasmic end of TM3 and the ICL2 were critical for MC3R function. We also reported for the first time biased signaling in MC3R.

Coagulation factor VII variants resistant to inhibitory antibodies

SummaryReplacement therapy is currently used to prevent and treat bleeding episodes in coagulation factor deficiencies. However, structural differences between the endogenous and therapeutic proteins might increase the risk for immune complications. This study was aimed at identifying factor (F)VII variants resistant to inhibitory antibodies developed after treatment with recombinant activated factor VII (rFVIIa) in a FVII-deficient patient homozygous for the p.A354V-p.P464Hfs mutation, which predicts trace levels of an elongated FVII variant in plasma. We performed fluorescent bead-based binding, ELISA-based competition as well as fluorogenic functional (activated FX and thrombin generation) assays in plasma and with recombinant proteins. We found that antibodies displayed higher affinity for the active than for the zymogen FVII (half-maximal binding at 0.54 ± 0.04 and 0.78 ± 0.07 BU/ml, respectively), and inhibited the coagulation initiation phase with a second-order kinetics. Isotypic analysis showed a polyclonal response with a large predominance of IgG1. We hypothesised that structural differences in the carboxyl-terminus between the inherited FVII and the therapeutic molecules contributed to the immune response. Intriguingly, a naturally-occurring, poorly secreted and 5-residue truncated FVII (FVII-462X) escaped inhibition. Among a series of truncated rFVII molecules, we identified a well-secreted and catalytically competent variant (rFVII-464X) with reduced binding to antibodies (half-maximal binding at 0.198 ± 0.003 BU/ml) as compared to the rFVII-wt (0.032 ± 0.002 BU/ml), which led to a 40-time reduced inhibition in activated FX generation assays. Taken together our results provide a paradigmatic example of mutation-related inhibitory antibodies, strongly support the FVII carboxyl-terminus as their main target and identify inhibitor-resistant FVII variants.

Mgr3p and Mgr1p Are Adaptors for the Mitochondrial i-AAA Protease Complex

By screening yeast knockouts for their dependence upon the mitochondrial genome, we identified Mgr3p, a protein that associates with the i-AAA protease complex in the mitochondrial inner membrane. Mgr3p and Mgr1p, another i-AAA-interacting protein, form a subcomplex that bind to the i-AAA subunit Yme1p. We find that loss of Mgr3p, like the lack of Mgr1p, reduces proteolysis by Yme1p. Mgr3p and Mgr1p can bind substrate even in the absence of Yme1p, and both proteins are needed for maximal binding of an unfolded substrate by the i-AAA complex. We speculate that Mgr3p and Mgr1p function in an adaptor complex that targets substrates to the i-AAA protease for degradation.

Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity

ABSTRACT CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of the repR promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5′-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 μM, respectively. In this work, we used a SELEX procedure to study copR operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated copR operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3′ positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated.

Structural determinants for activation and block of CFTR-mediated chloride currents by apigenin

Apigenin (4′,5,7-trihydroxyflavone) is an activator of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl−currents across epithelia at low concentrations and a blocker at high concentrations. We determined the roles of structural components of apigenin for both stimulation and block of Cl−currents across Calu-3 epithelia. The half-maximal binding affinity of apigenin for current stimulation ( Ks) was 9.1 ± 1.3 μM, and the rank-order of molecular structures was 7-hydroxyl > pyrone = 4′-hydroxyl > 5-hydroxyl. Both the 7-hydroxyl and the 4′-hydroxyl served as H-bond acceptors, whereas the 5-hydroxyl was an H-bond donor. The half-maximal binding affinity of apigenin during current block was 74 ± 11 μM. Blocked Cl−currents were structurally determined by 7-hydroxyl = 4′-hydroxyl > pyrone > 5-hydroxyl. Prestimulation of tissues with forskolin significantly affected activation kinetics and binding characteristics. After forskolin stimulation, Kswas 4.1 ± 0.9 μM, which was structurally determined by pyrone > all hydroxyls > single hydroxyls. In contrast, block of Cl−current by apigenin was not affected by forskolin stimulation. We conclude that apigenin binds to a stimulatory and an inhibitory binding site, which are distinguished by their affinities and the molecular interactions during binding.

