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1.
Nat Commun ; 10(1): 4427, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562314

RESUMO

Insulin and IGF-1 actions in vascular smooth muscle cells (VSMC) are associated with accelerated arterial intima hyperplasia and restenosis after angioplasty, especially in diabetes. To distinguish their relative roles, we delete insulin receptor (SMIRKO) or IGF-1 receptor (SMIGF1RKO) in VSMC and in mice. Here we report that intima hyperplasia is attenuated in SMIRKO mice, but not in SMIGF1RKO mice. In VSMC, deleting IGF1R increases homodimers of IR, enhances insulin binding, stimulates p-Akt and proliferation, but deleting IR decreases responses to insulin and IGF-1. Studies using chimeras of IR(extracellular domain)/IGF1R(intracellular-domain) or IGF1R(extracellular domain)/IR(intracellular-domain) demonstrate homodimer IRα enhances insulin binding and signaling which is inhibited by IGF1Rα. RNA-seq identifies hyaluronan synthase2 as a target of homo-IR, with its expression increases by IR activation in SMIGF1RKO mice and decreases in SMIRKO mice. Enhanced intima hyperplasia in diabetes is mainly due to insulin signaling via homo-IR, associated with increased Has2 expression.


Assuntos
Diabetes Mellitus/metabolismo , Hiperplasia/metabolismo , Resistência à Insulina/fisiologia , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animais , Modelos Animais de Doenças , Artéria Femoral/lesões , Artéria Femoral/metabolismo , Artéria Femoral/patologia , Homozigoto , Hialuronan Sintases/metabolismo , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Receptor IGF Tipo 1/química , Receptor de Insulina/química , Transdução de Sinais
2.
Cell Mol Life Sci ; 76(17): 3433-3447, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30980109

RESUMO

Enhancement of insulin-like growth factor 1 receptor (IGF-IR) degradation by heat shock protein 90 (HSP90) inhibitor is a potential antitumor therapeutic strategy. However, very little is known about how IGF-IR protein levels are degraded by HSP90 inhibitors in pancreatic cancer (PC). We found that the HSP90α inhibitor NVP-AUY922 (922) effectively downregulated and destabilized the IGF-1Rß protein, substantially reduced the levels of downstream signaling molecules (p-AKT, AKT and p-ERK1/2), and resulted in growth inhibition and apoptosis in IGF-1Rß-overexpressing PC cells. Preincubation with a proteasome or lysosome inhibitor (MG132, 3 MA or CQ) mainly led to IGF-1Rß degradation via the lysosome degradation pathway, rather than the proteasome-dependent pathway, after PC cells were treated with 922 for 24 h. These results might be associated with the inhibition of pancreatic cellular chymotrypsin-peptidase activity by 922 for 24 h. Interestingly, 922 induced autophagic flux by increasing LC3II expression and puncta formation. However, knockdown of the crucial autophagy component AGT5 and the chemical inhibitor 3 MA-blocked 922-induced autophagy did not abrogate 922-triggered IGF-1Rß degradation. Furthermore, 922 could enhance chaperone-mediated autophagy (CMA) activity and promote the association between HSP/HSC70 and IGF-1Rß or LAMP2A in coimmunoprecipitation and immunofluorescence analyses. Silencing of LAMP2A to inhibit CMA activity reversed 922-induced IGF-1Rß degradation, suggesting that IGF-1Rß degradation by 922 was partially dependent on the CMA pathway rather than macroautophagy. This finding is mirrored by the identification of the KFERQ-like motif in IGF-1Rß. These observations support the potential application of 922 for IGF-1Rß-overexpressing PC therapy and first identify the role of the CMA pathway in IGF-1Rß degradation by an HSP90 inhibitor.


