Direct Binding Studies Research Articles

A CYCLIC PEPTIDE TARGETS GLIOBLASTOMA BY BINDING TO ABERRANTLY EXPOSED SNAP25.

Disease-specific changes in tumors and other diseased tissues are an important target of research because they provide clues on the pathophysiology of the disease as well as uncovering potentially useful markers for diagnosis and treatment. Here, we report a new cyclic peptide, CESPLLSEC (CES), that specifically accumulated (homed) in intracranial U87MG and the WT-GBM model of glioblastoma from intravenous (IV) injection, associating with the vasculature. Affinity chromatography of U87MG tumor extracts on insolubilized CES peptide identified Synaptosomal Associated Protein 25 (SNAP25) as a candidate target molecule (receptor) for CES. Several results supported the identification of SNAP25 as the CES receptor. IV-injected FAM-CES colocalized with SNAP25 in the tumors, and direct binding studies showed specific CES peptide binding to recombinant human SNAP25. A CES peptide-drug conjugate designed for photodynamic therapy showed selective cytotoxicity to SNAP25+ glioblastoma cell lines. Specific accumulation of systemically injected anti-SNAP25 antibody in U87MG glioblastoma, and labeling of intact U87MG cells with anti-SNAP in flow cytometry showed that SNAP25 is available from the circulation but not in normal tissues and that it is present at the cell surface. Using an array of ECM proteins and surface plasmon resonance revealed that SNAP25 binds moderately to collagen V and strongly to collagen VI. Modeling studies suggested that CES and collagen VI compete for the same binding site on SNAP25. Our results introduce CES as a valuable targeting peptide for drug delivery, and its receptor SNAP25 as a possible molecular marker of interest for glioblastoma.

Staphylococcal peroxidase inhibitor (SPIN): Residue-level investigation of the helical bundle domain

Myeloperoxidase is a critical component of the antibacterial arsenal of neutrophils, whereby it consumes H2O2 as an oxidant to convert halogen and pseudohalogen anions into cytotoxic hypohalous acids. Following phagocytosis by neutrophils, the human pathogen Staphylococcus aureus secretes a potent myeloperoxidase inhibitory protein, called SPIN, as part of its immune evasion repertoire. The matured S. aureus SPIN polypeptide consists of only 73 residues yet contains two functional domains: whereas the 60 residue C-terminal helical bundle domain is responsible for MPO binding, the 13 residue N-terminal domain is required to inhibit MPO. Previous studies have informed understanding of the SPIN N-terminal domain, but comparatively little is known about the helical domain insofar as the contribution of individual residues is concerned. To address this limitation, we carried out a residue-level structure/function investigation on the helical bundle domain of S. aureus SPIN. Using sequence conservation and existing structures of SPIN bound to human MPO as a guide, we selected residues L49, E50, H51, E52, Y55, and Y75 for interrogation by site-directed mutagenesis. We found that loss of L49 or E52 reduced SPIN activity by roughly an order of magnitude, but that loss of Y55 or H51 caused progressively greater loss of inhibitory potency. Direct binding studies by SPR showed that loss of inhibitory potency in these SPIN mutants resulted from a diminished initial interaction between the inhibitor and MPO. Together, our studies provide new insights into the structure/function relationships of SPIN and identify positions Y55 and H51 as critical determinants of SPIN function.

Tissue factor binds to and inhibits interferon-α receptor 1 signaling

Tissue factor (TF), which is a member of the cytokine receptor family, promotes coagulation and coagulation-dependent inflammation. TF also exerts protective effects through unknown mechanisms. Here, we showed that TF bound to interferon-α receptor 1 (IFNAR1) and antagonized its signaling, preventing spontaneous sterile inflammation and maintaining immune homeostasis. Structural modeling and direct binding studies revealed binding of the TF C-terminal fibronectin III domain to IFNAR1, which restricted the expression of interferon-stimulated genes (ISGs). Podocyte-specific loss of TF in mice (PodΔF3) resulted in sterile renal inflammation, characterized by JAK/STAT signaling, proinflammatory cytokine expression, disrupted immune homeostasis, and glomerulopathy. Inhibiting IFNAR1 signaling or loss of Ifnar1 expression in podocytes attenuated these effects in PodΔF3 mice. As a heteromer, TF and IFNAR1 were both inactive, while dissociation of the TF-IFNAR1 heteromer promoted TF activity and IFNAR1 signaling. These data suggest that the TF-IFNAR1 heteromer is a molecular switch that controls thrombo-inflammation.

