RESUMO
Multivalent display of receptor-engaging antibodies or ligands can enhance their activity. Instead of achieving multivalency by attachment to preexisting scaffolds, here we unite form and function by the computational design of nanocages in which one structural component is an antibody or Fc-ligand fusion and the second is a designed antibody-binding homo-oligomer that drives nanocage assembly. Structures of eight nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage, respectively, closely match the corresponding computational models. Antibody nanocages targeting cell surface receptors enhance signaling compared with free antibodies or Fc-fusions in death receptor 5 (DR5)-mediated apoptosis, angiopoietin-1 receptor (Tie2)-mediated angiogenesis, CD40 activation, and T cell proliferation. Nanocage assembly also increases severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc-angiotensin-converting enzyme 2 (ACE2) fusion proteins.
Assuntos
Anticorpos/química , Anticorpos/imunologia , Nanoestruturas , Engenharia de Proteínas , Transdução de Sinais , Angiopoietinas/química , Angiopoietinas/imunologia , Angiopoietinas/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Antígenos CD40/química , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Simulação por Computador , Genes Sintéticos , Humanos , Fragmentos Fc das Imunoglobulinas/química , Ativação Linfocitária , Modelos Moleculares , Ligação Proteica , Receptor TIE-2/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Coronary artery disease is the most common cause of death globally and is linked to a number of risk factors including serum low density lipoprotein, high density lipoprotein, triglycerides and lipoprotein(a). Recently two proteins, angiopoietin-like protein 3 and 4, have emerged from genetic studies as being factors that significantly modulate plasma triglyceride levels and coronary artery disease. The exact function and mechanism of action of both proteins remains to be elucidated, however, mutations in these proteins results in up to 34% reduction in coronary artery disease and inhibition of function results in reduced plasma triglyceride levels. Here we report the crystal structures of the fibrinogen-like domains of both proteins. These structures offer new insights into the reported loss of function mutations, the mechanisms of action of the proteins and open up the possibility for the rational design of low molecular weight inhibitors for intervention in coronary artery disease.
Assuntos
Proteína 4 Semelhante a Angiopoietina/química , Proteínas Semelhantes a Angiopoietina/química , Doença da Artéria Coronariana/genética , Conformação Proteica , Proteína 3 Semelhante a Angiopoietina , Proteína 4 Semelhante a Angiopoietina/genética , Proteínas Semelhantes a Angiopoietina/genética , Angiopoietinas/química , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/patologia , Cristalografia por Raios X , Humanos , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Mutação com Perda de Função/genética , Domínios Proteicos/genética , Fatores de Risco , Triglicerídeos/sangueRESUMO
OBJECTIVE: Several members of the angiopoietin-like (ANGPTL) family of proteins, including ANGPTL3 and ANGPTL8, regulate lipoprotein lipase (LPL) activity. Deficiency in either ANGPTL3 or ANGPTL8 reduces plasma triglyceride levels and increases LPL activity, whereas overexpression of either protein does the opposite. Recent studies suggest that ANGPTL8 may functionally interact with ANGPTL3 to alter clearance of plasma triglycerides; however, the nature of this interaction has remained elusive. We tested the hypothesis that ANGPTL8 forms a complex with ANGPTL3 and that this complex is necessary for the inhibition of vascular LPL by ANGPTL3. METHODS: We analyzed the interactions of ANGPTL3 and ANGPTL8 with each other and with LPL using co-immunoprecipitation, western blotting, lipase activity assays, and the NanoBiT split-luciferase system. We also used adenovirus injection to overexpress ANGPTL3 in mice that lacked ANGPTL8. RESULTS: We found that ANGPTL3 or ANGPTL8 alone could only inhibit LPL at concentrations that far exceeded physiological levels, especially when LPL was bound to its endothelial cell receptor/transporter GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1). Physical interaction was observed between ANGPTL3 and ANGPTL8 when the proteins were co-expressed, and co-expression with ANGPTL3 greatly enhanced the secretion of ANGPTL8. Importantly, ANGPTL3-ANGPTL8 complexes had a dramatically increased ability to inhibit LPL compared to either protein alone. Adenovirus experiments showed that 2-fold overexpression of ANGPTL3 significantly increased plasma triglycerides only in the presence of ANGPTL8. Protein interaction assays showed that ANGPTL8 greatly increased the ability of ANGPTL3 to bind LPL. CONCLUSIONS: Together, these data indicate that ANGPTL8 binds to ANGPTL3 and that this complex is necessary for ANGPTL3 to efficiently bind and inhibit LPL.