Assuntos
Autofagia/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Isoxazóis/farmacologia , Receptor IGF Tipo 1/metabolismo , Resorcinóis/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Sequência de Aminoácidos , Proteína 5 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/antagonistas & inibidores , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/parasitologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/genética , Alinhamento de Sequência , Transdução de Sinais/efeitos dos fármacos
3.
Eur J Pharmacol ; 853: 93-102, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30878387

RESUMO

Vascular smooth muscle cell (VSMC) proliferation plays a critical role in arterial remodeling during various vascular diseases including atherosclerosis and hypertension. Tanshinone I, a major component of Salvia miltiorrhiza, exerts protective effects against cardiovascular diseases. In this study, we investigated the effects of tanshinone I on VSMC proliferation, as well as the underlying mechanisms. We found that this compound inhibited the proliferation of VSMCs in a dose-dependent manner, based on 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and 5-ethynyl-2'-deoxyuridine (EdU) assays. Western blotting demonstrated that tanshinone I inhibited the expression of proliferation-related proteins, including cyclin-dependent kinase 4 (CDK4), cyclin D3, and cyclin D1, in a dose-dependent manner. Molecular docking showed that this compound docked to the inhibitor-binding site of the insulin-like growth factor 1 (IGF-1) receptor (IGF-1R), and the binding energy between tanshinone I and IGF-1R was -9.021 kcal/mol. Molecular dynamic simulations showed that the IGF-1R-tanshinone I binding was stable. We also found that tanshinone I dose-dependently inhibited IGF-1R activation and its downstream molecules, insulin receptor substrate (IRS)-1, phosphatidylinositol-3-Kinase (PI3K), Akt, glycogen synthase kinase-3 beta (GSK3ß), mammalian target of rapamycin (mTOR), 70S6K, and ribosomal protein S6 (RPS6). Notably, activation of IGF-1R by recombinant IGF-1 rescued the activity of IGF-1R and its downstream molecules, and the proliferation of tanshinone I-treated VSMC. In addition, blocking PI3K signaling with LY294002 showed the important role of this pathway in tanshinone I-mediated suppression of VSMC proliferation. Collectively, these data demonstrated that tanshinone I might inhibit VSMC proliferation by inhibiting IGF-1R/PI3K signaling.


Assuntos
/farmacologia , Músculo Liso Vascular/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , /metabolismo , Proliferação de Células/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Receptor IGF Tipo 1/química
4.
Nano Lett ; 19(3): 1560-1569, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30789273

RESUMO

Bioactive peptides derived from proteins generally need to be folded into secondary structures to activate downstream signaling pathways. However, synthetic peptides typically form random-coils, thus losing their bioactivities. Here, we show that by introducing a self-assembling peptide motif and using different preparation pathways, a peptide from insulin-like growth factor-I (IGF-1) can be folded into an α-helix and ß-sheet. The ß-sheet one exhibits a low dissociation constant to the IGF-1 receptor (IGF-1R, 11.5 nM), which is only about 3 times higher than that of IGF-1 (4.3 nM). However, the α-helical one and the peptide without self-assembling motif show weak affinities to IGF-1R ( KD = 179.1 and 321.6 nM, respectively). At 10 nM, the ß-sheet one efficiently activates the IGF-1 downstream pathway, significantly enhancing HUVEC proliferation and preventing cell apoptosis. The ß-sheet peptide shows superior performance to IGF-1 in vivo, and it improves ischemic hind-limb salvage by significantly reducing muscle degradation and enhancing limb vascularization. Our study provides a useful strategy to constrain peptides into different conformations, which may lead to the development of supramolecular nanomaterials mimicking biofunctional proteins.


Assuntos
Fator de Crescimento Insulin-Like I/química , Nanofibras/química , Peptídeos/química , Receptor IGF Tipo 1/química , Apoptose/genética , Proliferação de Células/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Nanoestruturas/química , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Transdução de Sinais/genética
5.
Eur J Endocrinol ; 179(2): 85-95, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29789409

