Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its cognate receptor MET play several essent... more Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in post-natal life of epithelial organs such as liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search for HGF/SF mimics for therapy. In this work, we describe the rational design, production and characterisation of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF placed in tandem. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide a...
Auto-antibodies are classically associated with autoimmune diseases, where they are an integral p... more Auto-antibodies are classically associated with autoimmune diseases, where they are an integral part of diagnostic panels. However, recent evidence is accumulating on the presence of auto-antibodies against single or selected panels of auto-antigens in many types of cancer. Auto-antibodies might initially represent an epiphenomenon derived from the inflammatory environment induced by the tumor. However, their effect on tumor evolution can be crucial, as is discussed in this paper. It has been demonstrated that some of these auto-antibodies can be used for early detection and cancer staging, as well as for monitoring of cancer regression during treatment and follow up. Interestingly, certain auto-antibodies were found to promote cancer progression and metastasis, while others contribute to the body’s defense against it. Moreover, auto-antibodies are of a polyclonal nature, which means that often several antibodies are involved in the response to a single tumor antigen. Dissection of ...
Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB10... more Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apolipoprotein (a) [apo(a)]. The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analogue ε-aminocaproic acid (EACA). Interestingly, KIV-2 presents a Lysine Binding Site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to...
Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibod... more Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter k D , indicating attractive interactions that are beneficial for crystallizat...
NK1 is a splice variant of the polypeptide growth factor HGF/SF that consists of the N terminal (... more NK1 is a splice variant of the polypeptide growth factor HGF/SF that consists of the N terminal (N) and first kringle (K) domains and retains receptor binding and signalling. While NK1 behaves as a monomer in solution, two independent crystallographic structures have previously shown an identical, tightly packed dimer. Here we report a novel orthorhombic crystal form of NK1 at 2.5 A resolution in which four NK1 protomers are packed in two distinct dimers in the asymmetric unit. Although the basic architecture of the new NK1 dimers is similar to the two described earlier, the new crystal form demonstrates extensive hinge movement between the N and K domain that leads to re-orientation of the receptor-binding sites. The hinge bending is evidence of the paucity of strong interactions between domains within the protomer, in contrast to the extensive interactions between protomers in the dimer. These observations are consistent with domain swapping in the dimer, such that the interdomain interactions of the monomer are replaced by equivalent interprotomer interactions in the dimer and offer a route for protein engineering of NK1 variants which may act as receptor antagonists.
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatm... more Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatment. The Hepatocyte Growth Factor/Scatter Factor (HGF/SF), through its receptor MET, is one of the most potent survival-promoting factors for motor neurons (MN) and is known as a modulator of immune cell function. We recently developed a novel recombinant MET agonist optimized for therapy, designated K1K1. K1K1 was ten times more potent than HGF/SF in preventing MN loss in an in vitro model of ALS. Treatments with K1K1 delayed the onset of muscular impairment and reduced MN loss and skeletal muscle denervation of superoxide dismutase 1 G93A (SOD1G93A) mice. This effect was associated with increased levels of phospho-extracellular signal-related kinase (pERK) in the spinal cord and sciatic nerves and the activation of non-myelinating Schwann cells. Moreover, reduced activated microglia and astroglia, lower T cells infiltration and increased interleukin 4 (IL4) levels were found in the lum...
SMARCA4 chromatin remodelling factor is mutated in 11% of Coffin-Siris syndrome (CSS) patients an... more SMARCA4 chromatin remodelling factor is mutated in 11% of Coffin-Siris syndrome (CSS) patients and in almost all small-cell carcinoma of the ovary hypercalcaemic type (SCCOHT) tumours. Missense mutations with gain-of-function or dominant-negative effects are associated with CSS, whereas inactivating mutations, leading to loss of SMARCA4 expression, have been exclusively found in SCCOHT. We applied whole-exome sequencing to study a 15-year-old patient with mild CSS who concomitantly developed SCCOHT at age 13 years. Interestingly, our patient also showed congenital microphthalmia, which has never previously been reported in CSS patients. We detected a de novo germline heterozygous nonsense mutation in exon 19 of SMARCA4 (c.2935C > T;p.Arg979*), and a somatic frameshift mutation in exon 6 (c.1236_1236delC;p.Gln413Argfs*88), causing complete loss of SMARCA4 immunostaining in the tumour. The immunohistochemical findings are supported by the observation that the c.2935C > T mutant transcript was detected by reverse transcription polymerase chain reaction at a much lower level than the wild-type allele in whole blood and the lymphoblastoid cell line of the proband, confirming nonsense-mediated mRNA decay. Accordingly, immunoblotting demonstrated that there was approximately half the amount of SMARCA4 protein in the proband's cells as in controls. This study suggests that SMARCA4 constitutional mutations associated with CSS are not necessarily non-truncating, and that haploinsufficiency may explain milder CSS phenotypes, as previously reported for haploinsufficient ARID1B. In addition, our case supports the dual role of chromatin remodellers in developmental disorders and cancer, as well as the involvement of SMARCA4 in microphthalmia, confirming previous findings in mouse models and the DECIPHER database. Finally, we speculate that mild CSS might be under-recognized in a proportion of SCCOHT patients harbouring SMARCA4 mutations.
Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, m... more Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, many aspects of the role of androgens in adult teleost spermatogenesis have remained elusive. Since oestrogens inhibit androgen synthesis, we used an oestrogen-induced androgen depletion model to identify androgen-dependent stages during adult zebrafish spermatogenesis. Exposure to 10 nM 17β-oestradiol (E2) in vivo at least halved the mass of differentiating germ cells (from type B spermatogonia to spermatids), while type A spermatogonia accumulated. Studies on the cellular dynamics revealed that a reduction of spermatogonial proliferation together with an inhibition of their differentiation to type B spermatogonia were the basis for the oestrogen-mediated disturbance of spermatogenesis. The capacity of the zebrafish testis to produce 11-ketotestosterone as well as the expression of steroidogenesis-related genes was markedly decreased after in vivo oestrogen exposure. Moreover, the androg...
Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal h... more Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in -strands 2 (Lys 74), 4 (Tyr 124 , Asn 129 , and Thr 130), and 5 (Asp 150 and Asp 153) of the FSHR ECD identified in the human FSH⅐FSHR ECD crystal structure as contact sites with the common glycoprotein hormone ␣-subunit, and on noncontact residues in -strands 2 (Ser 78) and 8 (Asp 224 and Ser 226) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported ␣-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH⅐FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.
Crosstalk between signal transduction pathways provides a complex intracellular avenue for fine t... more Crosstalk between signal transduction pathways provides a complex intracellular avenue for fine tuning of hormone-induced signals. Over the last few years, we have studied the signaling mechanisms of three locust adipokinetic hormones (AKHs), which control mobilization of energy reserves from insect fat body as fuels for flight and transduce their signals via adenylyl cyclaseand phospholipase C-(PLC) dependent pathways. In this study, we examine possible crosstalk between these signaling routes. We show that cAMP does not affect basal and AKH-stimulated inositol phosphate (InsP n) production. Incubation of fat body with aluminium fluoride, an activator of G proteins, increased InsP n levels by 77%, whereas cholera toxin and pertussis toxin were ineffective. This implies that fat body PLC is not activated by G␥, but possibly by G q ␣. The involvement of this G protein in AKH signaling was demonstrated by our observation that the GPAntagonist-2A, which antagonizes G q , attenuated glycogen phosphorylase activation by AKH-I. As plasma membrane Ca 2 + channels may constitute another target for cAMP-mediated modulation, we studied the type of channels involved in AKH signaling using a variety of L-, N-and T-type Ca 2 + channel inhibitors. None of these blocked AKH-induced glycogen phosphorylase activation, suggesting that voltage-dependent Ca 2 + channels do not mediate AKH-induced Ca 2 + influx.
Proceedings of the National Academy of Sciences of the United States of America, Jan 3, 2010
Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proa... more Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.
This study aimed to improve, using the zebrafish model, our understanding of the distinct roles o... more This study aimed to improve, using the zebrafish model, our understanding of the distinct roles of pituitary gonadotropins FSH and LH in regulating testis functions in teleost fish. We report, for the first time in a vertebrate species, that zebrafish Leydig cells as well as Sertoli cells express the mRNAs for both gonadotropin receptors (fshr and lhcgr). Although Leydig cell fshr expression has been reported in other piscine species and may be a common feature of teleost fish, Sertoli cell lhcgr expression has not been reported previously and might be related to the undifferentiated gonochoristic mode of gonadal sex differentiation in zebrafish. Both recombinant zebrafish (rzf) gonadotropins (i.e. rzfLH and rzfFSH) stimulated androgen release in vitro and in vivo, with rzfFSH being significantly more potent than rzfLH. Forskolin-induced adenylate cyclase activation mimicked, whereas the protein kinase A inhibitor H-89 significantly reduced, the gonadotropin-stimulated androgen release. Therefore, we conclude that both FSH receptor and LH/choriogonadotropin receptor signaling are predominantly mediated through the cAMP/protein kinase A pathway to promote steroid production. Despite this similarity, other downstream mechanisms seem to differ. For example, rzfFSH up-regulated the testicular mRNA levels of a number of steroidogenesis-related genes both in vitro and in vivo, whereas rzfLH or human chorionic gonadotropin did not. Although not fully understood at present, these differences could explain the capacity of FSH to support both steroidogenesis and spermatogenesis on a long-term basis, whereas LH-stimulated steroidogenesis might be a more acute process, possibly restricted to periods during which peak steroid levels are required. (Endocrinology 151: 2349-2360, 2010) T he pituitary gonadotropins LH and FSH play critical roles in regulating male reproduction across vertebrates (1-3). In mammals, the specific activities of both gonadotropins are clearly defined, given the highly specific interactions between each hormone and its respective receptor, LH/choriogonadotropin receptor (Lhcgr) and FSH receptor (Fshr). In addition, Lhcgr and Fshr expression in mammalian testis is restricted to Leydig and Sertoli cells, respectively (1, 2, 4). Hence, LH predominantly controls Leydig cell steroidogenesis, whereas FSH regulates Sertoli cell functions (2, 4-6). In teleost fish, conversely, the biological activities of FSH and LH seem to be broader because both are strong steroidogenic hormones (7-16
Arteriosclerosis, Thrombosis, and Vascular Biology, 2009
Objectives— Several findings argue for a protective effect of high-density lipoproteins (HDLs) ag... more Objectives— Several findings argue for a protective effect of high-density lipoproteins (HDLs) against endothelial dysfunction. The molecular mechanisms underlying this protective effect are not fully understood, although recent works suggest that the actions of HDL on the endothelium are initiated by multiple interactions between HDLs (lipid or protein moiety) and cell surface receptors. We previously showed that the mitochondrial related F 1 -ATPase is a cell surface receptor for HDLs and their main atheroprotective apolipoprotein (apoA-I). Herein we test the hypothesis that the cell surface F 1 -ATPase may contribute to the ability of apoA-I and HDLs to maintain endothelial cell survival. Methods and Results— Cell imaging and binding assays confirmed the presence of the F 1 -ATPase at the surface of human umbilical vein endothelial cells (HUVECs) and its ability to bind apoA-I. Cell surface F 1 -ATPase activity (ATP hydrolysis into ADP) was stimulated by apoA-I and was inhibited ...