Assuntos
Proteínas Semelhantes a Angiopoietina/metabolismo , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/metabolismo , Proteína 3 Semelhante a Angiopoietina , Proteína 8 Semelhante a Angiopoietina , Angiopoietinas/química , Animais , Células CHO , Proteínas de Transporte/metabolismo , Cricetulus , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hormônios Peptídicos/metabolismo , Ligação Proteica , Ratos , Receptores de Lipoproteínas/metabolismoRESUMO
Angptl4 (Angiopoietin-like 4) is a circulating protein secreted by white and brown adipose tissues and the liver. Structurally, Angptl4 contains an N-terminal coiled-coil domain (CCD) connected to a C-terminal fibrinogen-like domain (FLD) via a cleavable linker, and both full-length Angptl4 and its individual domains circulate in the bloodstream. Angptl4 inhibits extracellular lipoprotein lipase (LPL) activity and stimulates the lipolysis of triacylglycerol stored by adipocytes in the white adipose tissue (WAT). The former activity is furnished by the CCD, but the Angptl4 domain responsible for stimulating adipocyte lipolysis is unknown. We show here that the purified FLD of Angptl4 is sufficient to stimulate lipolysis in mouse primary adipocytes and that increasing circulating FLD levels in mice through adenovirus-mediated overexpression (Ad-FLD) not only induces WAT lipolysis in vivo but also reduces diet-induced obesity without affecting LPL activity. Intriguingly, reduced adiposity in Ad-FLD mice was associated with increased oxygen consumption, fat utilization, and the expression of thermogenic genes (Ucp1 and Ppargc1a) in subcutaneous WAT. Moreover, Ad-FLD mice exhibited increased glucose tolerance. Chronically enhancing WAT lipolysis could produce ectopic steatosis because of an overflow of lipids from the WAT to peripheral tissues; however, this did not occur when Ad-FLD mice were fed a high-fat diet. Rather, these mice had reductions in both circulating triacylglycerol levels and the mRNA levels of lipogenic genes in the liver and skeletal muscle. We conclude that separating the FLD from the CCD-mediated LPL-inhibitory activity of full-length Angptl4 reveals lipolytic and thermogenic properties with therapeutic relevance to obesity and diabetes.
Assuntos
Gordura Abdominal/metabolismo , Angiopoietinas/metabolismo , Metabolismo Energético , Lipólise , Modelos Biológicos , Regulação para Cima , Gordura Abdominal/citologia , Gordura Abdominal/patologia , Tecido Adiposo Bege/citologia , Tecido Adiposo Bege/metabolismo , Tecido Adiposo Bege/patologia , Adiposidade , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/sangue , Angiopoietinas/química , Angiopoietinas/genética , Animais , Células Cultivadas , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Mutação , Obesidade/sangue , Obesidade/metabolismo , Obesidade/patologia , Obesidade/prevenção & controle , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismoRESUMO
Lipoprotein lipase (LPL) is responsible for the hydrolysis of triglycerides from circulating lipoproteins. Whereas most identified mutations in the LPL gene are deleterious, one mutation, LPLS447X, causes a gain of function. This mutation truncates two amino acids from LPL's C-terminus. Carriers of LPLS447X have decreased VLDL levels and increased HDL levels, a cardioprotective phenotype. LPLS447X is used in Alipogene tiparvovec, the gene therapy product for individuals with familial LPL deficiency. It is unclear why LPLS447X results in a serum lipid profile more favorable than that of LPL. In vitro reports vary as to whether LPLS447X is more active than LPL. We report a comprehensive, biochemical comparison of purified LPLS447X and LPL dimers. We found no difference in specific activity on synthetic and natural substrates. We also did not observe a difference in the Ki for ANGPTL4 inhibition of LPLS447X relative to that of LPL. Finally, we analyzed LPL-mediated uptake of fluorescently labeled lipoprotein particles and found that LPLS447X enhanced lipoprotein uptake to a greater degree than LPL did. An LPL structural model suggests that the LPLS447X truncation exposes residues implicated in LPL binding to uptake receptors.