Angiopoietin-2 binds to multiple interactive sites within von Willebrand factor

BackgroundBiosynthesis of von Willebrand factor (VWF) in endothelial cells drives the formation of storage-organelles known as Weibel-Palade bodies (WPBs). WPBs also contain several other proteins, including angiopoietin-2 (Ang-2). ObjectivesAt present, the molecular basis of the VWF–Ang-2 interaction is poorly understood. Here, we used immunosorbent-binding assays and specific recombinant VWF fragments to analyze VWF–Ang-2 interactions. ResultsWe found that VWF bound to immobilized Ang-2 most efficiently (half-maximal binding at 0.5 ± 0.1 μg/mL) under conditions of high CaCl2 (10 mM) and slightly acidic pH (6.4-7.0). Interestingly, several isolated recombinant VWF domains (A1/Fc, A2/Fc, D4/Fc, and D’D3-HPC4) displayed dose-dependent binding to immobilized Ang-2. Binding appeared specific, as antibodies against D’D3, A1, and A2 significantly reduced the binding of these domains to Ang-2. Complexes between VWF and Ang-2 in plasma could be detected by immunoprecipitation- and immunosorbent assays. Unexpectedly, control experiments also revealed complexes between VWF and angiopoietin-1 (Ang-1), a protein structurally homologous to Ang-2. Furthermore, direct binding studies showed dose-dependent binding of VWF to immobilized Ang-1 (half-maximal binding at 1.8 ± 1.0 μg/mL). Interestingly, rather than competing for Ang-1 binding, Ang-2 enhanced the binding of VWF to Ang-1 about 3-fold. Competition experiments further revealed that binding to VWF does not prevent Ang-1 and Ang-2 from binding to Tie-2. ConclusionOur data show that both Ang-1 and Ang-2 bind to VWF, seemingly using different interactive sites. Ang-2 modulates the binding of VWF to Ang-1, the (patho)-physiological consequences of which remain to be investigated.

MUC1-C is a master regulator of MICA/B NKG2D ligand and exosome secretion in human cancer cells

BackgroundThe MUC1-C protein evolved in mammals to protect barrier tissues from loss of homeostasis; however, MUC1-C promotes oncogenesis in association with chronic inflammation. Aberrant expression of MUC1-C in cancers has...

Tunable ultraviolet laser induces high signal-to-noise ratio in intrinsic fluorescence titration for protein- ligand interaction studies

Protein intrinsic fluorescence emitted by tryptophan residues is widely used for macromolecular mechanism studies, such as the protein binding by ligands and enzyme kinetics. However, modifications in fluorescence intensity can be weak using the intrinsic fluorescence in many proteins containing few tryptophan residues, or with their weak involvement in the binding. A low signal to noise ratio is a main drawback, due to the weak intensity of Xenon lamp illumination in the spectrofluorometer at the ultraviolet (UV) range, in addition to the small intrinsic fluorescence modifications with ligand binding, in many cases. We report herein the montage and the utilization of a laser light source in a spectrofluorometer in the UV range for the titration of protein intrinsic fluorescence. A significantly stronger modification of the protein intrinsic fluorescence was obtained with the titrations represented by E. coli Arginyl tRNA synthetase and human 17β-hydroxysteroid dehydrogenase type 7 with their natural ligands using a wavelength-tunable UV laser source. Also, a higher signal to noise ratio was obtained with the laser source. Finally, a similar KD was obtained with the titration using the two light sources. These results are supported by the direct binding study of equilibrium dialysis, showing that the laser source yields a reliable binding analysis. Thus, the utilization of a laser source in the intrinsic fluorescence titration and related usage can give a significant opening for the wider applications of the spectroscopy and photo technology to many new proteins with limited intrinsic fluorescence signals, while their mechanisms are critical to understand biochemistry and will facilitate medical applications. This study includes fundamental, applied, and interdisciplinary areas of physics.

Chemical, Analytical and Pharmacokinetic Characterisation of RO7304898, an API Consisting of Two Rapidly Interconverting Diastereoisomers.

