Trypanosoma cruzi bromodomain factor 2 (BDF2) binds to acetylated histones and is accumulated after UV irradiation

Histone tail post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) regulate many cellular processes. Among these modifications, phosphorylation, methylation and acetylation have already been described in trypanosomatid histones. Bromodomains, together with chromodomains and histone-binding SANT domains, were proposed to be responsible for “histone code” reading. The Trypanosoma cruzi genome encodes four coding sequences (CDSs) that contain a bromodomain, named TcBDF1-4. Here we show that one of those, TcBDF2, is expressed in discrete regions inside the nucleus of all the parasite life cycle stages and binds H4 and H2A purified histones from T. cruzi. Immunolocalization experiments using both anti-histone H4 acetylated peptides and anti-TcBDF2 antibodies determined that TcBDF2 co-localizes with histone H4 acetylated at lysines K10 and K14. TcDBF2 and K10 acetylated H4 interaction was confirmed by co-immunoprecipitation. It is also shown that TcBDF2 was accumulated after UV irradiation of T. cruzi epimastigotes. These results suggest that TcBDF2 could be taking part in a chromatin remodelling complex in T. cruzi.     

Homodimerization Controls the Fibroblast Growth Factor 9 Subfamily's Receptor Binding and Heparan Sulfate-Dependent Diffusion in the Extracellular Matrix

Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20''s receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.Fibroblast growth factor (FGF) signaling plays pleiotropic roles throughout the life spans of mammalian organisms, ranging from germ cell maturation, mesoderm induction, body plan formation, and organogenesis during embryonic development to serum phosphate homeostasis and glucose, bile acid, lipid, and cholesterol metabolism in the adult (3, 23, 27, 28, 57, 60, 62). The diversity of FGF signaling is underscored by virtue of the fact that aberrant FGF signaling leads to a wide array of human diseases, including skeletal and olfactory/reproductive syndromes, phosphate wasting disorders, and cancer (16, 60, 67). Recent data also implicate dysregulated FGF signaling in the etiology of neurodegenerative disorders, such as major depressive disorder and Parkinson''s disease (10, 63, 64).Based on pairwise sequence homology and phylogeny, the 18 bona fide mammalian FGFs (FGF1 to FGF10 and FGF16 to FGF23) are divided into six subfamilies (45). Five FGF subfamilies have high-to-moderate affinity for pericellular heparan sulfate (HS) glycosaminoglycans and thus diffuse locally within tissues to act in a paracrine fashion, whereas the poor affinity of the FGF19 subfamily for HS enables this subfamily to act in an endocrine manner (28, 38). All FGFs share a core homology region of about 120 amino acids, which fold into 12 antiparallel β strands (β1 to β12) that are arranged into three sets of four-stranded β sheets (β-trefoil fold) (39). The globular FGF core domain is flanked by highly divergent N- and C-terminal extensions, which are the principal regions responsible for the different biology of FGFs.FGFs exert their diverse actions by binding and activating FGF receptors (FGFRs) in an HS-dependent fashion (51, 53, 69). There are four distinct mammalian FGFR genes (FGFR1 to FGFR4), each coding for a single-pass transmembrane tyrosine kinase receptor whose ectodomain consists of three immunoglobulin-like domains (D1 to D3) connected by flexible linkers and whose intracellular domain contains the conserved tyrosine kinase domain flanked by the juxtamembrane (JM) and C-terminal regions (38). The 210-amino-acid-long D2-D3 segment of the ectodomain is both necessary and sufficient for ligand binding (20, 51, 52, 58, 70).FGF signaling is tightly regulated by spatial and temporal expression of ligands, receptors, HS cofactors, and most critically by means of FGF-FGFR binding specificity. The tissue-specific alternative splicing in the D3 domain of FGFR1 to FGFR3 is the main mechanism by which FGF-FGFR binding specificity is regulated. This splicing event gives rise to epithelial “b” isoforms (FGFR1b to FGFR3b) and mesenchymal “c” isoforms (FGFR1c to FGFR3c) (24, 25, 47, 68), which differ from one another at the primary sequences of their key ligand binding regions and thus in their FGF binding specificity/promiscuity profiles. Most FGFs are also expressed in either epithelial or mesenchymal tissues and exhibit specificity for FGFR isoforms expressed in the opposite tissues. This results in the establishment of a bidirectional signaling loop between the epithelium and mesenchyme that is essential for organogenesis and tissue homeostasis. It is well established that FGF7 and FGF10, which are expressed exclusively in the mesenchyme, activate specifically FGFR2b to mediate mesenchymal-to-epithelial signaling in the lung, prostate, and lacrimal, mammary, and salivary glands (19, 29, 35, 36, 59). Several lines of genetic and biochemical evidence suggest that the members of the FGF9 subfamily, which includes FGF9, FGF16, and FGF20, convey the reciprocal signaling from the epithelium to the mesenchyme. In the prostate, the epithelial-specific FGF9 has been shown to activate mesenchymal FGFR3c isoforms (25). In the heart, FGF9, FGF16, and FGF20 in the epicardium and endocardium stimulate myocardial proliferation and differentiation in vivo, acting redundantly through FGFR1c and FGFR2c (32). Analysis of FGF9-deficient mice has identified FGF9 as a reciprocal epithelial-to-mesenchymal signal required for morphogenesis of the lung, cecum, small intestine, and inner ear (14, 49, 65, 71). In addition, studies in zebra fish show that FGF16 and FGF20 are apical ectodermal ridge factors that are required for pectoral fin bud outgrowth and, in general, for cell proliferation and differentiation of the mesenchyme (41, 66).In light of the key role of the FGF9 subfamily in tissue homeostasis, it is essential to investigate the molecular mechanisms by which the activity of this subfamily is regulated. Our previous structural and in vitro studies of FGF9 showed that homodimerization masks FGF9''s key receptor binding sites, suggesting that ligand dimerization may autoinhibit FGF9''s biologic activity (50). In this report, we show that, like FGF9, FGF20 also homodimerizes in the crystal and in solution. Characterization of the dimer interface mutations in vitro and in living cells demonstrates that ligand homodimerization autoinhibits FGF9 and FGF20 signaling by suppressing both receptor binding and HS-dependent diffusion in the extracellular matrix (ECM). Our study is the first to implicate ligand dimerization as an autoregulatory mechanism in growth factor bioactivity.     