Assuntos
HDL-Colesterol/química , LDL-Colesterol/química , Lipase Lipoproteica/química , Mutação , Receptores de Lipoproteínas/química , Triglicerídeos/química , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/química , Angiopoietinas/genética , Angiopoietinas/metabolismo , Animais , Transporte Biológico , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , VLDL-Colesterol/química , VLDL-Colesterol/metabolismo , Expressão Gênica , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/genética , Hiperlipidemias/patologia , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Receptores de Lipoproteínas/genética , Receptores de Lipoproteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Serina/metabolismo , Especificidade por Substrato , Triglicerídeos/metabolismoRESUMO
Angiopoietin-like protein 3 (ANGPTL3) is a secreted protein that regulates lipid, glucose and energy metabolism. This study was conducted to better understand the effect of ANGPTL3 on important economic traits in cattle. First, transcript profiles for ANGPTL3 were measured in nine different Jiaxian cattle tissues. Second, polymorphisms were identified in the complete coding region and promoter region of the bovine ANGPTL3 gene in 707 cattle samples. Finally, an association study was carried out utilizing these single nucleotide polymorphisms (SNPs) to determine the effect of these SNPs on the growth and meat quality traits. Quantitative real-time PCR analysis showed that ANGPTL3 was mainly expressed in the liver. The promoter of the bovine ANGPTL3 contained several putative transcription factor binding sites (SF1, HNF-1, LXRα, NFκß, HNF-3 and C/EBP). In total, four SNPs of the bovine ANGPTL3 gene were identified by direct sequencing. SNP1 (rs469906272: g.-38T>C) was identified in the promoter, SNP2 (rs451104723:g.104A>T) and SNP3 (rs482516226: g.509A>G) were identified in exon 1, and SNP4 (rs477165942: g.8661T>C) was identified in exon 6. Changes in predicted protein structures due to non-synonymous SNPs were analyzed. Haplotype frequencies and linkage disequilibrium were also investigated. Analysis of four SNPs in cattle from different native Chinese breeds (Nanyang (NY) and Jiaxian (JX)) and commercial breeds (Angus (AG), Hereford (HF), Limousin (LM), Luxi (LX), Simmental (ST) and Jinnan (JN)) revealed a significant association with growth traits (including: BW and hipbone width) and meat quality traits (including: Warner-Bratzler shear force and ribeye area). Therefore, implementation of these four mutations in selection indices in the beef industry may be beneficial in selecting individuals with superior growth and meat quality traits.
Assuntos
Angiopoietinas/química , Bovinos/crescimento & desenvolvimento , Bovinos/genética , Carne/normas , Modelos Moleculares , Polimorfismo de Nucleotídeo Único/genética , Angiopoietinas/metabolismo , Animais , Bovinos/metabolismo , Perfilação da Expressão Gênica , Haplótipos/genética , Desequilíbrio de Ligação , Conformação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Especificidade da EspécieRESUMO
Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms. Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal when glycosylated but was strongly activated in the absence of its glycosylation. Specificity of each sensor was assessed in HEK cells with either the T2 or T3 enzymes deleted. Although the sensors are based on specific substrates of the T2 and T3 enzymes, elements in or near the enzyme recognition sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases.
Assuntos
Corantes Fluorescentes/química , N-Acetilgalactosaminiltransferases/fisiologia , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/química , Técnicas Biossensoriais , Inibidores Enzimáticos/farmacologia , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/química , Corantes Fluorescentes/metabolismo , Glicosilação , Células HEK293 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/fisiologia , Microscopia de Fluorescência , N-Acetilgalactosaminiltransferases/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismoAssuntos
Angiopoietinas/química , Angiopoietinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Animais , HumanosRESUMO
A better understanding of the interaction between extrinsic factors and surface receptors on stem cells will greatly benefit stem cell research and applications. Recently, we showed that several angiopoietin-like proteins (Angptls) bind and activate the immune inhibitory receptor human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) to support ex vivo expansion of hematopoietic stem cells (HSCs) and leukemia development. However, the molecular basis for the interaction between Angptls and LILRB2 was unclear. Here, we demonstrate that Angptl2 expressed in mammalian cells forms high-molecular-weight species and that ligand multimerization is required for activation of LILRB2 for downstream signaling. A novel motif in the first and fourth Ig domains of LILRB2 was identified that is necessary for the receptor to be bound and activated by Angptl2. The binding of Angptl2 to LILRB2 is more potent than and not completely overlapped with the binding of another ligand, HLA-G. Immobilized anti-LILRB2 antibodies induce a more potent activation of LILRB2 than Angptl2, and we developed a serum-free culture containing defined cytokines and immobilized anti-LILRB2 that supports a net expansion of repopulating human cord blood HSCs. Our elucidation of the mode of Angptl binding to LILRB2 enabled the development of a new approach for ex vivo expansion of human HSCs.