Exploration of the chemical, analytical and pharmacokinetic properties of the API, RO7304898, an allosteric EGFR inhibitor, intended to be developed as a mixture of two rapidly interconverting diastereoisomers with composition ratio of approximately 1:1. Assessment of diastereoisomer stereochemistry, interconversion rates, binding to EGFR protein, metabolic stability and in vivo PK in Wistar-Han rats was conducted. The two diastereoisomers of the API undergo fast interconversion at physiologically relevant pH and direct EGFR binding studies revealed diastereoisomer B to be the active moiety. Pharmacokinetic studies in rat revealed a low-moderate total plasma clearance of the API along with similar plasma concentration-time profiles for diastereoisomers A and B, and the diastereoisomeric ratio reached stable equilibrium favoring formation of the potent diastereoisomer B. In in vitro incubations, the API was metabolically stable in plasma and hepatocyte suspension incubations in all species tested except that of rat hepatocytes. Additionally, only small species differences in the A:B composition were observed in vitro with the potent diastereoisomer B being the predominant form. We demonstrated that the API, a mixture of two diastereoisomers; A (impotent) and B (potent), undergoes rapid interconversion which is faster than the apparent distribution and elimination rates of the individual diastereoisomers in vivo in rat, serving to diminish concerns that separate diastereoisomer effects may occur in subsequent pharmacologic and pivotal toxicological studies. Whilst vigilant monitoring of the diastereoisomeric ratio will need to be continued, this data adds confidence on the development pathway for this API to the clinic.

Group B Streptococcus Surface Protein β: Structural Characterization of a Complement Factor H-Binding Motif and Its Contribution to Immune Evasion.

The β protein from group B Streptococcus (GBS) is a ∼132-kDa, cell-surface exposed molecule that binds to multiple host-derived ligands, including complement factor H (FH). Many details regarding this interaction and its significance to immune evasion by GBS remain unclear. In this study, we identified a three-helix bundle domain within the C-terminal half of the B75KN region of β as the major FH-binding determinant and determined its crystal structure at 2.5 Å resolution. Analysis of this structure suggested a role in FH binding for a loop region connecting helices α1 and α2, which we confirmed by mutagenesis and direct binding studies. Using a combination of protein cross-linking and mass spectrometry, we observed that B75KN bound to complement control protein (CCP)3 and CCP4 domains of FH. Although this binding site lies within a complement regulatory region of FH, we determined that FH bound by β retained its decay acceleration and cofactor activities. Heterologous expression of β by Lactococcus lactis resulted in recruitment of FH to the bacterial surface and a significant reduction of C3b deposition following exposure to human serum. Surprisingly, we found that FH binding by β was not required for bacterial resistance to phagocytosis by neutrophils or killing of bacteria by whole human blood. However, loss of the B75KN region significantly diminished bacterial survival in both assays. Although our results show that FH recruited to the bacterial surface through a high-affinity interaction maintains key complement-regulatory functions, they raise questions about the importance of FH binding to immune evasion by GBS as a whole.

Preclinical Evaluation of eFT226, a Novel, Potent and Selective eIF4A Inhibitor with Anti-Tumor Activity in B-Cell Malignancies

