RESUMO
The insulin/insulin-like growth factor signalling axis is an evolutionary ancient and highly conserved hormonal system involved in the regulation of metabolism, growth and lifespan in animals. Human insulin is stored in the pancreas, while insulin-like growth factor-1 (IGF-1) is maintained in blood in complexes with IGF-binding proteins (IGFBP1-6). Insect insulin-like polypeptide binding proteins (IBPs) have been considered as IGFBP-like structural and functional homologues. Here, we report structures of the Drosophila IBP Imp-L2 in its free form and bound to Drosophila insulin-like peptide 5 and human IGF-1. Imp-L2 contains two immunoglobulin-like fold domains and its architecture is unrelated to human IGFBPs, suggesting a distinct strategy for bioavailability regulation of insulin-like hormones. Similar hormone binding modes may exist in other insect vectors, as the IBP sequences are highly conserved. Therefore, these findings may open research routes towards a rational interference of transmission of diseases such as malaria, dengue and yellow fevers.
Assuntos
Proteínas de Drosophila/química , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/química , Animais , Disponibilidade Biológica , Drosophila , Humanos , Insulina/farmacocinética , Fator de Crescimento Insulin-Like I/química , Conformação ProteicaRESUMO
Insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively) are protein hormones involved not only in normal growth and development but also in life span regulation and cancer. They exert their functions mainly through the IGF-1R or by binding to isoform A of the insulin receptor (IR-A). The development of IGF-1 and IGF-2 antagonists is of great clinical interest. Mutations of A4 and A8 sites of human insulin lead to disproportionate effects on hormone IR binding and activation. Here, we systematically modified IGF-1 sites 45, 46, and 49 and IGF-2 sites 45 and 48, which correspond, or are close, to insulin sites A4 and A8. The IGF-1R and IR-A binding and autophosphorylation potencies of these analogues were characterized. They retained the main IGF-1R-related properties, but the hormones with His49 in IGF-1 and His48 in IGF-2 showed significantly higher affinities for IR-A and for IR-B, being the strongest IGF-1- and IGF-2-like binders of these receptors ever reported. All analogues activated IR-A and IGF-1R without major discrepancies in their binding affinities. This study revealed that IR-A and IGF-1R contain specific sites, likely parts of their so-called sites 2', which can interact differently with specifically modified IGF analogues. Moreover, a clear importance of IGF-2 site 44 for effective hormone folding was also observed. These findings may facilitate novel and rational engineering of new hormone analogues for IR-A and IGF-1R studies and for potential medical applications.
Assuntos
Fator de Crescimento Insulin-Like II/química , Fator de Crescimento Insulin-Like I/química , Receptor de Insulina/química , Receptores de Somatomedina/genética , Evolução Molecular , Humanos , Insulina/química , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like II/genética , Ligantes , Mutação , Fosforilação , Ligação Proteica , Isoformas de Proteínas , Receptor IGF Tipo 1 , Receptor de Insulina/metabolismo , Receptores de Somatomedina/química , Transdução de SinaisRESUMO
Human insulin-like growth factor 1 (IGF-1) is a 70 amino acid protein hormone, with key impact on growth, development, and lifespan. The physiological and clinical importance of IGF-1 prompted challenging chemical and biological trials toward the development of its analogs as molecular tools for the IGF-1 receptor (IGF1-R) studies and as new therapeutics. Here, we report a new method for the total chemical synthesis of IGF-1 analogs, which entails the solid-phase synthesis of two IGF-1 precursor chains that is followed by the CuI-catalyzed azide-alkyne cycloaddition ligation and by biomimetic formation of a native pattern of disulfides. The connection of the two IGF-1 precursor chains by the triazole-containing moieties, and variation of its neighboring sequences (Arg36 and Arg37), was tolerated in IGF-1R binding and its activation. These new synthetic IGF-1 analogs are unique examples of disulfide bonds' rich proteins with intra main-chain triazole links. The methodology reported here also presents a convenient synthetic platform for the design and production of new analogs of this important human hormone with non-standard protein modifications.