Assuntos
Angiopoietinas/química , Angiopoietinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/genética , Animais , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Xenoenxertos , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Imunológicos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Myocardin (Myocd) and Myocd-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). RPEL motifs are monomeric globular actin (G-actin) binding elements that regulate MRTF localization and activity. However, the function of the RPEL motif in Myocd is largely unknown because of its low affinity for G-actin. Here, we demonstrated that the Myocd RPEL motif bound to actin-related protein 5 (Arp5) instead of conventional actin, resulting in a significant suppression of Myocd activity. In addition, Arp5 bound to a DNA binding domain of SRF via its C-terminal sequence and prevented the association of the Myocd-SRF complex with the promoter regions of smooth muscle genes. Well-differentiated smooth muscle cells mainly expressed a specific splicing variant of arp5; therefore, the protein level of Arp5 was markedly reduced by partial messenger RNA decay and translational suppression. In dedifferentiated smooth muscle cells, Arp5 knockdown restored the differentiated phenotype via Myocd activation. Thus, Arp5 is a key regulator of Myocd activity.
Assuntos
Angiopoietinas/fisiologia , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Processamento Alternativo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteína 6 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/química , Angiopoietinas/metabolismo , Sítios de Ligação , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/análise , Fenótipo , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de Sequência , Transativadores/análiseRESUMO
The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition.
Assuntos
Receptor TIE-2/fisiologia , Receptores da Família Eph/fisiologia , Transdução de Sinais , Angiopoietinas/química , Angiopoietinas/metabolismo , Sítios de Ligação , Ativação Enzimática , Ligantes , Modelos Moleculares , Estrutura Terciária de Proteína , Receptor TIE-2/química , Receptor TIE-2/metabolismo , Receptores da Família Eph/química , Receptores da Família Eph/metabolismoRESUMO
Apolipoproteins (apo) C-I and C-III are known to inhibit lipoprotein lipase (LPL) activity, but the molecular mechanisms for this remain obscure. We present evidence that either apoC-I or apoC-III, when bound to triglyceride-rich lipoproteins, prevent binding of LPL to the lipid/water interface. This results in decreased lipolytic activity of the enzyme. Site-directed mutagenesis revealed that hydrophobic amino acid residues centrally located in the apoC-III molecule are critical for attachment to lipid emulsion particles and consequently inhibition of LPL activity. Triglyceride-rich lipoproteins stabilize LPL and protect the enzyme from inactivating factors such as angiopoietin-like protein 4 (angptl4). The addition of either apoC-I or apoC-III to triglyceride-rich particles severely diminished their protective effect on LPL and rendered the enzyme more susceptible to inactivation by angptl4. These observations were seen using chylomicrons as well as the synthetic lipid emulsion Intralipid. In the presence of the LPL activator protein apoC-II, more of apoC-I or apoC-III was needed for displacement of LPL from the lipid/water interface. In conclusion, we show that apoC-I and apoC-III inhibit lipolysis by displacing LPL from lipid emulsion particles. We also propose a role for these apolipoproteins in the irreversible inactivation of LPL by factors such as angptl4.