Translational control of oncoprotein expression has been implicated in the pathogenesis of multiple solid tumors and hematological malignancies. Protein synthesis is tightly regulated largely at the initiation stage through the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4A, an RNA helicase, is an essential component of the translation initiation complex and selectively regulates a subset of mRNAs based on the sequence and structure of the 5‘-untranslated region (UTR). B-cell receptor signaling activates eIF4A resulting in the selective upregulation of oncogenes involved in cell proliferation, survival and metastasis. B-cell malignancies are often associated with dysregulation of oncogenes or anti-apoptotic proteins (e.g., c-MYC, CCND1/3, BCL2 and MCL-1) that contain structured 5‘-UTRs and require enhanced eIF4A activity for translation.eFT226 is a potent and selective eIF4A inhibitor that promotes eIF4A binding to specific mRNA sequences and interferes with the assembly of the eIF4F initiation complex. eFT226 selectively inhibits translation of mRNAs containing longer 5‘-UTRs, an increased frequency of uORFs (upstream open reading frame), and polypurine and/or G-quadraplex recognition motifs. The sequence dependency of eFT226 translational inhibition was evaluated in an in vitro translation assay demonstrating potent inhibition of reporter constructs containing a polypurine motif in the 5‘-UTR (IC50 ~2 nM). Direct binding studies also confirmed the formation of a stable ternary complex between eFT226, eIF4A and mRNA oligonucleotides containing polypurine motifs. This results in selective and translational downregulation of a subset of genes that are important for tumor growth and survival. eFT226 showed potent anti-proliferative activity (EC50 < 15 nM) and an induction of apoptosis against a panel of B-cell lymphoma cell lines. Sensitivity to eFT226 was associated with dose-dependent decreases in the oncogenic drivers c-MYC, CCND1/3, BCL2 or MCL-1 in ABC (activated B-Cell) and GCB (germinal center B-cell) DLBCL (Diffuse Large B-cell Lymphoma) subtypes and Burkitt's lymphoma. eFT226 has good pharmacokinetic properties that result in potent in vivo efficacy with ≤ 1 mg/kg/week IV administration. Preclinical efficacy studies using eFT226 showed in vivo activity across hematological tumor models suggesting that eFT226 may be active in tumor types where overexpression or amplification of oncogenes such as c-MYC, CCND1/3, BCL2 or MCL-1 play a role in disease pathogenesis. These results demonstrate the potential of eFT226 for the treatment of B-cell malignancies. Clinical trials in patients with hematological and other malignancies are planned. DisclosuresThompson:eFFECTOR Therapeutics: Employment, Equity Ownership. Eam:eFFECTOR Therapeutics: Employment, Equity Ownership. Young:eFFECTOR Therapeutics: Employment, Equity Ownership. Fish:eFFECTOR Therapeutics: Employment, Equity Ownership. Chen:eFFECTOR Therapeutics: Employment, Equity Ownership. Barrera:eFFECTOR Therapeutics: Employment, Equity Ownership. Howard:eFFECTOR Therapeutics: Employment, Equity Ownership. Parra:eFFECTOR Therapeutics: Employment, Equity Ownership. Molter:eFFECTOR Therapeutics: Employment. Staunton:eFFECTOR Therapeutics: Employment, Equity Ownership. Hung:eFFECTOR Therapeutics: Employment, Equity Ownership. Parker:eFFECTOR Therapeutics: Employment, Equity Ownership. Chiang:eFFECTOR THERAPEUTICS: Employment, Equity Ownership. Wegerski:eFFECTOR Therapeutics: Employment, Equity Ownership. Nevarez:eFFECTOR Therapeutics: Employment, Equity Ownership. Clarine:eFFECTOR Therapeutics: Employment, Equity Ownership. Sperry:eFFECTOR THERAPEUTICS: Employment, Equity Ownership. Xiang:eFFECTOR Therapeutics: Employment, Equity Ownership. Tran:eFFECTOR Therapeutics: Employment, Equity Ownership. Nilewski:eFFECTOR Therapeutics: Employment, Equity Ownership. Packard:eFFECTOR Therapeutics: Employment, Equity Ownership. Michels:eFFECTOR Therapeutics: Employment, Equity Ownership. Sprengeler:eFFECTOR Therapeutics: Employment, Equity Ownership. Ernst:eFFECTOR Therapeutics: Employment, Equity Ownership. Reich:eFFECTOR Therapeutics: Employment, Equity Ownership. Webster:eFFECTOR THERAPEUTICS: Employment, Equity Ownership.

The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair.

Nucleotide excision repair (NER) in eukaryotes is orchestrated by the core form of the general transcription factor TFIIH, containing the helicases XPB, XPD and five ‘structural’ subunits, p62, p44, p34, p52 and p8. Recent cryo-EM structures show that p62 makes extensive contacts with p44 and in part occupies XPD’s DNA binding site. While p44 is known to regulate the helicase activity of XPD during NER, p62 is thought to be purely structural. Here, using helicase and adenosine triphosphatase assays we show that a complex containing p44 and p62 enhances XPD’s affinity for dsDNA 3-fold over p44 alone. Remarkably, the relative affinity is further increased to 60-fold by dsDNA damage. Direct binding studies show this preference derives from p44/p62’s high affinity (20 nM) for damaged ssDNA. Single molecule imaging of p44/p62 complexes without XPD reveals they bind to and randomly diffuse on DNA, however, in the presence of UV-induced DNA lesions these complexes stall. Combined with the analysis of a recent cryo-EM structure, we suggest that p44/p62 acts as a novel DNA-binding entity that enhances damage recognition in TFIIH. This revises our understanding of TFIIH and prompts investigation into the core subunits for an active role during DNA repair and/or transcription.