Clustering of synthetic biotinylated K1 domain from HGF/SF by streptavidin is sufficient to gener... more Clustering of synthetic biotinylated K1 domain from HGF/SF by streptavidin is sufficient to generate a potent MET agonist.
Progestagenic sex steroid hormones play critical roles in reproduction across vertebrates, includ... more Progestagenic sex steroid hormones play critical roles in reproduction across vertebrates, including teleost fish. To further our understanding of how progesterone modulates testis functions in fish, we set out to clone a progesterone receptor (pgr) cDNA exhibiting nuclear hormone receptor features from zebrafish testis. The open reading frame of pgr consists of 1854 bp, coding for a 617-amino acid-long protein showing the highest similarity with other piscine Pgr proteins. Functional characterization of the receptor expressed in mammalian cells revealed that zebrafish Pgr exhibited progesterone-specific, dose-dependent induction of reporter gene expression, with 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), a typical piscine progesterone, showing the highest potency. Expression of pgr mRNA: 1) appeared in embryos at 8 h after fertilization; 2) was significantly higher in developing ovary than in early transforming testis at 4 wk of age but vice versa in young adults at 12 wk of...
Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proa... more Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.
Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal h... more Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in-strands 2 (Lys 74), 4 (Tyr 124 , Asn 129 , and Thr 130), and 5 (Asp 150 and Asp 153) of the FSHR ECD identified in the human FSHFSHR ECD crystal structure as contact sites with the common glycoprotein hormone-subunit, and on noncontact residues in-strands 2 (Ser 78) and 8 (Asp 224 and Ser 226) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSHFSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.
NK1 is a splice variant of the polypeptide growth factor HGF/SF that consists of the N terminal (... more NK1 is a splice variant of the polypeptide growth factor HGF/SF that consists of the N terminal (N) and first kringle (K) domains and retains receptor binding and signalling. While NK1 behaves as a monomer in solution, two independent crystallographic structures have previously shown an identical, tightly packed dimer. Here we report a novel orthor-hombic crystal form of NK1 at 2.5 A ˚ resolution in which four NK1 proto-mers are packed in two distinct dimers in the asymmetric unit. Although the basic architecture of the new NK1 dimers is similar to the two described earlier, the new crystal form demonstrates extensive hinge movement between the N and K domain that leads to reorientation of the receptor-binding sites. The hinge bending is evidence of the paucity of strong interactions between domains within the protomer, in contrast to the extensive interactions between protomers in the dimer. These observations are consistent with domain swapping in the dimer, such that the interdomain interactions of the monomer are replaced by equivalent interprotomer interactions in the dimer and offer a route for protein engineering of NK1 variants which may act as receptor antagonists.
Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, m... more Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, many aspects of the role of androgens in adult teleost spermatogenesis have remained elusive. Since oestrogens inhibit androgen synthesis, we used an oestrogen-induced androgen depletion model to identify androgen-dependent stages during adult zebrafish spermato-genesis. Exposure to 10 nM 17b-oestradiol (E 2) in vivo at least halved the mass of differentiating germ cells (from type B spermatogonia to spermatids), while type A spermatogonia accumulated. Studies on the cellular dynamics revealed that a reduction of spermatogonial proliferation together with an inhibition of their differentiation to type B spermatogonia were the basis for the oestrogen-mediated disturbance of spermato-genesis. The capacity of the zebrafish testis to produce 11-ketotestosterone as well as the expression of steroidogen-esis-related genes was markedly decreased after in vivo oestrogen exposure. Moreover, the androgen-release response to recom-binant zebrafish Lh was lost after oestrogen exposure. We conclude that oestrogen exposure caused a state of androgen insufficiency in adult male zebrafish. Since the downregulation of the steroidogenic system as well as the disturbance of spermatogenesis in testicular explants exposed to E 2 ex vivo was much less severe than after in vivo exposure, the main inhibitory effect appears to be exerted via feedback inhibition of gonadotropin release. This experimental setup helped to identify spermatogonial proliferation and their differentiation as androgen targets in adult zebrafish spermatogenesis.
Crosstalk between signal transduction pathways provides a complex intracellular avenue for fine t... more Crosstalk between signal transduction pathways provides a complex intracellular avenue for fine tuning of hormone-induced signals. Over the last few years, we have studied the signaling mechanisms of three locust adipokinetic hormones (AKHs), which control mobilization of energy reserves from insect fat body as fuels for flight and transduce their signals via adenylyl cyclase-and phospholipase C-(PLC) dependent pathways. In this study, we examine possible crosstalk between these signaling routes. We show that cAMP does not affect basal and AKH-stimulated inositol phosphate (InsP n) production. Incubation of fat body with aluminium fluoride, an activator of G proteins, increased InsP n levels by 77%, whereas cholera toxin and pertussis toxin were ineffective. This implies that fat body PLC is not activated by G, but possibly by G q. The involvement of this G protein in AKH signaling was demonstrated by our observation that the GPAntagonist-2A, which antagonizes G q , attenuated glycogen phosphorylase activation by AKH-I. As plasma membrane Ca 2 + channels may constitute another target for cAMP-mediated modulation, we studied the type of channels involved in AKH signaling using a variety of L-, N-and T-type Ca 2 + channel inhibitors. None of these blocked AKH-induced glycogen phosphorylase activation, suggesting that voltage-dependent Ca 2 + channels do not mediate AKH-induced Ca 2 + influx.
Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its cognate receptor MET play several essent... more Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in post-natal life of epithelial organs such as liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search for HGF/SF mimics for therapy. In this work, we describe the rational design, production and characterisation of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF placed in tandem. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide a...