RESUMO

OBJECTIVE: The insulin-like growth factor1 receptor (IGF1R) is important in growth and development, and inactivating IGF1R mutations cause short stature and relatively high levels of serum IGF-I. We identified an unclassified IGF1RR1353H variant in a male with extreme tall height, very low levels of serum IGF-I and delayed and prolonged growth spurt. The index case's mother and three sons all carried the variant, but so far only the eldest son (age 18 years) presented with tall height. We hypothesized that the variant could constitute an activating mutation. DESIGN: The IGF1RR1353H variant was investigated in Igf1r-/- mouse embryonic fibroblasts (R-cells) by cell cycle, colony formation and transcriptome analyses. RESULTS: The IGF1RR1353H (R-1353) exhibited significantly increased cell proliferation, G1-S progression and colony formation in soft agar. RNA sequencing identified 195 differentially expressed genes between R-WT and R-1353 (adjusted P < 1E-100). Most genes were upregulated in R-1353, including the gene encoding the androgen receptor (AR). Gene expression profiling showed the most significant enrichment in extracellular matrix organization (P = 2.76E-7), collagen biosynthesis (P = 1.21E-5) and cell adhesion (P = 7.38E-5). Retrospective biochemical analysis of the index case revealed decreased testosterone and sex hormone-binding globulin levels, whereas LH and FSH were within normal ranges. This profile suggests an increased sensitivity to androgen, which is compatible with the enhanced expression of Ar in R-1353 cells. CONCLUSIONS: Our findings suggest that R1353H constitutes an activating IGF1R variant. The possible deregulation of collagen turnover and increased androgen sensitivity implicates an association to tall phenotype in male carriers.


Assuntos
Regulação para Baixo , Transtornos do Crescimento/genética , Fator de Crescimento Insulin-Like I/análise , Mutação Puntual , Receptor IGF Tipo 1/genética , Adulto , Substituição de Aminoácidos , Animais , Estatura , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Transtornos do Crescimento/sangue , Transtornos do Crescimento/metabolismo , Transtornos do Crescimento/fisiopatologia , Heterozigoto , Humanos , Masculino , Camundongos Knockout , Linhagem , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de RNA , Índice de Gravidade de Doença
6.
J Mol Graph Model ; 75: 316-321, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28628857

RESUMO

The insulin-like growth factor-1 receptor (IGF-1R) plays a key role in proliferation, growth, differentiation, and development of several human malignancies including breast and pancreatic adenocarcinoma. IGF-1R targeted immunotherapeutic approaches are particularly attractive, as they may potentially elicit even stronger antitumor responses than traditional targeted approaches. Cancer peptide vaccines can produce immunologic responses against cancer cells by triggering helper T cell (Th) or cytotoxic T cells (CTL) in association with Major Histocompatibility Complex (MHC) class I or II molecules on the cell surface of antigen presenting cells. In our previous study, we set a technique based on molecular docking in order to find the best MHC class I and II binder peptides using GOLD. In the present work, molecular docking analyses on a library consisting of 30 peptides mimicking discontinuous epitopes from IGF-1R extracellular domain identified peptides 249 and 86, as the best MHC binder peptides to both MHC class I and II molecules. The receptors most often targeted by peptide 249 are HLA-DR4, HLA-DR3 and HLA-DR2 and those most often targeted by peptide 86 are HLA-DR4, HLA-DP2 and HLA-DR3. These findings, based on bioinformatics analyses, can be conducted in further experimental analyses in cancer therapy and vaccine design.


Assuntos
Neoplasias da Mama/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Receptor IGF Tipo 1/imunologia , Vacinas de Subunidades/imunologia , Sequência de Aminoácidos , Sítios de Ligação , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Feminino , Humanos , Simulação de Acoplamento Molecular , Receptor IGF Tipo 1/química , Vacinas de Subunidades/química
7.
Nat Commun ; 8: 14892, 2017 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-28345670

RESUMO

Despite a high degree of homology, insulin receptor (IR) and IGF-1 receptor (IGF1R) mediate distinct cellular and physiological functions. Here, we demonstrate how domain differences between IR and IGF1R contribute to the distinct functions of these receptors using chimeric and site-mutated receptors. Receptors with the intracellular domain of IGF1R show increased activation of Shc and Gab-1 and more potent regulation of genes involved in proliferation, corresponding to their higher mitogenic activity. Conversely, receptors with the intracellular domain of IR display higher IRS-1 phosphorylation, stronger regulation of genes in metabolic pathways and more dramatic glycolytic responses to hormonal stimulation. Strikingly, replacement of leucine973 in the juxtamembrane region of IR to phenylalanine, which is present in IGF1R, mimics many of these signalling and gene expression responses. Overall, we show that the distinct activities of the closely related IR and IGF1R are mediated by their intracellular juxtamembrane region and substrate binding to this region.