The molecular basis of how buried human leukocyte antigen polymorphism modulates natural killer cell function

Micropolymorphisms within human leukocyte antigen (HLA) class I molecules can change the architecture of the peptide-binding cleft, leading to differences in peptide presentation and T cell recognition. The impact of such HLA variation on natural killer (NK) cell recognition remains unclear. Given the differential association of HLA-B*57:01 and HLA-B*57:03 with the control of HIV, recognition of these HLA-B57 allomorphs by the killer cell immunoglobulin-like receptor (KIR) 3DL1 was compared. Despite differing by only two polymorphic residues, both buried within the peptide-binding cleft, HLA-B*57:01 more potently inhibited NK cell activation. Direct-binding studies showed KIR3DL1 to preferentially recognize HLA-B*57:01, particularly when presenting peptides with positively charged position (P)Ω-2 residues. In HLA-B*57:01, charged PΩ-2 residues were oriented toward the peptide-binding cleft and away from KIR3DL1. In HLA-B*57:03, the charged PΩ-2 residues protruded out from the cleft and directly impacted KIR3DL1 engagement. Accordingly, KIR3DL1 recognition of HLA class I ligands is modulated by both the peptide sequence and conformation, as determined by the HLA polymorphic framework, providing a rationale for understanding differences in clinical associations.

Basonuclin 1 deficiency causes testicular premature aging: BNC1 cooperates with TAF7L to regulate spermatogenesis.

Basonuclin (BNC1) is expressed primarily in proliferative keratinocytes and gametogenic cells. However, its roles in spermatogenesis and testicular aging were not clear. Previously we discovered a heterozygous BNC1 truncation mutation in a premature ovarian insufficiency pedigree. In this study, we found that male mice carrying the truncation mutation exhibited progressively fertility loss and testicular premature aging. Genome-wide expression profiling and direct binding studies (by chromatin immunoprecipitation sequencing) with BNC1 in mouse testis identified several spermatogenesis-specific gene promoters targeted by BNC1 including kelch-like family member 10 (Klhl10), testis expressed 14 (Tex14), and spermatogenesis and centriole associated 1 (Spatc1). Moreover, biochemical analysis showed that BNC1 was associated with TATA-box binding protein-associated factor 7 like (TAF7L), a germ cell-specific paralogue of the transcription factor IID subunit TAF7, both in vitro and in testis, suggesting that BNC1 might directly cooperate with TAF7L to regulate spermatogenesis. The truncation mutation disabled nuclear translocation of the BNC1/TAF7L complex, thus, disturbing expression of related genes and leading to testicular premature aging. Similarly, expressions of BNC1, TAF7L, Y-box-binding protein 2 (YBX2), outer dense fiber of sperm tails 1 (ODF1), and glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS) were significantly decreased in the testis of men with non-obstructive azoospermia. The present study adds to the understanding of the physiology of male reproductive aging and the mechanism of spermatogenic failure in infertile men.