Auto-antibodies are classically associated with autoimmune diseases, where they are an integral p... more Auto-antibodies are classically associated with autoimmune diseases, where they are an integral part of diagnostic panels. However, recent evidence is accumulating on the presence of auto-antibodies against single or selected panels of auto-antigens in many types of cancer. Auto-antibodies might initially represent an epiphenomenon derived from the inflammatory environment induced by the tumor. However, their effect on tumor evolution can be crucial, as is discussed in this paper. It has been demonstrated that some of these auto-antibodies can be used for early detection and cancer staging, as well as for monitoring of cancer regression during treatment and follow up. Interestingly, certain auto-antibodies were found to promote cancer progression and metastasis, while others contribute to the body’s defense against it. Moreover, auto-antibodies are of a polyclonal nature, which means that often several antibodies are involved in the response to a single tumor antigen. Dissection of ...
Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB10... more Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apolipoprotein (a) [apo(a)]. The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analogue ε-aminocaproic acid (EACA). Interestingly, KIV-2 presents a Lysine Binding Site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to...
Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibod... more Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter k D , indicating attractive interactions that are beneficial for crystallizat...
NK1 is a splice variant of the polypeptide growth factor HGF/SF that consists of the N terminal (... more NK1 is a splice variant of the polypeptide growth factor HGF/SF that consists of the N terminal (N) and first kringle (K) domains and retains receptor binding and signalling. While NK1 behaves as a monomer in solution, two independent crystallographic structures have previously shown an identical, tightly packed dimer. Here we report a novel orthorhombic crystal form of NK1 at 2.5 A resolution in which four NK1 protomers are packed in two distinct dimers in the asymmetric unit. Although the basic architecture of the new NK1 dimers is similar to the two described earlier, the new crystal form demonstrates extensive hinge movement between the N and K domain that leads to re-orientation of the receptor-binding sites. The hinge bending is evidence of the paucity of strong interactions between domains within the protomer, in contrast to the extensive interactions between protomers in the dimer. These observations are consistent with domain swapping in the dimer, such that the interdomain interactions of the monomer are replaced by equivalent interprotomer interactions in the dimer and offer a route for protein engineering of NK1 variants which may act as receptor antagonists.
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatm... more Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatment. The Hepatocyte Growth Factor/Scatter Factor (HGF/SF), through its receptor MET, is one of the most potent survival-promoting factors for motor neurons (MN) and is known as a modulator of immune cell function. We recently developed a novel recombinant MET agonist optimized for therapy, designated K1K1. K1K1 was ten times more potent than HGF/SF in preventing MN loss in an in vitro model of ALS. Treatments with K1K1 delayed the onset of muscular impairment and reduced MN loss and skeletal muscle denervation of superoxide dismutase 1 G93A (SOD1G93A) mice. This effect was associated with increased levels of phospho-extracellular signal-related kinase (pERK) in the spinal cord and sciatic nerves and the activation of non-myelinating Schwann cells. Moreover, reduced activated microglia and astroglia, lower T cells infiltration and increased interleukin 4 (IL4) levels were found in the lum...
SMARCA4 chromatin remodelling factor is mutated in 11% of Coffin-Siris syndrome (CSS) patients an... more SMARCA4 chromatin remodelling factor is mutated in 11% of Coffin-Siris syndrome (CSS) patients and in almost all small-cell carcinoma of the ovary hypercalcaemic type (SCCOHT) tumours. Missense mutations with gain-of-function or dominant-negative effects are associated with CSS, whereas inactivating mutations, leading to loss of SMARCA4 expression, have been exclusively found in SCCOHT. We applied whole-exome sequencing to study a 15-year-old patient with mild CSS who concomitantly developed SCCOHT at age 13 years. Interestingly, our patient also showed congenital microphthalmia, which has never previously been reported in CSS patients. We detected a de novo germline heterozygous nonsense mutation in exon 19 of SMARCA4 (c.2935C > T;p.Arg979*), and a somatic frameshift mutation in exon 6 (c.1236_1236delC;p.Gln413Argfs*88), causing complete loss of SMARCA4 immunostaining in the tumour. The immunohistochemical findings are supported by the observation that the c.2935C > T mutant transcript was detected by reverse transcription polymerase chain reaction at a much lower level than the wild-type allele in whole blood and the lymphoblastoid cell line of the proband, confirming nonsense-mediated mRNA decay. Accordingly, immunoblotting demonstrated that there was approximately half the amount of SMARCA4 protein in the proband's cells as in controls. This study suggests that SMARCA4 constitutional mutations associated with CSS are not necessarily non-truncating, and that haploinsufficiency may explain milder CSS phenotypes, as previously reported for haploinsufficient ARID1B. In addition, our case supports the dual role of chromatin remodellers in developmental disorders and cancer, as well as the involvement of SMARCA4 in microphthalmia, confirming previous findings in mouse models and the DECIPHER database. Finally, we speculate that mild CSS might be under-recognized in a proportion of SCCOHT patients harbouring SMARCA4 mutations.
Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, m... more Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, many aspects of the role of androgens in adult teleost spermatogenesis have remained elusive. Since oestrogens inhibit androgen synthesis, we used an oestrogen-induced androgen depletion model to identify androgen-dependent stages during adult zebrafish spermatogenesis. Exposure to 10 nM 17β-oestradiol (E2) in vivo at least halved the mass of differentiating germ cells (from type B spermatogonia to spermatids), while type A spermatogonia accumulated. Studies on the cellular dynamics revealed that a reduction of spermatogonial proliferation together with an inhibition of their differentiation to type B spermatogonia were the basis for the oestrogen-mediated disturbance of spermatogenesis. The capacity of the zebrafish testis to produce 11-ketotestosterone as well as the expression of steroidogenesis-related genes was markedly decreased after in vivo oestrogen exposure. Moreover, the androg...
Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal h... more Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in -strands 2 (Lys 74), 4 (Tyr 124 , Asn 129 , and Thr 130), and 5 (Asp 150 and Asp 153) of the FSHR ECD identified in the human FSH⅐FSHR ECD crystal structure as contact sites with the common glycoprotein hormone ␣-subunit, and on noncontact residues in -strands 2 (Ser 78) and 8 (Asp 224 and Ser 226) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported ␣-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH⅐FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.
Crosstalk between signal transduction pathways provides a complex intracellular avenue for fine t... more Crosstalk between signal transduction pathways provides a complex intracellular avenue for fine tuning of hormone-induced signals. Over the last few years, we have studied the signaling mechanisms of three locust adipokinetic hormones (AKHs), which control mobilization of energy reserves from insect fat body as fuels for flight and transduce their signals via adenylyl cyclaseand phospholipase C-(PLC) dependent pathways. In this study, we examine possible crosstalk between these signaling routes. We show that cAMP does not affect basal and AKH-stimulated inositol phosphate (InsP n) production. Incubation of fat body with aluminium fluoride, an activator of G proteins, increased InsP n levels by 77%, whereas cholera toxin and pertussis toxin were ineffective. This implies that fat body PLC is not activated by G␥, but possibly by G q ␣. The involvement of this G protein in AKH signaling was demonstrated by our observation that the GPAntagonist-2A, which antagonizes G q , attenuated glycogen phosphorylase activation by AKH-I. As plasma membrane Ca 2 + channels may constitute another target for cAMP-mediated modulation, we studied the type of channels involved in AKH signaling using a variety of L-, N-and T-type Ca 2 + channel inhibitors. None of these blocked AKH-induced glycogen phosphorylase activation, suggesting that voltage-dependent Ca 2 + channels do not mediate AKH-induced Ca 2 + influx.
Proceedings of the National Academy of Sciences of the United States of America, Jan 3, 2010
Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proa... more Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.
This study aimed to improve, using the zebrafish model, our understanding of the distinct roles o... more This study aimed to improve, using the zebrafish model, our understanding of the distinct roles of pituitary gonadotropins FSH and LH in regulating testis functions in teleost fish. We report, for the first time in a vertebrate species, that zebrafish Leydig cells as well as Sertoli cells express the mRNAs for both gonadotropin receptors (fshr and lhcgr). Although Leydig cell fshr expression has been reported in other piscine species and may be a common feature of teleost fish, Sertoli cell lhcgr expression has not been reported previously and might be related to the undifferentiated gonochoristic mode of gonadal sex differentiation in zebrafish. Both recombinant zebrafish (rzf) gonadotropins (i.e. rzfLH and rzfFSH) stimulated androgen release in vitro and in vivo, with rzfFSH being significantly more potent than rzfLH. Forskolin-induced adenylate cyclase activation mimicked, whereas the protein kinase A inhibitor H-89 significantly reduced, the gonadotropin-stimulated androgen release. Therefore, we conclude that both FSH receptor and LH/choriogonadotropin receptor signaling are predominantly mediated through the cAMP/protein kinase A pathway to promote steroid production. Despite this similarity, other downstream mechanisms seem to differ. For example, rzfFSH up-regulated the testicular mRNA levels of a number of steroidogenesis-related genes both in vitro and in vivo, whereas rzfLH or human chorionic gonadotropin did not. Although not fully understood at present, these differences could explain the capacity of FSH to support both steroidogenesis and spermatogenesis on a long-term basis, whereas LH-stimulated steroidogenesis might be a more acute process, possibly restricted to periods during which peak steroid levels are required. (Endocrinology 151: 2349-2360, 2010) T he pituitary gonadotropins LH and FSH play critical roles in regulating male reproduction across vertebrates (1-3). In mammals, the specific activities of both gonadotropins are clearly defined, given the highly specific interactions between each hormone and its respective receptor, LH/choriogonadotropin receptor (Lhcgr) and FSH receptor (Fshr). In addition, Lhcgr and Fshr expression in mammalian testis is restricted to Leydig and Sertoli cells, respectively (1, 2, 4). Hence, LH predominantly controls Leydig cell steroidogenesis, whereas FSH regulates Sertoli cell functions (2, 4-6). In teleost fish, conversely, the biological activities of FSH and LH seem to be broader because both are strong steroidogenic hormones (7-16
Arteriosclerosis, Thrombosis, and Vascular Biology, 2009
Objectives— Several findings argue for a protective effect of high-density lipoproteins (HDLs) ag... more Objectives— Several findings argue for a protective effect of high-density lipoproteins (HDLs) against endothelial dysfunction. The molecular mechanisms underlying this protective effect are not fully understood, although recent works suggest that the actions of HDL on the endothelium are initiated by multiple interactions between HDLs (lipid or protein moiety) and cell surface receptors. We previously showed that the mitochondrial related F 1 -ATPase is a cell surface receptor for HDLs and their main atheroprotective apolipoprotein (apoA-I). Herein we test the hypothesis that the cell surface F 1 -ATPase may contribute to the ability of apoA-I and HDLs to maintain endothelial cell survival. Methods and Results— Cell imaging and binding assays confirmed the presence of the F 1 -ATPase at the surface of human umbilical vein endothelial cells (HUVECs) and its ability to bind apoA-I. Cell surface F 1 -ATPase activity (ATP hydrolysis into ADP) was stimulated by apoA-I and was inhibited ...
Clustering of synthetic biotinylated K1 domain from HGF/SF by streptavidin is sufficient to gener... more Clustering of synthetic biotinylated K1 domain from HGF/SF by streptavidin is sufficient to generate a potent MET agonist.