Assuntos
Expressão Gênica , Insulina/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Substituição de Aminoácidos , Animais , Sítios de Ligação , Proliferação de Células , Regulação da Expressão Gênica , Glicólise , Leucina/química , Camundongos , Mutagênese Sítio-Dirigida , Fenilalanina/química , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/genética
8.
J Anim Breed Genet ; 134(1): 34-42, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27112238

RESUMO

As a member of the somatotropic axis, insulin-like growth factor I receptor (IGF1R) seems to be a promising candidate gene. Two silent polymorphisms, identified by MspI and TaqI restriction enzymes, were selected within exon 2, encoding the majority of the putative ligand binding pocket. A total of 1169 cows of four pure breeds (Polish Holstein Friesian, Montbeliarde, Jersey and Holstein Friesian) were genotyped. The T (IGF1R/e2/MspI) and G (IGF1R/e2/TaqI) alleles were found to be prevalent. Three combinations of genotypes (TT/GG, TT/AG and CT/GG) were associated with the highest productivity (milk, protein and fat yields) among all breeds under study, as opposed to individuals carrying the worst CC/AA combination. In view of the specific structure of the ligand binding pocket and the significance of insulin-like growth factor I signalling promoting the development and differentiation in a variety of tissues (not only limited to mammary gland), the existence of missense mutation is unlikely. Potential mutations are likely limited to mRNA transcription and further post-transcriptional modifications. Further investigations should follow searching for the most useful IGF1R haplotypes, associated with higher milk production traits, exerting at the same time positive or neutral impact on health and welfare of individuals.


Assuntos
Bovinos/genética , Leite/química , Polimorfismo Genético , Receptor IGF Tipo 1/genética , Animais , Sítios de Ligação , Bovinos/classificação , Éxons , Lactação , Receptor IGF Tipo 1/química
9.
Tumour Biol ; 37(10): 14271-14290, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27592256

RESUMO

Receptor tyrosine kinases (RTKs) are transmembrane high-affinity surface receptors responsible for cell migration, adhesion, apoptosis, metabolism, and cell proliferation activities in various cancers. Minute aberration in the RTK signaling modulates the downstream signaling pathways that results in cancer. Ganoderic acid is a triterpene isolated from Ganoderma lucidum, which is renowned for its therapeutics effect, especially in cancer. The present study discusses receptor-based molecular docking of insulin receptor (IR), insulin-like growth factor receptor 1 (IGFR-1), vascular endothelial growth factor receptor-1 (VEGFR-1), vascular endothelial growth factor receptor-2 (VEGFR-2), and estrogen receptor (ER) with 50 isoforms of ganoderic acid along with natural inhibitors. These receptors were assessed for toxicity (ADMET) by using Maestro 9.6 (Schrödinger Inc). The calculated docking free energy yielded an excellent dock score for the ganoderic acid when docked with proteins IR, IGFR-1, VEGFR-1, VEGFR-2, and ER, suggesting its potential in combating cancer. Protein-ligand profile highlighted the binding interactions comprising lipophilic, hydrogen bonding, pi-pi stacking interactions, and noncovalent bonding which play a pivotal role in targeting cancer. In silico studies revealed structure of ganoderic acid A as best isoforms among 50 isoforms which exhibits biological activity in liver cancer cells. Ganoderic acids A significantly decrease the viability, proliferation, and oxidative stress in a dose-dependent manner in liver cancer cells.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Receptores de Superfície Celular/química , Triterpenos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Simulação por Computador , Humanos , Técnicas In Vitro , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Simulação de Acoplamento Molecular , Conformação Proteica , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Estrogênicos/química , Receptores Estrogênicos/metabolismo , Células Tumorais Cultivadas , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
10.
J Biol Chem ; 291(40): 21234-21245, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-27510031

RESUMO

Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains.