Abstract 2698: eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of lymphoma

Abstract Dysregulated messenger RNA (mRNA) translation drives the pathogenesis of multiple hematological malignancies. In lymphoma this includes the upregulation of key driver oncogenes and anti-apoptotic proteins (e.g., MYC, CCND1/3, BCL2 and MCL1) that contain a highly structured 5’-untranslated region (UTR) in their mRNA requiring enhanced eIF4A helicase activity for translation. eIF4A is a component of the eIF4F translation initiation complex and catalyzes the ATP-dependent unwinding of RNA duplexes and facilitates 43S ribosome scanning within the 5’-UTR. The activation of oncogenic signaling pathways, including RAS and PI3K, enhance eIF4A activity through phosphorylation of eIF4B, eIF4G and PDCD4 which facilitates formation of eIF4F and full activation of eIF4A. The PI3K/AKT/mTOR pathway is frequently activated in lymphoma, promoting the translation of oncogenes with complex 5’-UTRs that are required for tumor cell proliferation, survival and metastasis. eFT226 is a potent and sequence selective eIF4A1 inhibitor that promotes eIF4A1 binding to specific 5’-UTR polypurine and/or G-quadraplex recognition motifs leading to a selective block in ribosome mRNA scanning. The sequence dependency of eFT226 translational inhibition was evaluated in cell-based reporter assays demonstrating &amp;gt;100-fold greater sensitivity for reporter constructs containing a polypurine motif in the 5’-UTR (IC50 ~2 nM). Direct binding studies also confirmed the formation of a stable ternary complex with increased drug residence time between eFT226, eIF4A1 and RNA oligonucleotides containing polypurine motifs. The ability of eFT226 to inhibit MYC or MCL1 expression was found to be dependent on the presence of their respective 5’-UTR supporting a translational regulation mechanism dependent on recognition elements within the 5’-UTR. eFT226 shows potent anti-proliferative activity (GI50 &amp;lt; 15 nM) against a panel of B-cell lymphoma cell lines. Treatment with eFT226 leads to coordinated inhibition of MYC, CCND1/3, BCL2 or MCL1 protein expression resulting in significant anti-tumor activity. eFT226 has good pharmacokinetic properties and exhibits significant in vivo activity across a panel of diffuse large B cell lymphoma (DLBCL), and Burkitt lymphoma tumor models with ≤1 mg/kg/week IV administration. Further evaluation of predictive markers of sensitivity or resistance has shown that tumors with mTOR mediated activation of eIF4A are most sensitive to eFT226. In addition, tumors with PTEN mutations do not exhibit activated eIF4A and are generally resistant to induction of apoptosis by eFT226, resulting in reduced in vivo efficacy. The association of eFT226 activity with PI3K/mTOR pathway activation and mutational status provides a means to identify patient subsets during clinical development. Clinical trials in patients with lymphoma and other malignancies are planned. Citation Format: Peggy A. Thompson, Boreth Eam, Nathan P. Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Eric Sung, Ana Parra, Jocelyn Staunton, Gary G. Chiang, Christopher J. Wegerski, Andres Nevarez, Jeff Clarine, Samuel Sperry, Alan Xiang, Chinh Tran, Christian Nilewski, Garrick K. Packard, Theodore Michels, Paul A. Sprengeler, Justin T. Ernst, Siegfried H. Reich, Kevin R. Webster. eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2698.

Anti-Factor XII Autoantibodies in Patients with Recurrent Pregnancy Loss Recognize the Second Epidermal Growth Factor-Like Domain.

Summary Background Factor XII (FXII) deficiency and autoantibodies that bind to FXII (anti-FXII) have been described in patients with adverse pregnancy outcomes, including recurrent pregnancy loss. It has been reported that FXII functions not only as a coagulation protein but also as a growth factor. Objectives We studied the association between anti-FXII and the epidermal growth factor (EGF) system in patients with recurrent pregnancy loss. Patients/Methods We used synthetic peptides that span the second EGF-like domain in the heavy chain of FXII (EGF2) in inhibition and direct binding studies to determine if anti-FXII antibodies recognize EGF2. Furthermore, we examined whether anti-FXII antibodies, which recognize EGF2, also recognize recombinant EGF and heparin-binding EGF-like growth factor (HB-EGF). Results Among 100 patients with recurrent pregnancy loss, the plasma of 23 patients (23.0%) recognized the synthetic peptide ASQ41, which covers EGF2. Among the 23 anti-ASQ41-positive patients, plasma samples from 13 patients (56.5%) recognized the 22-residue segment C-terminal half of ASQ41. Among the 23 anti-ASQ41-positive patients, the plasma of 17 patients (73.9%) recognized recombinant human EGF. Affinity-purified anti-FXII antibodies, which recognize ASQ41, also recognized recombinant EGF family proteins such as EGF and HB-EGF. Conclusions The autoantibodies in patients with recurrent pregnancy loss recognized the EGF2 domain in FXII and other proteins of the EGF family. Since proteins in the EGF family play an important role in normal pregnancy, autoantibody-associated disruption of the EGF system may cause pregnancy loss.

4,6- O-Pyruvyl Ketal Modified N-Acetylmannosamine of the Secondary Cell Wall Polysaccharide of Bacillus anthracis Is the Anchoring Residue for Its Surface Layer Proteins.

The secondary cell wall polysaccharide (SCWP) of Bacillus anthracis plays a key role in the organization of the cell envelope of vegetative cells and is intimately involved in host-guest interactions. Genetic studies have indicated that it anchors S-layer and S-layer-associated proteins, which are involved in multiple vital biological functions, to the cell surface of B. anthracis. Phenotypic observations indicate that specific functional groups of the terminal unit of SCWP, including 4,6- O-pyruvyl ketal and acetyl esters, are important for binding of these proteins. These observations are based on genetic manipulations and have not been corroborated by direct binding studies. To address this issue, a synthetic strategy was developed that could provide a range of pyruvylated oligosaccharides derived from B. anthracis SCWP bearing base-labile acetyl esters and free amino groups. The resulting oligosaccharides were used in binding studies with a panel of S-layer and S-layer-associated proteins, which identified structural features of SCWP important for binding. A single pyruvylated ManNAc monosaccharide exhibited strong binding to all proteins, making it a promising structure for S-layer protein manipulation. The acetyl esters and free amine of SCWP did not significantly impact binding, and this observation is contrary to a proposed model in which SCWP acetylation is a prerequisite for association of some but not all S-layer and S-layer-associated proteins.