Progestagenic sex steroid hormones play critical roles in reproduction across vertebrates, includ... more Progestagenic sex steroid hormones play critical roles in reproduction across vertebrates, including teleost fish. To further our understanding of how progesterone modulates testis functions in fish, we set out to clone a progesterone receptor (pgr) cDNA exhibiting nuclear hormone receptor features from zebrafish testis. The open reading frame of pgr consists of 1854 bp, coding for a 617-amino acid-long protein showing the highest similarity with other piscine Pgr proteins. Functional characterization of the receptor expressed in mammalian cells revealed that zebrafish Pgr exhibited progesterone-specific, dose-dependent induction of reporter gene expression, with 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), a typical piscine progesterone, showing the highest potency. Expression of pgr mRNA: 1) appeared in embryos at 8 h after fertilization; 2) was significantly higher in developing ovary than in early transforming testis at 4 wk of age but vice versa in young adults at 12 wk of...
Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proa... more Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.
Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal h... more Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in-strands 2 (Lys 74), 4 (Tyr 124 , Asn 129 , and Thr 130), and 5 (Asp 150 and Asp 153) of the FSHR ECD identified in the human FSHFSHR ECD crystal structure as contact sites with the common glycoprotein hormone-subunit, and on noncontact residues in-strands 2 (Ser 78) and 8 (Asp 224 and Ser 226) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSHFSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.
NK1 is a splice variant of the polypeptide growth factor HGF/SF that consists of the N terminal (... more NK1 is a splice variant of the polypeptide growth factor HGF/SF that consists of the N terminal (N) and first kringle (K) domains and retains receptor binding and signalling. While NK1 behaves as a monomer in solution, two independent crystallographic structures have previously shown an identical, tightly packed dimer. Here we report a novel orthor-hombic crystal form of NK1 at 2.5 A ˚ resolution in which four NK1 proto-mers are packed in two distinct dimers in the asymmetric unit. Although the basic architecture of the new NK1 dimers is similar to the two described earlier, the new crystal form demonstrates extensive hinge movement between the N and K domain that leads to reorientation of the receptor-binding sites. The hinge bending is evidence of the paucity of strong interactions between domains within the protomer, in contrast to the extensive interactions between protomers in the dimer. These observations are consistent with domain swapping in the dimer, such that the interdomain interactions of the monomer are replaced by equivalent interprotomer interactions in the dimer and offer a route for protein engineering of NK1 variants which may act as receptor antagonists.
Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, m... more Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, many aspects of the role of androgens in adult teleost spermatogenesis have remained elusive. Since oestrogens inhibit androgen synthesis, we used an oestrogen-induced androgen depletion model to identify androgen-dependent stages during adult zebrafish spermato-genesis. Exposure to 10 nM 17b-oestradiol (E 2) in vivo at least halved the mass of differentiating germ cells (from type B spermatogonia to spermatids), while type A spermatogonia accumulated. Studies on the cellular dynamics revealed that a reduction of spermatogonial proliferation together with an inhibition of their differentiation to type B spermatogonia were the basis for the oestrogen-mediated disturbance of spermato-genesis. The capacity of the zebrafish testis to produce 11-ketotestosterone as well as the expression of steroidogen-esis-related genes was markedly decreased after in vivo oestrogen exposure. Moreover, the androgen-release response to recom-binant zebrafish Lh was lost after oestrogen exposure. We conclude that oestrogen exposure caused a state of androgen insufficiency in adult male zebrafish. Since the downregulation of the steroidogenic system as well as the disturbance of spermatogenesis in testicular explants exposed to E 2 ex vivo was much less severe than after in vivo exposure, the main inhibitory effect appears to be exerted via feedback inhibition of gonadotropin release. This experimental setup helped to identify spermatogonial proliferation and their differentiation as androgen targets in adult zebrafish spermatogenesis.
Crosstalk between signal transduction pathways provides a complex intracellular avenue for fine t... more Crosstalk between signal transduction pathways provides a complex intracellular avenue for fine tuning of hormone-induced signals. Over the last few years, we have studied the signaling mechanisms of three locust adipokinetic hormones (AKHs), which control mobilization of energy reserves from insect fat body as fuels for flight and transduce their signals via adenylyl cyclase-and phospholipase C-(PLC) dependent pathways. In this study, we examine possible crosstalk between these signaling routes. We show that cAMP does not affect basal and AKH-stimulated inositol phosphate (InsP n) production. Incubation of fat body with aluminium fluoride, an activator of G proteins, increased InsP n levels by 77%, whereas cholera toxin and pertussis toxin were ineffective. This implies that fat body PLC is not activated by G, but possibly by G q. The involvement of this G protein in AKH signaling was demonstrated by our observation that the GPAntagonist-2A, which antagonizes G q , attenuated glycogen phosphorylase activation by AKH-I. As plasma membrane Ca 2 + channels may constitute another target for cAMP-mediated modulation, we studied the type of channels involved in AKH signaling using a variety of L-, N-and T-type Ca 2 + channel inhibitors. None of these blocked AKH-induced glycogen phosphorylase activation, suggesting that voltage-dependent Ca 2 + channels do not mediate AKH-induced Ca 2 + influx.
Modern molecular imaging techniques have greatly improved tumor detection and post-treatment foll... more Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the human ether-à-go-go-related gene-1 (hERG1) potassium channel, and to obtain a proof of concept for their potential use for in vivo molecular imaging. The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the VH domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the "native" anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the in vivo distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses in vivo. In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.