Assuntos
Fator de Crescimento Insulin-Like I/análogos & derivados , Animais , Arginina/química , Química Click , Cobre/química , Reação de Cicloadição , Dissulfetos/química , Avaliação Pré-Clínica de Medicamentos/métodos , Fibroblastos , Humanos , Fator de Crescimento Insulin-Like I/síntese química , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Metionina/química , Camundongos , Células NIH 3T3/efeitos dos fármacos , Fosforilação , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Técnicas de Síntese em Fase Sólida , Triazóis/químicaRESUMO
Human insulin is a pivotal protein hormone controlling metabolism, growth, and aging and whose malfunctioning underlies diabetes, some cancers, and neurodegeneration. Despite its central position in human physiology, the in vivo oligomeric state and conformation of insulin in its storage granules in the pancreas are not known. In contrast, many in vitro structures of hexamers of this hormone are available and fall into three conformational states: T6, T3Rf3, and R6 As there is strong evidence for accumulation of neurotransmitters, such as serotonin and dopamine, in insulin storage granules in pancreatic ß-cells, we probed by molecular dynamics (MD) and protein crystallography (PC) if these endogenous ligands affect and stabilize insulin oligomers. Parallel studies independently converged on the observation that serotonin binds well within the insulin hexamer (site I), stabilizing it in the T3R3 conformation. Both methods indicated serotonin binding on the hexamer surface (site III) as well. MD, but not PC, indicated that dopamine was also a good site III ligand. Some of the PC studies also included arginine, which may be abundant in insulin granules upon processing of pro-insulin, and stable T3R3 hexamers loaded with both serotonin and arginine were obtained. The MD and PC results were supported further by in solution spectroscopic studies with R-state-specific chromophore. Our results indicate that the T3R3 oligomer is a plausible insulin pancreatic storage form, resulting from its complex interplay with neurotransmitters, and pro-insulin processing products. These findings may have implications for clinical insulin formulations.
Assuntos
Simulação por Computador , Células Secretoras de Insulina , Insulina , Modelos Biológicos , Neurotransmissores/metabolismo , Multimerização Proteica , Vesículas Secretórias , Serotonina/metabolismo , Humanos , Insulina/química , Insulina/metabolismo , Células Secretoras de Insulina/química , Células Secretoras de Insulina/metabolismo , Simulação de Dinâmica Molecular , Vesículas Secretórias/química , Vesículas Secretórias/metabolismoRESUMO
Heparan sulfate (HS) is a glycosaminoglycan that forms a key component of the extracellular matrix (ECM). Breakdown of HS is carried out by heparanase (HPSE), an endo-ß-glucuronidase of the glycoside hydrolase 79 (GH79) family. Overexpression of HPSE results in breakdown of extracellular HS and release of stored growth factors and hence is strongly linked to cancer metastasis. Here we present crystal structures of human HPSE at 1.6-Å to 1.9-Å resolution that reveal how an endo-acting binding cleft is exposed by proteolytic activation of latent proHPSE. We used oligosaccharide complexes to map the substrate-binding and sulfate-recognition motifs. These data shed light on the structure and interactions of a key enzyme involved in ECM maintenance and provide a starting point for the design of HPSE inhibitors for use as biochemical tools and anticancer therapeutics.
Assuntos
Glucuronidase/química , Glucuronidase/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Cristalografia por Raios X , Humanos , Ligação Proteica , Conformação ProteicaRESUMO
Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer's disease; aberrant signalling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor (IGF1R). Despite more than three decades of investigation, the three-dimensional structure of the insulin-insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal ß-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone-receptor recognition is novel within the broader family of receptor tyrosine kinases. We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titration calorimetry data that dissect the hormone-insulin receptor interface. Together, our findings provide an explanation for a wealth of biochemical data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogues.