Assuntos
Antígenos CD/química , Fator de Crescimento Insulin-Like II/química , Receptor IGF Tipo 1/química , Receptor de Insulina/química , Substituição de Aminoácidos , Antígenos CD/genética , Antígenos CD/metabolismo , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Mutação de Sentido Incorreto , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
PLoS One ; 11(8): e0161459, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27548502

RESUMO

The insulin-family proteins bind to their own receptors, but insulin-like growth factor II (IGF-II) can also bind to the A isoform of the insulin receptor (IR-A), activating unique and alternative signaling pathways from those of insulin. Although extensive studies of insulin have revealed that its activation is associated with the opening of the B chain-C terminal (BC-CT), the activation mechanism of the insulin-like growth factors (IGFs) still remains unknown. Here, we present the first comprehensive study of the insulin-family proteins comparing their activation process and mechanism using molecular dynamics simulations to reveal new insights into their specificity to the insulin receptor. We have found that all the proteins appear to exhibit similar stochastic dynamics in their conformational change to an active state. For the IGFs, our simulations show that activation involves two opening locations: the opening of the BC-CT section away from the core, similar to insulin; and the additional opening of the BC-CT section away from the C domain. Furthermore, we have found that these two openings occur simultaneously in IGF-I, but not in IGF-II, where they can occur independently. This suggests that the BC-CT section and the C domain behave as a unified domain in IGF-I, but as two independent domains in IGF-II during the activation process, implying that the IGFs undergo different activation mechanisms for receptor binding. The probabilities of the active and inactive states of the proteins suggest that IGF-II is hyperactive compared to IGF-I. The hinge residue and the hydrophobic interactions in the core are found to play a critical role in the stability and activity of IGFs. Overall, our simulations have elucidated the crucial differences and similarities in the activation mechanisms of the insulin-family proteins, providing new insights into the molecular mechanisms responsible for the observed differences between IGF-I and IGF-II in receptor binding.


Assuntos
Fator de Crescimento Insulin-Like II/química , Fator de Crescimento Insulin-Like I/química , Insulina/química , Simulação de Dinâmica Molecular , Receptor IGF Tipo 1/química , Receptor IGF Tipo 2/química , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Termodinâmica
12.
Mol Endocrinol ; 30(6): 600-13, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27082896

RESUMO

Many studies have provided evidence to demonstrate the beneficial renal effects of resveratrol (RESV) due to its antioxidant character and its capacity for activation of surtuin 1. However, the molecular mechanisms underlying the protective role of RESV against kidney injury are still incompletely understood. The present study used Lepr db/db (db/db) and Lepr db/m (db/m) mice as models to evaluate the effect of RESV on diabetic nephropathy (DN). RESV reduced proteinuria and attenuated the progress of renal fibrosis in db/db mice. Treatment with RESV markedly attenuated the diabetes-induced changes in renal superoxide dismutase copper/zinc, superoxide dismutase manganese, catalase, and malonydialdehyde as well as the renal expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), α-smooth muscle actin (α-SMA), and E-cadherin in db/db mice. The kidney expression of the IGF-1 receptor (IGF-1R) was increased in db/db mice, but the expression of 3-hydroxy-3-methylglutaryl reductase degradation (HRD1), a ubiquitin E3 ligase, was significantly decreased in the DN model. RESV treatment dramatically decreased IGF-1R and increased HRD1 expressions, consistent with data obtained with HKC-8 cells. HRD1 physically interacted with IGF-1R in HKC-8 cells and liquid chromatography and tandem mass spectrometry (LC-MS/MS) data supported the concept that IGF-1R is one of the HRD1 substrates. HRD1 promoted the IGF-1R ubiquitination for degradation in HKC-8 cells, and the down-regulation of HRD1 reversed the protective effects of RESV in HKC-8 cells. In summary, we have demonstrated that RESV reduces proteinuria and attenuates the progression of renal fibrosis in db/db mice. These protective effects of RESV on DN were associated with the up-regulation of HRD1, induced by RESV, and the promotion of IGF-1R ubiquitination and degradation.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Rim/patologia , Substâncias Protetoras/uso terapêutico , Receptor IGF Tipo 1/metabolismo , Estilbenos/uso terapêutico , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Biomarcadores/metabolismo , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Cromatografia Líquida , Regulação para Baixo/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/química , Resveratrol , Estilbenos/farmacologia , Espectrometria de Massas em Tandem , Fator de Crescimento Transformador beta/farmacologia , Ubiquitinação/efeitos dos fármacos
13.
Mol Nutr Food Res ; 60(9): 1912-23, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27028006