Identification and characterization of novel recombinant vaccine antigens for immunization against genital Chlamydia trachomatis

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide, with over 91 million cases estimated annually. An effective subunit vaccine against Chlamydia may require a multivalent subunit cocktail of antigens in a single formulation for broad coverage of a heterogeneous major histocompatibility complex population. Herein, we describe the identification of novel C. trachomatis antigens by CD4+ and CD8+ T-cell expression cloning, serological expression cloning, and an in silico analysis of the C. trachomatis genome. These antigens elicited human CD4+ T-cell responses, and a subset proved to be immunogenic and protective when administered as immunoprophylactic vaccines against C. trachomatis challenge. Candidate vaccines consisting of the prioritized C. trachomatis antigens adjuvanted in a GlaxoSmithKline proprietary AS01B adjuvant were prioritized based on induction of solid protection against challenge in C57BL/6 and BALB/c mice with C. trachomatis . Some of the vaccines prevented bacterial shedding and colonization of the upper genital tract to varying degrees by mechanisms that may include CD4+ T cells.     

Discovery of a potent,selective and orally bioavailable 3,9-diazaspiro[5.5]undeca-2-one CCR5 antagonist

Replacement of the cyclic carbamate in our previously disclosed 1-oxa-3,9-diazaspiro[5.5]undecan-2-one template led to the discovery of two novel series of 3,9-diazaspiro[5.5]undecane and undeca-2-one CCR5 antagonists. The synthesis, SAR, and antiviral activities of these two series are described. One compound (32) was found to have attractive combination of antiviral potency, selectivity, and pharmacokinetic profile. The asymmetric synthesis of 32 was also accomplished and both enantiomers were equally potent.     

Discovery of the first known small-molecule inhibitors of heme-regulated eukaryotic initiation factor 2α (HRI) kinase

A series of indeno[1,2-c]pyrazoles were discovered to be the first known inhibitors of heme-regulated eukaryotic initiation factor 2α (HRI) kinase. The synthesis, structure–activity relationship profile, and in-vitro pharmacological characterization of this inaugural series of HRI kinase inhibitors are detailed.     

Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells

TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18^®:DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.     

Cryoelectron Microscopy Structure of Purified γ-Secretase at 12 Å Resolution

γ-Secretase, an integral membrane protein complex, catalyzes the intramembrane cleavage of the β-amyloid precursor protein (APP) during the neuronal production of the amyloid β-peptide. As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimer's disease. Existing biochemical studies conflict on the oligomeric assembly state of the protease complex, and its detailed structure is not known. Here, we report that purified active human γ-secretase in digitonin has a total molecular mass of ∼ 230 kDa when measured by scanning transmission electron microscopy. This result supports a complex that is monomeric for each of the four component proteins. We further report the three-dimensional structure of the γ-secretase complex at 12 Å resolution as obtained by cryoelectron microscopy and single-particle image reconstruction. The structure reveals several domains on the extracellular side, three solvent-accessible low-density cavities, and a potential substrate-binding surface groove in the transmembrane region of the complex.