With a 5-year recurrence rate of 30–78\%, urothelial cell carcinoma (UCC) rates amongst the highe... more With a 5-year recurrence rate of 30–78\%, urothelial cell carcinoma (UCC) rates amongst the highest of all solid malignancies. Consequently, after transurethral resection, patients are subjugated to life-long endoscopic surveillance. A multimodal near-infrared (NIR) fluorescence-based imaging strategy can improve diagnosis, resection and surveillance, hence increasing quality of life. Methods: Expression of urokinase plasminogen activator receptor (uPAR) and epithelial cell adhesion molecule (EpCAM) are determined on paraffin-embedded human UCC using immunohistochemistry and on UCC cell lines by flow cytometry. MNPR-101, a humanised monoclonal antibody targeting uPAR is conjugated to IRDye800CW and binding is validated in vitro using surface plasmon resonance and cell-based binding assays. In vivo NIR fluorescence and photoacoustic three-dimensional (3D) imaging are performed with subcutaneously growing human UM-UC-3luc2 cells in BALB/c-nude mice. The translational potential is confirmed in a metastasising UM-UC-3luc2 orthotopic mouse model. Infliximab-IRDye800CW and rituximab-IRDye800CW are used as controls. Results: UCCs show prominent uPAR expression at the tumour-stroma interface and EpCAM on epithelial cells. uPAR and EpCAM are expressed by 6/7 and 4/7 UCC cell lines, respectively. In vitro, MNPR-101-IRDye800CW has a picomolar affinity for domain 2-3 of uPAR. In vivo fluorescence imaging with MNPR-101-IRDye800CW, specifically delineates both subcutaneous and orthotopic tumours with tumour-to-background ratios reaching as high as 6.8, differing significantly from controls (p < 0.0001). Photoacoustic 3D in depth imaging confirms the homogenous distribution of MNPR-101-IRDye800CW through the tumour. Conclusions: MNPR-101-IRDye800CW is suitable for multimodal imaging of UCC, awaiting clinical translation.
Lanthionine synthetase component C-like protein 2 (LANCL2) has been identified as the mammalian r... more Lanthionine synthetase component C-like protein 2 (LANCL2) has been identified as the mammalian receptor mediating the functional effects of the universal stress hormone abscisic acid (ABA) in mammals. ABA stimulates insulin independent glucose uptake in myocytes and adipocytes via LANCL2 binding in vitro, improves glucose tolerance in vivo and induces brown fat activity in vitro and in vivo. The emerging role of the ABA/LANCL2 system in glucose and lipid metabolism makes it an attractive target for pharmacological interventions in diabetes mellitus and the metabolic syndrome. The aim of this study was to investigate the presence of ABA binding site(s) on LANCL2 and identify the amino acid residues involved in ABA binding. Equilibrium binding assays ([3H]-ABA saturation binding and surface plasmon resonance analysis) suggested multiple ABA-binding sites, prompting us to perform a computational study that indicated one putative high-affinity and two low-affinity binding sites. Site-directed mutagenesis (single mutant R118I, triple mutants R118I/R22I/K362I and R118I/S41A/E46I) and equilibrium binding experiments on the mutated LANCL2 proteins identified a high-affinity ABA-binding site involving R118, with a KD of 2.6 nM ± 1.2 nM, as determined by surface plasmon resonance. Scatchard plot analysis of binding curves from both types of equilibrium binding assays revealed a Hill coefficient >1, suggesting cooperativity of ABA binding to LANCL2. Identification of the high-affinity ABA-binding site is expected to allow the design of ABA agonists/antagonists, which will help to understand the role of the ABA/LANCL2 system in human physiology and disease.
mAbs, either mono- or bispecific (bsAb), represent one of the most successful approaches to treat... more mAbs, either mono- or bispecific (bsAb), represent one of the most successful approaches to treat many types of malignancies. However, there are certain limitations to the use of full length mAbs for clinical applications, which can be overcome by engineered antibody fragments. The aim of this study was to develop a small bsAb, in the format of a single-chain diabody (scDb), to efficiently target two proteins, the hERG1 potassium channel and the β1 subunit of integrin receptors, which specifically form a macromolecular complex in cancer cells. We provide evidence that the scDb we produced binds to the hERG1/β1 complex in cancer cells and tissues, but does not bind to the hERG1 channel in nonpathologic tissues, in particular the heart. The scDb-hERG1-β1 (i) downregulates the formation of the hERG1/β1 complex, (ii) inhibits Akt phosphorylation and HIF-1α expression, and (iii) decreases cell survival, proliferation, and migration in vitro. These effects only occur in cancer cells (either colon, pancreatic, or breast), but not in normal cells. In vivo, the scDb-hERG1-β1 shows a good pharmacokinetic profile, with a half-life of 13.5 hours and no general, cardiac, or renal toxicity when injected intravenously up to the dose of 8 mg/kg. The scDb-hERG1-β1 accumulates into subcutaneous xenografted tumors, arising from either colon or pancreatic human cancer cells, and induces a reduction of tumor growth and vascularization. Overall, the scDb-hERG1-β1 represents an innovative single-chain bispecific antibody for therapeutic applications in solid cancers that overexpress the hERG1/β1 integrin signaling complex.
Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB10... more Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apo(a). The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analog, ε-aminocaproic acid. Interestingly, KIV-2 presents a lysine binding site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to be negligible in surface plasmon resonance experiments; and with the LBS being nonfunctional, we propose to rename it “pseudo-LBS”. Further investigation of the protein by computational small-molecule docking allowed us to identify a possible heparin-binding site away from the LBS, which was confirmed by specific reverse charge mutations abolishing heparin binding. This study opens new possibilities to define the pathogenesis of Lp(a)-related diseases and to facilitate the design of specific therapeutic drugs.
Depending on the linker length between the VH and the VL domain, single-chain Fv (scFv) antibody ... more Depending on the linker length between the VH and the VL domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter kD, indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion.