RESUMO

SCOPE: Tannin-rich fruits have been evaluated as alternative prevention strategies for colorectal cancer based on their anti-inflammatory properties. This study compared tannin-rich preparations from mango (rich in gallotannins) and pomegranate (rich in ellagitannins) in the dextran sodium sulfate-induced colitis model. METHODS AND RESULTS: In rats, mango and pomegranate beverages decreased intestinal inflammation and the levels of pro-inflammatory cytokines in mucosa and serum. The mango beverage suppressed the ratio of phosphorylated/total protein expression of the IGF-1R-AKT/mTOR axis and downregulated mRNA expression of Igf1, Insr, and pik3cv. Pomegranate decreased p70S6K and RPS6, as well as Rps6ka2, Map2k2, and Mapk1 mRNA. In silico modeling indicated a high binding of docked of gallic acid to the catalytic domain of IGF-1R, which may suppress the activity of the enzyme. Ellagic acid docked effectively into the catalytic domains of both IGF-1R and EGFR. In vitro assays with lipopolysaccharide-treated CCD-18Co cells using polyphenolic extracts from each beverage, as well as pure compounds, corroborated the predictions made in silico. CONCLUSION: Mango polyphenols inhibited the IGF-1R- AKT/mTOR axis, and pomegranate polyphenols downregulate the mTOR downstream pathway through reductions in ERK1/2. These results suggest that extracts rich in gallo- and ellagitannins act on different molecular targets in the protection against ulcerative colitis.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Colite/tratamento farmacológico , Lythraceae/química , Mangifera/química , Animais , Anti-Inflamatórios não Esteroides/química , Colite/induzido quimicamente , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sucos de Frutas e Vegetais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Simulação de Acoplamento Molecular , Polifenóis/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
14.
Cell Signal ; 28(6): 595-605, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26931381

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth factor (IGF)-1 receptor, a receptor tyrosine kinase, also plays an essential role in Kaposi's sarcoma growth and survival. In this study we examined the effect of the constitutively active viral receptor ORF74 on human IGF-1R signaling. Constitutive and CXCL1-induced ORF74 signaling did not transactivate IGF-1R. In contrast, IGF-1 stimulated phospholipase C (PLC) activation in an ORF74-dependent manner without affecting chemokine binding to ORF74. Inhibition of constitutive ORF74 activity by mutagenesis or the inverse agonist CXCL10, or neutralizing IGF-1R with an antibody or silencing IGF-1R expression using siRNA inhibited PLC activation by IGF-1. Transactivation of ORF74 in response to IGF-1 occurred independently of Src, PI3K, and secreted ORF74 ligands. Furthermore, tyrosine residues in the carboxyl-terminus and intracellular loop 2 of ORF74 are not essential for IGF-1-induced PLC activation. Interestingly, PLC activation in response to IGF-1 is specific for ORF74 as IGF-1 was unable to activate PLC in cells expressing the constitutively active human cytomegalovirus (HCMV)-encoded GPCR US28. Interestingly, IGF-1 does not induce ß-arrestin recruitment to ORF74. The proximity ligation assay revealed close proximity between ORF74 and IGF-1R on the cell surface, but a physical interaction was not confirmed by co-immunoprecipitation. Unmasking IGF-1R signaling to PLC in response to IGF-1 is a previously unrecognized action of ORF74.