Assuntos
Insulina/química , Insulina/metabolismo , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Animais , Sítios de Ligação , Calorimetria , Bovinos , Linhagem Celular , Cristalografia por Raios X , Humanos , Leucina/metabolismo , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Reprodutibilidade dos TestesRESUMO
Cbl is a member of the large family of LysR-type transcriptional regulators (LTTRs) common in bacteria and found also in Archaea and algal chloroplasts. The function of Cbl is required in Escherichia coli for expression of sulphate starvation-inducible (ssi) genes, associated with the biosynthesis of cysteine from organic sulphur sources (sulphonates). Here, we report the crystal structure of the cofactor-binding domain of Cbl (c-Cbl) from E. coli. The overall fold of c-Cbl is very similar to the regulatory domain (RD) of another LysR family member, CysB. The RD is composed of two subdomains enclosing a cavity, which is expected to bind effector molecules. We have constructed and analysed several full-length Cbl variants bearing single residue substitutions in the RD that affect cofactor responses. Using in vivo and in vitro transcription assays, we demonstrate that pssuE, a Cbl responsive promoter, is down-regulated not only by the cofactor, adenosine phosphosulphate (APS), but also by thiosulphate, and, that the same RD determinants are important for the response to both cofactors. We also demonstrate the effects of selected site-directed mutations on Cbl oligomerization and discuss these in the context of the structure. Based on the crystal structure and molecular modelling, we propose a model for the interaction of Cbl with adenosine phosphosulphate.
Assuntos
Adenosina Fosfossulfato/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Modelos Moleculares , Tiossulfatos/química , Fatores de Transcrição/química , Sítios de Ligação , Cristalografia por Raios X , Regulação para Baixo , Proteínas de Escherichia coli/genética , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Secundária de Proteína , Fatores de Transcrição/genéticaRESUMO
Cbl (CysB-like protein) is a member of the family of LysR-type transcriptional regulators (LTTRs) and controls genes engaged in sulfur assimilation in Escherichia coli. It has been postulated that adenosine 5-phosphosulfate (APS) is responsible for abolishing Cbl-activated transcription from the ssu promoter (Bykowski et al., 2002). To elucidate the structural basis of Cbl function and to confirm the role of APS as an anti-inducer, the cofactor-binding domain of Cbl (c-Cbl, MW = 26 kDa) was cloned, purified and crystallized in the presence of APS. The crystals belong to space group C222(1), but show substantial variation of the unit-cell parameters and diffraction anisotropy. Despite this, X-ray data extending to 3.0 A resolution have been collected and solution of the structure by molecular replacement is in progress.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Fatores de Transcrição/química , Adenosina Fosfossulfato/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Cristalização , Cristalografia por Raios X , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/isolamento & purificação , Ligação Proteica/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Síncrotrons , Fatores de Transcrição/biossíntese , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Gankyrin is a 25-kDa hepatocellular carcinoma-associated protein that mediates protein-protein interactions in cell cycle control and protein degradation. It has been reported to form complexes with cyclin-dependent kinase 4, retinoblastoma protein, the S6b ATPase subunit of the 19 S regulator of the 26 S proteasome, and Mdm2, an E3 ubiquitin ligase involved in p53 degradation. It is the first protein described to bind both to the 26 S proteasome and to proteins in other complexes containing cyclin-dependent kinase(s) and p53 ubiquitylating activities, thus providing a mechanism for delivering cell cycle regulating machinery and ubiquitylated substrates to the proteasome for degradation. Gankyrin contains a 33-residue motif known as the ankyrin repeat that occurs five and a half to six times in the sequence. As a step toward understanding gankyrin interactions with its protein partners we have determined its three-dimensional crystal structure to 2.0-A resolution. It reveals that the entire 226-residue gankyrin polypeptide folds into seven ankyrin repeat elements. The ankyrin repeats, consisting of an antiparallel beta-hairpin followed by a perpendicularly oriented helix-loop-helix, pack side-by-side, creating an extended curved structure with a groove running across the long concave surface. Comparison with the structures of other ankyrin repeat proteins suggests that interactions with partner proteins are mediated by residues situated on this concave surface.