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatm... more Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatment. The Hepatocyte Growth Factor/Scatter Factor (HGF/SF), through its receptor MET, is one of the most potent survival-promoting factors for motor neurons (MN) and is known as a modulator of immune cell function. We recently developed a novel recombinant MET agonist optimized for therapy, designated K1K1. K1K1 was ten times more potent than HGF/SF in preventing MN loss in an in vitro model of ALS. Treatments with K1K1 delayed the onset of muscular impairment and reduced MN loss and skeletal muscle denervation of superoxide dismutase 1 G93A (SOD1G93A) mice. This effect was associated with increased levels of phospho-extracellular signal-related kinase (pERK) in the spinal cord and sciatic nerves and the activation of non-myelinating Schwann cells. Moreover, reduced activated microglia and astroglia, lower T cells infiltration and increased interleukin 4 (IL4) levels were found in the lumbar spinal cord of K1K1 treated mice. K1K1 treatment also prevented the infiltration of T cells in skeletal muscle of SOD1G93A mice. All these protective effects were lost on long-term treatment suggesting a mechanism of drug tolerance. These data provide a rational justification for further exploring the long-term loss of K1K1 efficacy in the perspective of providing a potential treatment for ALS.
Cancer is one of the main causes of death worldwide and early detection is crucial for effective ... more Cancer is one of the main causes of death worldwide and early detection is crucial for effective treatment. Scientists have therefore focused on the identification of circulating cancer-specific molecules, so-called markers, that can be detected with non- or less-invasive techniques. One attractive emerging marker is represented by circulating antibodies against molecules that are specific for the tumor cells and not produced by normal cells. Many such auto-antibodies have been discovered, but still not much is known about their significance and role in either promoting or inhibiting tumor growth. The aim of this review is to summarize the current knowledge on auto-antibodies in different cancer types and on their current or possible utilization for effective cancer management. Reference to autoimmune diseases will also be made, as they share with cancer the presence of such auto-antibodies. Auto-antibodies are classically associated with autoimmune diseases, where they are an integral part of diagnostic panels. However, recent evidence is accumulating on the presence of auto-antibodies against single or selected panels of auto-antigens in many types of cancer. Auto-antibodies might initially represent an epiphenomenon derived from the inflammatory environment induced by the tumor. However, their effect on tumor evolution can be crucial, as is discussed in this paper. It has been demonstrated that some of these auto-antibodies can be used for early detection and cancer staging, as well as for monitoring of cancer regression during treatment and follow up. Interestingly, certain auto-antibodies were found to promote cancer progression and metastasis, while others contribute to the body’s defense against it. Moreover, auto-antibodies are of a polyclonal nature, which means that often several antibodies are involved in the response to a single tumor antigen. Dissection of these antibody specificities is now possible, allowing their identification at the genetic, structural, and epitope levels. In this review, we report the evidence available on the presence of auto-antibodies in the main cancer types and discuss some of the open issues that still need to be addressed by the research community.
Abscisic acid (ABA) is a plant hormone also present and active in animals. In mammals, ABA regula... more Abscisic acid (ABA) is a plant hormone also present and active in animals. In mammals, ABA regulates blood glucose levels by stimulating insulin-independent glucose uptake and metabolism in adipocytes and myocytes through its receptor LANCL2. The objective of this study was to investigate whether another member of the LANCL protein family, LANCL1, also behaves as an ABA receptor and, if so, which functional effects are mediated by LANCL1. ABA binding to human recombinant LANCL1 was explored by equilibrium-binding experiments with [3H]ABA, circular dichroism, and surface plasmon resonance. Rat L6 myoblasts overexpressing either LANCL1 or LANCL2, or silenced for the expression of both proteins, were used to investigate the basal and ABA-stimulated transport of a fluorescent glucose analog (NBDG) and the signaling pathway downstream of the LANCL proteins using Western blot and qPCR analysis. Finally, glucose tolerance and sensitivity to ABA were compared in LANCL2−/− and wild-type (WT) siblings. Human recombinant LANCL1 binds ABA with a Kd between 1 and 10 μM, depending on the assay (i.e., in a concentration range that lies between the low and high-affinity ABA binding sites of LANCL2). In L6 myoblasts, LANCL1 and LANCL2 similarly, i) stimulate both basal and ABA-triggered NBDG uptake (4-fold), ii) activate the transcription and protein expression of the glucose transporters GLUT4 and GLUT1 (4-6-fold) and the signaling proteins AMPK/PGC-1α/Sirt1 (2-fold), iii) stimulate mitochondrial respiration (5-fold) and the expression of the skeletal muscle (SM) uncoupling proteins sarcolipin (3-fold) and UCP3 (12-fold). LANCL2−/− mice have a reduced glucose tolerance compared to WT. They spontaneously overexpress LANCL1 in the SM and respond to chronic ABA treatment (1 μg/kg body weight/day) with an improved glycemia response to glucose load and an increased SM transcription of GLUT4 and GLUT1 (20-fold) of the AMPK/PGC-1α/Sirt1 pathway and sarcolipin, UCP3, and NAMPT (4- to 6-fold). LANCL1 behaves as an ABA receptor with a somewhat lower affinity for ABA than LANCL2 but with overlapping effector functions: stimulating glucose uptake and the expression of muscle glucose transporters and mitochondrial uncoupling and respiration via the AMPK/PGC-1α/Sirt1 pathway. Receptor redundancy may have been advantageous in animal evolution, given the role of the ABA/LANCL system in the insulin-independent stimulation of cell glucose uptake and energy metabolism.
Hepatocyte growth factor/scatter factor (HGF/SF) and its cognate receptor MET play several essent... more Hepatocyte growth factor/scatter factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in postnatal life of epithelial organs such as the liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search of HGF/SF mimics for therapy. In this work, we describe the rational design, production, and characterization of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF in tandem orientation. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide applications in acute and chronic diseases of the liver and other epithelial organs dependent of MET activation.
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Papers by Hugo de Jonge