Assuntos
Receptor IGF Tipo 1/metabolismo , Receptores de Quimiocinas/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Proteínas Virais/metabolismo , Células HEK293 , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptor IGF Tipo 1/química , Tirosina/metabolismo
15.
Org Biomol Chem ; 14(9): 2698-705, 2016 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-26839188

RESUMO

The interaction of IGF-II with the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R) has recently been identified as potential therapeutic target for the treatment of cancer. Understanding the interactions of IGF-II with these receptors is required for the development of potential anticancer therapeutics. This work describes an efficient convergent synthesis of native IGF-II and two non-native IGF-II analogues with coumarin fluorescent probes incorporated at residues 19 and 28. These fluorescent analogues bind with nanomolar affinities to the IGF-1R and are suitable for use in fluorescence resonance energy transfer (FRET) studies. From these studies the F19Cou IGF-II and F28Cou IGF-II proteins were identified as good probes for investigating the binding interactions of IGF-II with the IGF-1R and its other high affinity binding partners.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Fluorescência , Fator de Crescimento Insulin-Like II/química , Receptor IGF Tipo 1/química , Sítios de Ligação , Fator de Crescimento Insulin-Like II/análogos & derivados , Estrutura Molecular
16.
Sci Rep ; 6: 19431, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26792393

RESUMO

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone's B-chain C-terminus for its IR-B specificity.


Assuntos
Insulina/química , Insulina/metabolismo , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Alquinos/química , Azidas/química , Reação de Cicloadição , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estabilidade Proteica , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/metabolismo , Relação Estrutura-Atividade
17.
Sheng Wu Gong Cheng Xue Bao ; 32(5): 693-701, 2016 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-29019206

RESUMO

The length of IGF1R 3'UTR is greater than 7 kb. The structure of IGF1R 3'UTR is complex, with multiple binding sites of miRNAs. IGF1R is involved in the regulation of MAPK and PI3K/AKT signaling pathways and theformation and development of tumors. Bioinformatics analysis can reveal the structure features of IGF1R, which provides ideas for further research. The analysis shows that the binding sites between IGF1R and miRNAs have the highest mutation rate in Neuroblastoma. We analyzed the structure of 3'UTR, miRNAs binding sites, physical and chemical properties, hydrophilic-hydrophobic property, glycosylation and phosphorylation sites, secondary structure and tertiary structure modeling of IGF1R. The locations and names of amino acids interacting in IGF1R and IGF1 were obtained by molecular docking. Therefore, if IGF1R 3'UTR is mutated, the capacity of IGF1R combined with miRNAs will reduce and the IGF1R expression will be up-regulated, and the function of miRNAs will be repressed. We can change the sites of IGF1R to combine with IGF1 to repress the function of IGF1R and IGF1. Then the function of IGF1R will be repressed.


Assuntos
Biologia Computacional , Simulação de Acoplamento Molecular , Receptor IGF Tipo 1/química , Sítios de Ligação , Humanos , Fator de Crescimento Insulin-Like I , MicroRNAs/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais
18.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 31(9): 1211-5, 2015 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-26359102