Assuntos
Apolipoproteína C-III/química , Apolipoproteína C-I/química , Lipase Lipoproteica/química , Triglicerídeos/química , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/química , Angiopoietinas/genética , Angiopoietinas/metabolismo , Animais , Apolipoproteína C-I/genética , Apolipoproteína C-I/metabolismo , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Bovinos , Emulsões , Humanos , Lipólise/fisiologia , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Mutagênese Sítio-Dirigida , Triglicerídeos/genética , Triglicerídeos/metabolismoRESUMO
Patients with metabolic syndrome are at high risk for developing atherosclerotic cardiovascular diseases and diabetes. In addition to total cholesterol, LDL-C and HDL-C, elevated plasma triglycerides (TG) are increasingly recognized as an independent risk factor for cardiovascular diseases. Recently 3 groups independently reported the identification and characterization of a novel blood lipid regulator, Lipasin/RIFL/ANGPTL8, which here is referred to as Lipasin for its lipoprotein lipase inhibition effect and for being a circulating factor denoted by 'in'. Being highly enriched in the liver, Lipasin is a hepatocyte-derived circulating factor that regulates plasma TG levels. Lipasin is nutritionally regulated, as its mRNA levels in liver and fat as well as its protein level in serum are reduced by fasting. Mice deficient for Lipasin have lower serum TG levels; conversely, its adenovirus-mediated overexpression increases serum TG levels, in part, through promoting ANGPTL3 cleavage, releasing its N-terminal domain that inhibits lipoprotein lipase. Lipasin sequence variations are associated with LDL-C and HDL-C concentrations in humans. Being lipogenic, Lipasin is highly induced during adipogenesis. Levels of Lipasin and ANGPTL4 show opposite changes in response to fasting or cold environment. Lipasin, a novel but atypical ANGPTL family member, is emerging as a critical lipid regulator and a potential drug target.
Assuntos
Adipogenia , Metabolismo dos Lipídeos , Lipase Lipoproteica/antagonistas & inibidores , Hormônios Peptídicos/metabolismo , Proteína 3 Semelhante a Angiopoietina , Proteína 4 Semelhante a Angiopoietina , Proteína 8 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/química , Angiopoietinas/metabolismo , Animais , HDL-Colesterol/sangue , HDL-Colesterol/metabolismo , LDL-Colesterol/sangue , LDL-Colesterol/metabolismo , Temperatura Baixa , Humanos , Síndrome Metabólica/complicações , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Camundongos , Hormônios Peptídicos/química , Hormônios Peptídicos/genética , Conformação Proteica , Triglicerídeos/sangue , Triglicerídeos/metabolismoRESUMO
Angiopoietin-like proteins (ANGPTLs) play major roles in the trafficking and metabolism of lipids. Inactivation of ANGPTL3, a gene located in an intron of DOCK7, results in very low levels of LDL-cholesterol (C), HDL-C and triglyceride (TAG). We identified another ANGPTL family member, ANGPTL8, which is located in the corresponding intron of DOCK6. A variant in this family member (rs2278426, R59W) was associated with lower plasma LDL-C and HDL-C levels in three populations. ANGPTL8 is expressed in liver and adipose tissue, and circulates in plasma of humans. Expression of ANGPTL8 was reduced by fasting and increased by refeeding in both mice and humans. To examine the functional relationship between the two ANGPTL family members, we expressed ANGPTL3 at physiological levels alone or together with ANGPTL8 in livers of mice. Plasma TAG level did not change in mice expressing ANGPTL3 alone, whereas coexpression with ANGPTL8 resulted in hypertriglyceridemia, despite a reduction in circulating ANGPTL3. ANGPTL8 coimmunoprecipitated with the N-terminal domain of ANGPTL3 in plasma of these mice. In cultured hepatocytes, ANGPTL8 expression increased the appearance of N-terminal ANGPTL3 in the medium, suggesting ANGPTL8 may activate ANGPTL3. Consistent with this scenario, expression of ANGPTL8 in Angptl3(-/-) mice failed to promote hypertriglyceridemia. Thus, ANGPTL8, a paralog of ANGPTL3 that arose through duplication of an ancestral DOCK gene, regulates postprandial TAG and fatty acid metabolism by controlling activation of its progenitor, and perhaps other ANGPTLs. Inhibition of ANGPTL8 provides a new therapeutic strategy for reducing plasma lipoprotein levels.
Assuntos
Angiopoietinas/fisiologia , Sequência de Aminoácidos , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/química , Angiopoietinas/metabolismo , Animais , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Proteínas Ativadoras de GTPase , Fatores de Troca do Nucleotídeo Guanina/genética , Hipertrigliceridemia/fisiopatologia , Íntrons , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Triglicerídeos/sangueRESUMO
Homing of colorectal cancer (CRC) cells to the liver is a non-random process driven by a crosstalk between tumour cells and components of the host tissue. Here we report the isolation of a liver metastasis-specific peptide ligand (CGIYRLRSC) that binds a complex of E-cadherin and α(6) integrin on the surface of CRC cells. We identify angiopoietin-like 6 protein as a peptide-mimicked natural ligand enriched in hepatic blood vessels of CRC patients. We demonstrate that an interaction between hepatic angiopoietin-like 6 and tumoural α(6) integrin/E-cadherin drives liver homing and colonization by CRC cells, and that CGIYRLRSC inhibits liver metastasis through interference with this ligand/receptor system. Our results indicate a mechanism for metastasis whereby a soluble factor accumulated in normal vessels functions as a specific ligand for circulating cancer cells. Consistently, we show that high amounts of coexpressed α(6) integrin and E-cadherin in primary tumours represent a poor prognostic factor for patients with advanced CRC.
