Venom Peptides, Polyphenols and Alkaloids: Are They the Next Antidiabetics That Will Preserve ß-Cell Mass and Function in Type 2 Diabetes?

Improvement of insulin secretion by pancreatic ß-cells and preservation of their mass are the current challenges that future antidiabetic drugs should meet for achieving efficient and long-term glycemic control in patients with type 2 diabetes (T2D). The successful development of glucagon-like peptide 1 (GLP-1) analogues, derived from the saliva of a lizard from the Helodermatidae family, has provided the proof of concept that antidiabetic drugs directly targeting pancreatic ß-cells can emerge from venomous animals. The literature reporting on the antidiabetic effects of medicinal plants suggests that they contain some promising active substances such as polyphenols and alkaloids, which could be active as insulin secretagogues and ß-cell protectors. In this review, we discuss the potential of several polyphenols, alkaloids and venom peptides from snake, frogs, scorpions and cone snails. These molecules could contribute to the development of new efficient antidiabetic medicines targeting ß-cells, which would tackle the progression of the disease.

Macrophage-Colony-Stimulating Factor Receptor Enhances Prostate Cancer Cell Growth and Aggressiveness In Vitro and In Vivo and Increases Osteopontin Expression.

Prostate cancer is a major public health concern and one of the most prevalent forms of cancer worldwide. The definition of altered signaling pathways implicated in this complex disease is thus essential. In this context, abnormal expression of the receptor of Macrophage Colony-Stimulating Factor-1 (M-CSF or CSF-1) has been described in prostate cancer cells. Yet, outcomes of this expression remain unknown. Using mouse and human prostate cancer cell lines, this study has investigated the functionality of the wild-type CSF-1 receptor in prostate tumor cells and identified molecular mechanisms underlying its ligand-induced activation. Here, we showed that upon CSF-1 binding, the receptor autophosphorylates and activates multiple signaling pathways in prostate tumor cells. Biological experiments demonstrated that the CSF-1R/CSF-1 axis conferred significant advantages in cell growth and cell invasion in vitro. Mouse xenograft experiments showed that CSF-1R expression promoted the aggressiveness of prostate tumor cells. In particular, we demonstrated that the ligand-activated CSF-1R increased the expression of spp1 transcript encoding for osteopontin, a key player in cancer development and metastasis. Therefore, this study highlights that the CSF-1 receptor is fully functional in a prostate cancer cell and may be a potential therapeutic target for the treatment of prostate cancer.

A novel agonist for the HGF receptor MET promotes differentiation of human pluripotent stem cells into hepatocyte-like cells.

Hepatocyte growth factor (HGF) is the natural ligand of the MET receptor tyrosine kinase. This ligand-receptor couple is essential for the maturation process of hepatocytes. Previously, the rational design of a synthetic protein based on the assembly of two K1 domains from HGF led to the production of a potent and stable MET receptor agonist. In this study, we compared the effects of K1K1 with HGF during the differentiation of hepatocyte progenitors derived from human induced pluripotent stem cells (hiPSCs). In vitro, K1K1, in the range of 20 to 200 nM, successfully substituted for HGF and efficiently activated ERK downstream signaling. Analysis of the levels of hepatocyte markers showed typical liver mRNA and protein expression (HNF4α, albumin, alpha-fetoprotein, CYP3A4) and phenotypes. Although full maturation was not achieved, the results suggest that K1K1 is an attractive candidate MET agonist suitable for replacing complex and expensive HGF treatments to induce hepatic differentiation of hiPSCs.

Redox-Controlled Chemical Protein Synthesis: Sundry Shades of Latency.