Assuntos
Quinases Ciclina-Dependentes/química , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Oncogênicas/química , Proteínas Proto-Oncogênicas , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Elétrons , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Plasmídeos/metabolismo , Complexo de Endopeptidases do Proteassoma , Conformação Proteica , Dobramento de ProteínaRESUMO
Gankyrin is an oncoprotein overexpressed in hepatocarcinoma cells that binds to the cell-cycle regulator CDK4 and the S6b ATPase subunit of the regulatory component of the proteasome. It belongs to the family of ankyrin-repeat proteins that appear to mediate protein-protein interactions in diverse biochemical processes. Gankyrin has been crystallized from polyethylene glycol solutions and diffraction data have been obtained from these crystals that extend to 2.1 A spacing.
Assuntos
Proteínas Oncogênicas/química , Cristalização/métodos , Humanos , Polietilenoglicóis , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas , Difração de Raios X/métodosRESUMO
Eubacteria and eukaryotic cellular organelles have membrane-bound ATP-dependent proteases, which degrade misassembled membrane protein complexes and play a vital role in membrane quality control. The bacterial protease FtsH also degrades an interesting subset of cytoplasmic regulatory proteins, including sigma(32), LpxC, and lambda CII. The crystal structure of the ATPase module of FtsH has been solved, revealing an alpha/beta nucleotide binding domain connected to a four-helix bundle, similar to the AAA modules of proteins involved in DNA replication and membrane fusion. A sulfate anion in the ATP binding pocket mimics the beta-phosphate group of an adenine nucleotide. A hexamer form of FtsH has been modeled, providing insights into possible modes of nucleotide binding and intersubunit catalysis.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Escherichia coli/enzimologia , Proteínas de Membrana/química , Metaloendopeptidases/química , Conformação Proteica , Proteases Dependentes de ATP , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Proteínas de Escherichia coli , Modelos Biológicos , Modelos Moleculares , Estrutura Molecular , Dobramento de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Alinhamento de SequênciaRESUMO
FtsH is a membrane-anchored ATP-dependent protease that degrades misfolded or misassembled membrane proteins as well as a subset of cytoplasmic regulatory proteins. It belongs to the family of AAA(+) ATPases with roles in diverse cellular processes. The ATPase domain of FtsH from Escherichia coli has been crystallized from ammonium sulfate solutions and crystals diffracting to 1.5 A resolution have been obtained.
Assuntos
Proteínas de Bactérias/química , Escherichia coli/enzimologia , Proteínas de Membrana/química , Proteases Dependentes de ATP , Cristalização , Cristalografia por Raios X , Proteínas de Escherichia coli , Proteínas de Choque Térmico/química , Conformação Proteica , Estrutura Terciária de Proteína , Serina Endopeptidases/químicaRESUMO
The activation function 2/ligand-dependent interaction between nuclear receptors and their coregulators is mediated by a short consensus motif, the so-called nuclear receptor (NR) box. Nuclear receptors exhibit distinct preferences for such motifs depending both on the bound ligand and on the NR box sequence. To better understand the structural basis of motif recognition, we characterized the interaction between estrogen receptor alpha and the NR box regions of the p160 coactivator TIF2. We have determined the crystal structures of complexes between the ligand-binding domain of estrogen receptor alpha and 12-mer peptides from the Box B2 and Box B3 regions of TIF2. Surprisingly, the Box B3 module displays an unexpected binding mode that is distinct from the canonical LXXLL interaction observed in other ligand-binding domain/NR box crystal structures. The peptide is shifted along the coactivator binding site in such a way that the interaction motif becomes LXXYL rather than the classical LXXLL. However, analysis of the binding properties of wild type NR box peptides, as well as mutant peptides designed to probe the Box B3 orientation, suggests that the Box B3 peptide primarily adopts the "classical" LXXLL orientation in solution. These results highlight the potential difficulties in interpretation of protein-protein interactions based on co-crystal structures using short peptide motifs.