Assuntos
Angiopoietinas/metabolismo , Caderinas/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Integrina alfa6/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Motivos de Aminoácidos , Proteína 6 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/química , Angiopoietinas/genética , Animais , Vasos Sanguíneos , Caderinas/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Feminino , Humanos , Integrina alfa6/genética , Fígado/irrigação sanguínea , Fígado/metabolismo , Camundongos , Camundongos Nus , Metástase Neoplásica , Ligação ProteicaRESUMO
Angiopoietin-like protein 4 (Angptl4), a potent regulator of plasma triglyceride metabolism, binds to lipoprotein lipase (LPL) through its N-terminal coiled-coil domain (ccd-Angptl4) inducing dissociation of the dimeric enzyme to inactive monomers. In this study, we demonstrate that fatty acids reduce the inactivation of LPL by Angptl4. This was the case both with ccd-Angptl4 and full-length Angptl4, and the effect was seen in human plasma or in the presence of albumin. The effect decreased in the sequence oleic acid > palmitic acid > myristic acid > linoleic acid > linolenic acid. Surface plasmon resonance, isothermal titration calorimetry, fluorescence, and chromatography measurements revealed that fatty acids bind with high affinity to ccd-Angptl4. The interactions were characterized by fast association and slow dissociation rates, indicating formation of stable complexes. The highest affinity for ccd-Angptl4 was detected for oleic acid with a subnanomolar equilibrium dissociation constant (K(d)). The K(d) values for palmitic and myristic acid were in the nanomolar range. Linoleic and linolenic acid bound with much lower affinity. On binding of fatty acids, ccd-Angptl4 underwent conformational changes resulting in a decreased helical content, weakened structural stability, dissociation of oligomers, and altered fluorescence properties of the Trp-38 residue that is located close to the putative LPL-binding region. Based on these results, we propose that fatty acids play an important role in modulating the effects of Angptl4.
Assuntos
Angiopoietinas/metabolismo , Ácidos Graxos/metabolismo , Lipase Lipoproteica/metabolismo , Multimerização Proteica , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/química , Angiopoietinas/genética , Animais , Sítios de Ligação , Bovinos , Ácidos Graxos/química , Humanos , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Camundongos , Plasma/química , Plasma/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Albumina Sérica/química , Albumina Sérica/genética , Albumina Sérica/metabolismo , Especificidade por Substrato , Ressonância de Plasmônio de SuperfícieRESUMO
The metabolic syndrome, a common disorder including glucose intolerance and dyslipidemia, poses a major public health issue. Patients with high blood lipids, such as triglycerides, are at high risk in developing atherosclerotic cardiovascular diseases. To identify genes involved in metabolism, we performed RNA-seq experiments on the liver and fat in mice treated with a high-fat diet or fasting, and identified Gm6484 (named Lipasin) as a novel nutritionally regulated gene. Human LIPASIN is liver specific, while the mouse one is enriched in the liver and fat, including both brown and white adipose tissues. Obesity increases liver Lipasin, whereas fasting reduces its expression in fat. ANGPTL3 (Angiopoietin-like 3) and ANGPTL4 are critical regulators of blood lipids. LIPASIN shares homology with ANGPTL3's N-terminal domain that is needed for lipid regulation, and with ANGPTL4's N-terminal segment that mediates lipoprotein lipase (LPL) binding. Lipasin overexpression by adenoviruses in mice increases serum triglyceride levels, and a recombinant Lipasin inhibits LPL activity. Therefore, a potential mechanism for Lipasin-mediated triglyceride elevation is through reduced triglyceride clearance by LPL inhibition. Lipasin is thus a novel nutritionally-regulated liver-enriched factor that plays a role in lipid metabolism.