Suppression of isoproterenol-induced cardiotoxicity in rats by raspberry ketone via activation of peroxisome proliferator activated receptor-α

The peroxisome proliferator-activated receptor-α (PPAR-α) controls the lipid and glucose metabolism and also affects inflammation, cell proliferation and apoptosis during cardiovascular disease. Raspberry ketone (RK) is a red raspberry (Rubusidaeus, Family-Rosaceae) plant constituent, which activates PPAR-α. This study was conducted to assess the cardioprotective action of RK against isoproterenol (ISO)-induced cardiotoxicity. Wistar rats were randomly divided into six groups (six rats/group). Rats were orally administered with RK (50, 100 and 200 mg/kg, respectively) and fenofibrate (standard, 80 mg/kg) for 28 days and ISO was administered (85 mg/kg, subcutaneously) on 27th and 28th day. Administration of ISO in rats significantly altered hemodynamic and electrocardiogram patterns, total antioxidant capacity, PPAR-α, and apolipoprotein C-III levels. These myocardial aberrations were further confirmed during infarct size, heart weight to body weight ratio and immunohistochemical assessments (caspase-3 and nuclear factor-κB). RK pretreatment (100 and 200 mg/kg) significantly protected rats against oxidative stress, inflammation, and dyslipidemia caused by ISO as demonstrated by change in hemodynamic, biochemical and histological parameters. The results so obtained were quite comparable with fenofibrate. Moreover, RK was found to have binding affinity with PPAR-α, as confirmed by docking analysis. PPAR-α expression and concentration was also found increased in presence of RK which gave impression that RK probably showed cardioprotection via PPAR-α activation, however direct binding study of RK with PPAR-α is needed to confirm this assumption.

Ubiquitin Receptor RPN13 Mediates the Inhibitory Interaction of Diphenyldihaloketones CLEFMA and EF24 With the 26S Proteasome.

The proteasome is a validated target in drug discovery for diseases associated with unusual proteasomal activity. Here we report that two diphenyldihaloketones, CLEFMA and EF24, inhibit the peptidase activity of the 26S proteasome. The objective of this study was to investigate interaction of these compounds with the proteasome and identify a putative target within the protein components of the 26S proteasome. We employed standard fluorogenic peptide-based proteasome activity assay for trypsin-like, chymotrypsin-like, and caspase-like activities of human purified 26S proteasome in cell-free conditions. GFPu-1 and HUVEC cells were used as proteasome reporter cells. Direct binding studies used purified 19S, 20S, 26S, and recombinant RPN13-Pru for interaction with biotinylated analogs of CLEFMA and EF24. The reaction mixtures were subjected to horizontal gel electrophoresis, streptavidin-blotting, pull-down assays, and immunoblotting. The identity of the interacting protein was determined by 2D gel electrophoresis and LC-MS/MS. Drug affinity responsive target stability technique was utilized to examine if CLEFMA binding confers protection to RPN13 against thermolysin-catalyzed proteolysis. We found that trypsin-and chymotrypsin-like activities of the 26S proteasome were reduced significantly by both compounds. The compounds also reduced the proteolytic activity in GFPu-1 and HUVEC cells, resulting in accumulation of ubiquitinated proteins without affecting the autophagy process. From direct binding assays a 43 kDa protein in the 26S proteasome was found to be the interacting partner. This protein was identified by tandem mass spectroscopy as regulatory particle subunit 13 (RPN13), a ubiquitin receptor in the 19S regulatory particle. Furthermore, binding of CLEFMA to RPN13 did not protect latter from thermolysin-mediated proteolysis. Together, this study showed diphenyldihaloketones as potential proteasome inhibitors for treatment of diseases with perturbed proteasome function. The results also unraveled RPN13 as a unique target of CLEFMA and EF24. As a result, these compounds inhibit both trypsin-like and chymotrypsin-like proteasome activities.