RESUMO

OBJECTIVE: To construct the prokaryotic expression vectors of extracellular domain (ß-ED) and intracellular protein kinase domain (ß-PKD) of insulin-like growth factor 1 receptor beta (IGF1R-ß) subunit, purify the fusion proteins GST-IGF1R ß-ED and GST-IGF1R ß-PKD, and detect their activities. METHODS: Human GST-IGF1R ß-ED and GST-IGF1R ß-PKD coding regions were amplified from human mammary cDNA library by PCR and cloned into the prokaryotic expression vector pGEX-KG. The fusion proteins GST-IGF1R ß-ED and GST-IGF1R ß-PKD were expressed in E.coli Rossate and purified by GST-Sepharose 4B beads. The expression of the fusion proteins were detected by Western blotting. The interactions of the proteins with mediator of epidermal growth factor receptor-2 (ERBB2)-driven cell motility (MEMO) protein were identified by GST pull-down assay. RESULTS: GST-IGF1R ß-ED and GST-IGF1R ß-PKD recombinant plasmids were successfully cloned. Double enzyme digestion and sequencing confirmed that the inserted fragments were identical to the target ones. The fusion proteins were successfully induced in Rossate and Western blotting showed the expression as expected. GST pull-down assay revealed that GST-IGF1R ß-PKD could interact with MEMO in vitro. CONCLUSION: GST-IGF1R ß-ED and GST-IGF1R ß-PKD were successfully cloned and purified. In addition, GST-IGF1R ß-PKD could interact with MEMO in vitro, which demonstrated the good activity of the purified proteins.


Assuntos
Receptor IGF Tipo 1/genética , Proteínas Recombinantes de Fusão/biossíntese , Escherichia coli/genética , Humanos , Ferroproteínas não Heme/genética , Plasmídeos , Estrutura Terciária de Proteína , Subunidades Proteicas , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/isolamento & purificação
19.
Sci Rep ; 5: 12055, 2015 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-26170015

RESUMO

Vimentin, an intermediate filament protein, is generally recognised as an intracellular protein. Previously, we reported that vimentin was secreted from astrocytes and promoted axonal growth. The effect of extracellular vimentin in neurons was a new finding, but its signalling pathway was unknown. In this study, we aimed to determine the signalling mechanism of extracellular vimentin that facilitates axonal growth. We first identified insulin-like growth factor 1 receptor (IGF1R) as a receptor that is highly phosphorylated by vimentin stimulation. IGF1R blockades diminished vimentin- or IGF1-induced axonal growth in cultured cortical neurons. IGF1, IGF2 and insulin were not detected in the neuron culture medium after vimentin treatment. The combined drug affinity responsive target stability method and western blotting analysis showed that vimentin and IGF1 interacted with IGF1R directly. In addition, immunoprecipitation and western blotting analyses confirmed that recombinant IGF1R bound to vimentin. The results of a molecular dynamics simulation revealed that C-terminal residues (residue number 330-407) in vimentin are the most appropriate binding sites with IGF1R. Thus, extracellular vimentin may be a novel ligand of IGF1R that promotes axonal growth in a similar manner to IGF1. Our results provide novel findings regarding the role of extracellular vimentin and IGF1R in axonal growth.


Assuntos
Axônios/metabolismo , Espaço Extracelular/metabolismo , Receptor IGF Tipo 1/metabolismo , Vimentina/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Axônios/efeitos dos fármacos , Sítios de Ligação , Células Cultivadas , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Ratos , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/química , Vimentina/química , Vimentina/farmacologia
20.
Structure ; 23(7): 1271-82, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26027733

RESUMO

The homodimeric insulin and type 1 insulin-like growth factor receptors (IR and IGF-1R) share a common architecture and each can bind all three ligands within the family: insulin and insulin-like growth factors I and II (IGF-I and IFG-II). The receptor monomers also assemble as heterodimers, the primary ligand-binding sites of which each comprise the first leucine-rich repeat domain (L1) of one receptor type and an α-chain C-terminal segment (αCT) of the second receptor type. We present here crystal structures of IGF-I bound to such a hybrid primary binding site and of a ligand-free version of an IR αCT peptide bound to an IR L1 plus cysteine-rich domain construct (IR310.T). These structures, refined at 3.0-Å resolution, prove congruent to respective existing structures of insulin-complexed IR310.T and the intact apo-IR ectodomain. As such, they provide key missing links in the emerging, but sparse, repertoire of structures defining the receptor family.


Assuntos
Fator de Crescimento Insulin-Like I/química , Receptor IGF Tipo 1/química , Receptor de Insulina/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína
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