Assuntos
Metabolismo dos Lipídeos , Fígado/metabolismo , Proteínas/metabolismo , Triglicerídeos/sangue , Sequência de Aminoácidos , Proteína 3 Semelhante a Angiopoietina , Proteína 4 Semelhante a Angiopoietina , Proteína 8 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/química , Angiopoietinas/genética , Angiopoietinas/metabolismo , Animais , Sequência Conservada , Dislipidemias/metabolismo , Evolução Molecular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fenômenos Fisiológicos da Nutrição , Hormônios Peptídicos , Proteínas/genéticaRESUMO
Several angiopoietin-like (ANGPTL) molecules have been implicated in enhancement of ex-vivo expansion of murine and human (hu) hematopoietic stem cells, but there are no reports on hematopoietic progenitor cells (HPCs). We assessed purified recombinant endotoxin-free hu ANGPTL-2 Coiled-Coil (CC), -3, -3CC, -3 fibrinogen-like domain (FLD), -4, -4CC, -5CC, -6 and -7 for effects on proliferation and survival of HPCs from hu cord blood (CB). None of the ANGPTL molecules stimulated CB HPC proliferation, or enhanced or inhibited colony formation of CB HPC stimulated by various growth factors. However, ANGPTL-2CC, -3, and -3CC significantly enhanced survival of HPC (CFU-GM, BFU-E, CFU-GEMM) subjected to delayed addition of growth factors. Survival enhancing effects of ANGPTL-3 were neutralized by purified anti-ANGPTL-3, but not by anti-ANGPTL-4, -6, or -7. ANGPTL-2CC, -3, and -3CC, but not -4, -6, or -7 also enhanced replating capacity of single CB CFU-GEMM colonies, an estimate of the self-renewal capabilities of HPCs, by greater than 2 fold. Effects of at least ANGPTL-3CC may in part be mediated through phosphorylation of ERK. The ANGPTL molecules did not influence ex-vivo expansion of hu CB CD34(+) cells, alone, or in combination with SCF, TPO, Flt3-ligand, with or without IL-3. Thus, among ANGPTL family members, ANGPTL-2 and -3 had enhancing activities on human HPC survival and replating activity, effects requiring the CC domain of the ANGPTL molecules. This information is of relevance to hu HPC regulation.
Assuntos
Angiopoietinas/genética , Sangue Fetal/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteínas Recombinantes/genética , Transdução de Sinais/fisiologia , Proteína 2 Semelhante a Angiopoietina , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/antagonistas & inibidores , Angiopoietinas/química , Angiopoietinas/farmacologia , Anticorpos Neutralizantes/farmacologia , Antígenos CD34/biossíntese , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Eritropoetina/farmacologia , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologiaRESUMO
Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.
Assuntos
Angiopoietinas/metabolismo , Inibidores Enzimáticos/metabolismo , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/metabolismo , Pró-Proteína Convertases/metabolismo , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/química , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Furina/metabolismo , Humanos , Lipase/metabolismo , Lipase Lipoproteica/química , Camundongos , Ligação Proteica , Receptores de Lipoproteínas , Serina Endopeptidases/metabolismo , Especificidade por SubstratoRESUMO
PURPOSE OF REVIEW: Angiopoietin-like proteins (Angptls) comprise a family of secreted glycoproteins with high homology to angiopoietins which are important regulators of angiogenesis. Angptl3 and Angptl4 inhibit lipoprotein lipase in mice. This article reviews recent human studies on Angptls and their effects on lipoprotein profiles and cardiovascular disease. RECENT FINDINGS: Population-based studies have indicated that loss-of-function mutations of Angptl3, Angptl4, and Angptl5 are associated with a low triglyceride concentration. Angptl3 concentration is positively correlated with HDL-cholesterol, but not with triglyceride, suggesting that Angptl3 regulates HDL metabolism by inhibiting endothelial lipase. Angptl4 concentration is correlated positively with triglycerides in patients with metabolic syndrome, although most studies have failed to find such a correlation. Angptl6 has no effects on lipoprotein profiles. Angptl3 is associated with atherosclerosis in coronary, carotid, and femoral arteries. Conflicting results have been obtained regarding whether the E40K variant (a loss-of-function mutation of Angptl4) is associated with an increased risk for cardiovascular disease, which may occur due to the lipid-independent actions of Angptl4. SUMMARY: Angptl3, Angptl4, and possibly Angptl5 are regulators of lipoprotein metabolism in humans. Whether inhibition of these Angptls will be useful for dyslipidemia to prevent cardiovascular disease remains to be elucidated in future studies.