The last two decades have witnessed the rise in power of chemical protein synthesis to the point where it now constitutes an established corpus of synthetic methods efficiently complementing biological approaches. One factor explaining this spectacular evolution is the emergence of a new class of chemoselective reactions enabling the formation of native peptide bonds between two unprotected peptidic segments, also known as native ligation reactions. In recent years, their application has fueled the production of homogeneous batches of large and highly decorated protein targets with a control of their composition at the atomic level. In doing so, native ligation reactions have provided the means for successful applications in chemical biology, medicinal chemistry, materials science, and nanotechnology research.The native chemical ligation (NCL) reaction has had a major impact on the field by enabling the chemoselective formation of a native peptide bond between a C-terminal peptidyl thioester and an N-terminal cysteinyl peptide. Since its introduction in 1994, the NCL reaction has been made the object of significant improvements and its scope and limitations have been thoroughly investigated. Furthermore, the diversification of peptide segment assembly strategies has been essential to access proteins of increasing complexity and has had to overcome the challenge of controlling the reactivity of ligation partners.One hallmark of NCL is its dependency on thiol reactivity, including for its catalysis. While Nature constantly plays with the redox properties of biological thiols for the regulation of numerous biochemical pathways, such a control of reactivity is challenging to achieve in synthetic organic chemistry and, in particular, for those methods used for assembling peptide segments by chemical ligation. This Account covers the studies conducted by our group in this area. A leading theme of our research has been the conception of controllable acyl donors and cysteine surrogates that place the chemoselective formation of amide bonds by NCL-like reactions under the control of dichalcogenide-based redox systems. The dependency of the redox potential of dichalcogenide bonds on the nature of the chalcogenides involved (S, Se) has appeared as a powerful means for diversifying the systems, while allowing their sequential activation for protein synthesis. Such a control of reactivity mediated by the addition of harmless redox additives has greatly facilitated the modular and efficient preparation of multiple targets of biological relevance. Taken together, these endeavors provide a practical and robust set of methods to address synthetic challenges in chemical protein synthesis.

Dimerization of kringle 1 domain from hepatocyte growth factor/scatter factor provides a potent MET receptor agonist.

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.

Fast Protein Modification in the Nanomolar Concentration Range Using an Oxalyl Amide as Latent Thioester.

We show that latent oxalyl thioester surrogates are a powerful means to modify peptides and proteins in highly dilute conditions in purified aqueous media or in mixtures as complex as cell lysates. Designed to be shelf-stable reagents, they can be activated on demand to enable ligation reactions with peptide concentrations as low as a few hundred nM at rates approaching 30 M-1 s-1 .

Catalysis of Hydrazone and Oxime Peptide Ligation by Arginine.

Hydrazone and oxime peptide ligations are catalyzed by arginine. The catalysis is assisted intramolecularly by the side-chain guanidinium group. Hydrazone ligation in the presence of arginine proceeds efficiently in phosphate buffer at neutral pH but is particularly powerful in bicarbonate/CO2 buffer. In addition to acting as a catalyst, arginine prevents the aggregation of proteins during ligation. With its dual properties as a nucleophilic catalyst and a protein aggregation inhibitor, arginine hydrochloride is a useful addition to the hydrazone/oxime ligation toolbox.

A cysteine selenosulfide redox switch for protein chemical synthesis.

The control of cysteine reactivity is of paramount importance for the synthesis of proteins using the native chemical ligation (NCL) reaction. We report that this goal can be achieved in a traceless manner during ligation by appending a simple N-selenoethyl group to cysteine. While in synthetic organic chemistry the cleavage of carbon-nitrogen bonds is notoriously difficult, we describe that N-selenoethyl cysteine (SetCys) loses its selenoethyl arm in water under mild conditions upon reduction of its selenosulfide bond. Detailed mechanistic investigations show that the cleavage of the selenoethyl arm proceeds through an anionic mechanism with assistance of the cysteine thiol group. The implementation of the SetCys unit in a process enabling the modular and straightforward assembly of linear or backbone cyclized polypeptides is illustrated by the synthesis of biologically active cyclic hepatocyte growth factor variants.

Transformation of Receptor Tyrosine Kinases into Glutamate Receptors and Photoreceptors.

Receptor tyrosine kinases (RTKs) are key regulators of cellular functions in metazoans. In vertebrates, RTKs are mostly activated by polypeptides but are not naturally sensitive to amino acids or light. Taking inspiration from Venus kinase receptors (VKRs), an atypical family of RTKs found in nature, we have transformed the human insulin (hIR) and hepatocyte growth factor receptor (hMET) into glutamate receptors by replacing their extracellular binding domains with the ligand-binding domain of metabotropic glutamate receptor type 2 (mGluR2). We then imparted light sensitivity through covalent attachment of a synthetic glutamate-based photoswitch via a self-labelling SNAP tag. By employing a Xenopus laevis oocyte kinase activity assay, we demonstrate how these chimeric RTKs, termed light-controlled human insulin receptor (LihIR) and light-controlled human MET receptor (LihMET), can be used to exert optical control over the insulin or MET signaling pathways. Our results outline a potentially general strategy to convert RTKs into photoreceptors.