Differential Binding to and Regulation of JAK2 by the SH2 Domain and N-Terminal Region of SH2-Bβ

ABSTRACT SH2-Bβ has been shown to bind via its SH2 (Src homology 2) domain to tyrosyl-phosphorylated JAK2 and strongly activate JAK2. In this study, we demonstrate the existence of an additional binding site(s) for JAK2 within the N-terminal region of SH2-Bβ (amino acids 1 to 555) and the ability of this region of SH2-B to inhibit JAK2. Four lines of evidence support the existence of this additional binding site(s). In a glutathione S-transferase pull-down assay, wild-type SH2-Bβ and SH2-Bβ(R555E) with a defective SH2 domain bind to both tyrosyl-phosphorylated JAK2 from growth hormone (GH)-treated cells and non-tyrosyl-phosphorylated JAK2 from control cells, whereas the SH2 domain of SH2-Bβ binds only to tyrosyl-phosphorylated JAK2 from GH-treated cells. Similarly, JAK2 is present in αSH2-B immunoprecipitates in the absence and presence of GH, with GH substantially increasing the coprecipitation of JAK2 with SH2-B. When coexpressed in COS cells, SH2-Bβ coimmunoprecipitates not only wild-type, tyrosyl-phosphorylated JAK2 but also kinase-inactive, non-tyrosyl-phosphorylated JAK2(K882E), although to a lesser extent. ΔC555 (amino acids 1 to 555 of SH2-Bβ) that lacks most of the SH2 domain binds similarly to wild-type JAK2 and kinase-inactive JAK2(K882E). Experiments using a series of N- and C-terminally truncated SH2-Bβ constructs indicate that the pleckstrin homology (PH) domain (amino acids 269 to 410) and amino acids 410 to 555 are necessary for maximal binding of SH2-Bβ to inactive JAK2, but neither region alone is sufficient for maximal binding. The SH2 domain of SH2-Bβ is necessary and sufficient for the stimulatory effect of SH2-Bβ on JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B. In contrast, ΔC555 lacking the SH2 domain, and to a lesser extent the PH domain alone, inhibits JAK2. ΔC555 also blocks JAK2-mediated tyrosyl phosphorylation of Stat5B in COS cells and GH-stimulated nuclear accumulation of Stat5B in 3T3-F442A cells. These data indicate that in addition to the SH2 domain, SH2-Bβ has one or more lower-affinity binding sites for JAK2 within amino acids 269 to 555. The interaction via this site(s) in SH2-B with inactive JAK2 seems likely to increase the local concentration of SH2-Bβ around JAK2, thereby facilitating binding of the SH2 domain to ligand-activated JAK2. This would result in a more rapid and robust cellular response to hormones and cytokines that activate JAK2. This interaction between inactive JAK2 and SH2-B may also help prevent abnormal activation of JAK2.

The pH 6 Antigen of Yersinia pestisBinds to β1-Linked Galactosyl Residues in Glycosphingolipids

ABSTRACT The Yersinia pestis pH 6 antigen was expressed by, and purified from, Escherichia coli containing cloned psa genes. By an enzyme-linked immunosorbence-based assay, purified pH 6 antigen bound to gangliotetraosylceramide (GM1A), gangliotriaosylceramide (GM2A), and lactosylceramide (LC) (designations follow the nomenclature of L. Svennerholm [J. Neurochem. 10:613–623, 1963]). Binding to GM1A, GM2A, and LC was saturable, with 50% maximal binding occurring at 498 ± 4, 390, and 196 ± 3 nM, respectively. Thin-layer chromatography (TLC) overlay binding confirmed that purified pH 6 antigen bound to GM1A, GM2A, and LC and also revealed binding to hydroxylated galactosylceramide. Intact E. coli cells which expressed the pH 6 antigen had a specificity similar to that of purified pH 6 in the TLC overlay assay except that nonhydroxylated galactosylceramide was also bound. The binding patterns observed indicate that the presence of β1-linked galactosyl residues in glycosphingolipids is the minimum determinant required for binding of the pH 6 antigen.

Functional characterization of albumin binding to the apical membrane of OK cells

We characterized binding of albumin to the apical membrane of opossum kidney (OK) cells using fluorescein isothiocyanate (FITC)-albumin (i.e., bovine serum albumin, BSA) as substrate. Functional analysis of binding data showed one specific binding site characterized by half-maximal binding (Michaelis constant, (Km) at 20 mg/l (300 nmol/l) and maximal binding capacity (Bmax) of 0.61 microgram/mg cellular protein. Excess of unlabeled albumin (BSA) inhibited binding at low concentrations of FITC-albumin completely but only partially at high concentrations. FITC-albumin binding was reversible and pH dependent. Km increased about sixfold when pH decreased from 7.4 to 5.0. The inhibitory effects of conalbumin, alpha-lactalbumin, and transferrin were significantly smaller compared with BSA. We conclude that OK cells express a high-affinity binding site for albumin on the apical membrane. This binding site is pH sensitive, binds albumin in the physiological range, and could be responsible for the effective receptor-mediated reabsorption of albumin in the proximal tubule.