A Heterozygous Truncating Mutation in BNC1 Causes Male Subfertility: BNC1 Cooperates with TAF7L to Regulate Spermatogenesis

Basonuclin (BNC1) is expressed primarily in proliferative keratinocytes and gametogenic cells. However, its role in spermatogenesis is not clear. Previously we discovered a heterozygous BNC1 truncating mutation in a premature ovarian insufficiency pedigree. In this study, we found that male mice carrying the truncating mutation exhibited sub-fertility. Genome-wide expression profiling and direct binding studies(ChIP-seq) with BNC1 in mouse testis identified several spermatogenesis specific gene promoters targeted by BNC1 including Klhl10, Tex14, and Spatc1. Moreover, biochemical analysis showed that BNC1 was associated with TAF7L, a germ-cell-specific paralogue of the TFIID subunit TAF7, both in vitro and in testis, suggesting that BNC1 might directly cooperate with TAF7L to regulate spermatogenesis. The truncating mutation disabled nucleal translocation of the BNC1/TAF7L complex, thus disturbing expression of related genes. Similarly, expressions of BNC1, TAF7L, YBX2, ODF1, and GAPDHS were significantly decreased in the testis of men with NOA.The present study adds to the understanding of the physiology of male reproduction and the mechanism of spermatogenic failure in infertile men. Funding: This work was supported by National Key Research and Development Program of China (2017YFC1001003), National Key Research and Development Plan 459 (2017YFC1001303), the National Basic Research Program of China (No.2013CB967404), the National Natural Science Foundation of China (No. 81471421, 81270664), The competent department of Science & Technology Project (No. 2017FZA7013) and the Science Foundation for Distinguished Young Scholars of Zhejiang Province (No.LR14H040001). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The Medical Ethics Committee of Zhejiang University, approved the study protocol.

Multivalent Binding of Precision Glycooligomers on Soft Glycocalyx Mimicking Hydrogels.

We present a synthetic approach toward soft, glycooligomer-functionalized microgel particles mimicking carbohydrate presenting cell surfaces and analyze their specific binding to a model lectin (Concanavalin A, ConA). Focusing on multivalent presentation, a series of sequence-controlled glycooligomers with varying spacing and number of mannose units was synthesized and analyzed for the resulting glycooligomer-ConA affinity. Both direct binding and inhibition studies show a higher affinity with increasing the number of sugar moieties, but they level off for higher valent systems, indicating steric hindrance. Furthermore, the results suggest that increasing the scaffold length tends to decrease binding due to entropic repulsion, which could be compensated by larger scaffolds able to address multiple ConA binding sites. These findings were consistent in all assays (adhesion, fluorescence, and ITC) regardless of binding partner immobilization, demonstrating that flexible ligands exert similar binding modes in solution and when attached to polymer networks, which is relevant for designing glyco-functionalized materials.

Truncation of ADAMTS13 by Plasmin Enhances Its Activity in Plasma

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) cleaves von Willebrand Factor (VWF) multimers to control their thrombogenicity. The fibrinolytic enzyme plasmin can cleave VWF in a similar manner. However, plasmin can also cleave ADAMTS13, which ultimately inactivates it. This leaves the overall role of plasmin in primary haemostasis uncertain.We investigated the combined molecular effects of plasmin on VWF and ADAMTS13. We first identified that plasmin destroys FRETS-VWF73 substrate by cleaving the ADAMTS13 binding region in a buffered system. We next investigated how plasmin affects both VWF and ADAMTS13 under static conditions in plasma by western blotting. We found that globular VWF is largely protected from plasmin cleavage. However, ADAMTS13 is rapidly cleaved under these conditions, suggesting inactivation. Surprisingly, we observed that plasmin enhances ADAMTS13 activity in a modified two-stage FRETS-VWF73 assay that protects FRETS-VWF73 substrate from degradation. In direct binding studies under the same conditions, we found that plasmin generates multiple C-terminally truncated forms of ADAMTS13 with VWF-binding capacity. In an effort to seek evidence for this mechanism in vivo, we analysed plasma from patients with systemic amyloidosis, which is hallmarked by a hyperfibrinolytic state. We found that their plasma contained increased levels of C-terminally truncated forms of ADAMTS13, which correlated with their hyperfibrinolytic state.We propose that truncation of ADAMTS13 by plasmin abolishes intramolecular self-association, which improves interaction with unfolded VWF.