The Role of the Conserved SUMO-2/3 Cysteine Residue on Domain Structure Investigated Using Protein Chemical Synthesis.

While the semi or total synthesis of ubiquitin or polyubiquitin conjugates has attracted a lot of attention the past decade, the preparation of small ubiquitin-like modifier (SUMO) conjugates is much less developed. We describe hereinafter some important molecular features to consider when preparing SUMO-2/3 conjugates by chemical synthesis using the native chemical ligation and extended methods. In particular, we clarify the role of the conserved cysteine residue on SUMO-2/3 domain stability and properties. Our data reveal that SUMO-2 and -3 proteins behave differently from the Cys → Ala modification with SUMO-2 being less impacted than SUMO-3, likely due to a stabilizing interaction occurring in SUMO-2 between its tail and the SUMO core domain. While the Cys → Ala modification has no effect on the enzyme-catalyzed conjugation, it shows a deleterious effect on the enzyme-catalyzed deconjugation process, especially with the SUMO-3 conjugate. Whereas it is often stated that SUMO-2 and SUMO-3 are structurally and functionally indistinguishable, here we show that these proteins have specific structural and biochemical properties. This information is important to consider when designing and preparing SUMO-2/3 conjugates, and should help in making progress in the understanding of the specific role of SUMO-2 and/or SUMO-3 modifications on protein structure and function.

Hypoxia leads to decreased autophosphorylation of the MET receptor but promotes its resistance to tyrosine kinase inhibitors.

The receptor tyrosine kinase MET and its ligand, the Hepatocyte Growth Factor/Scattor Factor (HGF/SF), are essential to the migration, morphogenesis, and survival of epithelial cells. In addition, dysregulation of MET signaling has been shown to promote tumor progression and invasion in many cancers. Therefore, HGF/SF and MET are major targets for chemotherapies. Improvement of targeted therapies requires a perfect understanding of tumor microenvironment that strongly modifies half-life, bio-accessibility and thus, efficacy of treatments. In particular, hypoxia is a crucial microenvironmental phenomenon promoting invasion and resistance to treatments. Under hypoxia, MET auto-phosphorylation resulting from ligand stimulation or from receptor overexpression is drastically decreased within minutes of oxygen deprivation but is quickly reversible upon return to normoxia. Besides a decreased phosphorylation of its proximal adaptor GAB1 under hypoxia, activation of the downstream kinases Erk and Akt is maintained, while still being dependent on MET receptor. Consistently, several cellular responses induced by HGF/SF, including motility, morphogenesis, and survival are effectively induced under hypoxia. Interestingly, using a semi-synthetic ligand, we show that HGF/SF binding to MET is strongly impaired during hypoxia but can be quickly restored upon reoxygenation. Finally, we show that two MET-targeting tyrosine kinase inhibitors (TKIs) are less efficient on MET signalling under hypoxia. Like MET loss of phosphorylation, this hypoxia-induced resistance to TKIs is reversible under normoxia. Thus, although hypoxia does not affect downstream signaling or cellular responses induced by MET, it causes immediate resistance to TKIs. These results may prove useful when designing and evaluation of MET-targeted therapies against cancer.

High-MET status in non-small cell lung tumors correlates with receptor phosphorylation but not with the serum level of soluble form.

OBJECTIVES: The receptor tyrosine kinase MET is essential to embryonic development and organ regeneration. Its deregulation is associated with tumorigenesis. While MET gene amplification and mutations leading to MET self-activation concern only a few patients, a high MET level has been found in about half of the non-small cell lung cancers (NSCLCs) tested. How this affects MET activation in tumors is unclear. Also uncertain is the prognostic value, in cancer, of a phenomenon well described in cell models: MET shedding, i.e. its cleavage by membrane proteases leading to release of a soluble fragment into the medium. MATERIALS AND METHODS: A prospective cohort of 39 NSCLC patients was constituted at diagnosis or soon after. Normal tissues, tumor tissues, and blood samples were obtained. This allowed, for the same patient, synchronous determination of (i) the MET level in the tumor, (ii) receptor phosphorylation, and (iii) the concentration of soluble MET fragment (sMET) in the serum. RESULTS: After confirming the adequacy of an ELISA for measuring the serum level of sMET, we found no correlation between this level and the concentration of MET in tumors, as evaluated by immunohistochemistry and western blotting. Nevertheless, all but one tumor displaying a high MET level also displayed receptor phosphorylation, restricted to a small number of tumor cells. CONCLUSION: Our results thus demonstrate that the serum level of sMET is not indicative of the amount of MET present in the tumor cells and cannot be used as a biomarker for therapeutic purposes. However, MET scoring of tumor biopsies could be a first step prior to determination of MET receptor activation in high-MET tumors.

A Central Cysteine Residue Is Essential for the Thermal Stability and Function of SUMO-1 Protein and SUMO-1 Peptide-Protein Conjugates.

SUMOylation constitutes a major post-translational modification (PTM) used by the eukaryote cellular machinery to modulate protein interactions of the targeted proteins. The small ubiquitin-like modifier-1 (SUMO-1) features a central and conserved cysteine residue (Cys52) that is located in the hydrophobic core of the protein and in tight contact with Phe65, suggesting the occurrence of an S/π interaction. To investigate the importance of Cys52 on SUMO-1 thermal stability and biochemical properties, we produced by total chemical synthesis SUMO-1 or SUMO-1 Cys52Ala peptide-protein conjugates featuring a native isopeptidic bond between SUMO-1 and a peptide derived from p53 tumor suppressor protein. The Cys52Ala modification perturbed SUMO-1 secondary structure and resulted in a dramatic loss of protein thermal stability. Moreover, the cleavage of the isopeptidic bond by the deconjugating enzyme Upl1 was significantly less efficient than for the wild-type conjugate. Similarly, the in vitro SUMOylation of RanGap1 by E1/E2 conjugating enzymes was significantly less efficient with the SUMO-1 C52A analog compared to wild-type SUMO-1. These data demonstrate the critical role of Cys52 in maintaining SUMO-1 conformation and function and the importance of keeping this cysteine intact for the study of SUMO-1 protein conjugates.

One-pot chemical synthesis of small ubiquitin-like modifier protein-peptide conjugates using bis(2-sulfanylethyl)amido peptide latent thioester surrogates.

Small ubiquitin-like modifier (SUMO) post-translational modification (PTM) of proteins has a crucial role in the regulation of important cellular processes. This protocol describes the chemical synthesis of functional SUMO-peptide conjugates. The two crucial stages of this protocol are the solid-phase synthesis of peptide segments derivatized by thioester or bis(2-sulfanylethyl)amido (SEA) latent thioester functionalities and the one-pot assembly of the SUMO-peptide conjugate by a sequential native chemical ligation (NCL)/SEA native peptide ligation reaction sequence. This protocol also enables the isolation of a SUMO SEA latent thioester, which can be attached to a target peptide or protein in a subsequent step. It is compatible with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, and it gives access to homogeneous, reversible and functional SUMO conjugates that are not easily produced using living systems. The synthesis of SUMO-peptide conjugates on a milligram scale takes 20 working days.

Thiocarbamate-linked polysulfonate-peptide conjugates as selective hepatocyte growth factor receptor binders.

The capacity of many proteins to interact with natural or synthetic polyanions has been exploited for modulating their biological action. However, the polydispersity of these macromolecular polyanions as well as their poor specificity is a severe limitation to their use as drugs. An emerging trend in this field is the synthesis of homogeneous and well-defined polyanion-peptide conjugates, which act as bivalent ligands, with the peptide part bringing the selectivity of the scaffold. Alternately, this strategy can be used for improving the binding of short peptides to polyanion-binding protein targets. This work describes the design and first synthesis of homogeneous polysulfonate-peptide conjugates using thiocarbamate ligation for binding to the extracellular domain of MET tyrosine kinase receptor for hepatocyte growth factor.

Anti-apoptotic role of caspase-cleaved GAB1 adaptor protein in hepatocyte growth factor/scatter factor-MET receptor protein signaling.

The GRB2-associated binder 1 (GAB1) docking/scaffold protein is a key mediator of the MET-tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). Activated MET promotes recruitment and tyrosine phosphorylation of GAB1, which in turn recruits multiple proteins and mediates MET signaling leading to cell survival, motility, and morphogenesis. We previously reported that, without its ligand, MET is a functional caspase target during apoptosis, allowing the generation of a p40-MET fragment that amplifies apoptosis. In this study we established that GAB1 is also a functional caspase target by evidencing a caspase-cleaved p35-GAB1 fragment that contains the MET binding domain. GAB1 is cleaved by caspases before MET, and the resulting p35-GAB1 fragment is phosphorylated by MET upon HGF/SF binding and can interact with a subset of GAB1 partners, PI3K, and GRB2 but not with SHP2. This p35-GAB1 fragment favors cell survival by maintaining HGF/SF-induced MET activation of AKT and by hindering p40-MET pro-apoptotic function. These data demonstrate an anti-apoptotic role of caspase-cleaved GAB1 in HGF/SF-MET signaling.

Shedding-generated Met receptor fragments can be routed to either the proteasomal or the lysosomal degradation pathway.

The receptor tyrosine kinase Met and its ligand, the hepatocyte growth factor/scatter factor, are essential for embryonic development, whereas deregulation of Met signaling pathways is associated with tumorigenesis and metastasis. The presenilin-regulated intramembrane proteolysis (PS-RIP) is involved in ligand-independent downregulation of Met. This proteolytic process involves shedding of the Met extracellular domain followed by γ-secretase cleavage, generating labile intracellular fragments degraded by the proteasome. We demonstrate here that upon shedding both generated Met N- and C-terminal fragments are degraded directly in the lysosome, with C-terminal fragments escaping γ-secretase cleavage. PS-RIP and lysosomal degradation are complementary, because their simultaneous inhibition induces synergistic accumulation of fragments. Met N-terminal fragments associate with the high-affinity domain of HGF/SF, confirming its decoy activity which could be reduced through their routing to the lysosome at the expense of extracellular release. Finally, the DN30 monoclonal antibody inducing Met shedding promotes receptor degradation through induction of both PS-RIP and the lysosomal pathway. Thus, we demonstrate that Met shedding initiates a novel lysosomal degradation which participates to ligand-independent downregulation of the receptor.

Polysaccharide microarrays: application to the identification of heparan sulphate mimetics.

The interaction of polysaccharides with proteins modulates or triggers many biological effects. In particular, heparan sulphate proteoglycans (HSPGs) have multiple regulatory interactions with growth factors, enzymes, enzyme inhibitors, and some components of the extracellular matrix. The important role played by HSPGs has motivated the synthesis and selection of HSPG mimetics for modulating the biological activity of HS-binding proteins. We present hereinafter an efficient polysaccharide microarray method that allows the screening of HS-mimetic libraries towards HS-binding growth factors, a major class of polypeptides whose inhibition or potentiation is of high medical interest.

The glucose transporter 2 undergoes plasma membrane endocytosis and lysosomal degradation in a secretagogue-dependent manner.

In beta-cells of the pancreas, the glucose transporter (GLUT)-2 facilitative glucose transporter protein is localized to the plasma membrane and functions as part of the glucose sensing mechanism for the stimulation of insulin secretion. We observed that expressed GLUT2 protein in the cultured Min6B1 cell line undergoes enhanced endocytosis at high extracellular glucose concentrations that stimulate insulin secretion. Moreover, the internalized GLUT2 protein undergoes rapid degradation induced by chronic high-glucose or arginine stimulation but does not undergo plasma membrane recycling or accumulation in any microscopically apparent intracellular membrane compartment. The rapid degradation of GLUT2 was prevented by lysosomal inhibition (chloroquine) concomitant with the accumulation of GLUT2 in endomembrane structures. In contrast, neither endocytosis nor the lack of internal membrane localized GLUT2 remained completely unaffected by proteosomal inhibition (lactacystin) or an heat shock protein-90 inhibitor (geldanamycin). Moreover, the endocytosis and degradation of GLUT2 was specific for beta-cells because expression of GLUT2 in 3T3L1 adipocytes remained cell surface localized and did not display a rapid rate of degradation. Together, these data demonstrate that hyperglycemia directly affects beta-cell function and activates a trafficking pathway that results in the rapid endocytosis and degradation of the cell surface GLUT2 glucose transporter.