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1.
bioRxiv ; 2023 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-37398498

RESUMO

Axon pathfinding is controlled by attractive and repulsive molecular cues that activate receptors on the axonal growth cone, but the full repertoire of axon guidance molecules remains unknown. The vertebrate DCC receptor family contains the two closely related members DCC and Neogenin with prominent roles in axon guidance and three additional, divergent members - Punc, Nope, and Protogenin - for which functions in neural circuit formation have remained elusive. We identified a secreted Punc/Nope/Protogenin ligand, WFIKKN2, which guides mouse peripheral sensory axons through Nope-mediated repulsion. In contrast, WFIKKN2 attracts motor axons, but not via Nope. These findings identify WFIKKN2 as a bifunctional axon guidance cue that acts through divergent DCC family members, revealing a remarkable diversity of ligand interactions for this receptor family in nervous system wiring. One-Sentence Summary: WFIKKN2 is a ligand for the DCC family receptors Punc, Nope, and Prtg that repels sensory axons and attracts motor axons.

2.
Nat Commun ; 13(1): 5222, 2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064790

RESUMO

The trimeric serine protease HTRA1 is a genetic risk factor associated with geographic atrophy (GA), a currently untreatable form of age-related macular degeneration. Here, we describe the allosteric inhibition mechanism of HTRA1 by a clinical Fab fragment, currently being evaluated for GA treatment. Using cryo-EM, X-ray crystallography and biochemical assays we identify the exposed LoopA of HTRA1 as the sole Fab epitope, which is approximately 30 Å away from the active site. The cryo-EM structure of the HTRA1:Fab complex in combination with molecular dynamics simulations revealed that Fab binding to LoopA locks HTRA1 in a non-competent conformational state, incapable of supporting catalysis. Moreover, grafting the HTRA1-LoopA epitope onto HTRA2 and HTRA3 transferred the allosteric inhibition mechanism. This suggests a conserved conformational lock mechanism across the HTRA family and a critical role of LoopA for catalysis, which was supported by the reduced activity of HTRA1-3 upon LoopA deletion or perturbation. This study reveals the long-range inhibition mechanism of the clinical Fab and identifies an essential function of the exposed LoopA for activity of HTRA family proteases.


Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A , Degeneração Macular , Serina Endopeptidases , Cristalografia por Raios X , Epitopos , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Degeneração Macular/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
3.
Am J Ophthalmol ; 232: 49-57, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34214452

RESUMO

PURPOSE: FHTR2163 is a novel antigen-binding fragment (Fab) directed against high-temperature requirement protein A1 (HtrA1). HTRA1 inhibition may preserve retinal integrity and slow disease progression in geographic atrophy (GA) secondary to age-related macular degeneration (AMD). This study examined the safety, pharmacokinetics, immunogenicity, and changes in the HTRA1-specific substrate Dickkop-related protein 3 (DKK3) in patients with GA who received FHTR2163. DESIGN: Phase I, open-label, single ascending dose escalation and multiple-dose expansion study. METHODS: Adults aged ≥ 50 years with GA secondary to AMD with best corrected visual acuity ranging between Snellen 20/125 and 20/400 were enrolled. In the first stage, a single intravitreal injection of FHTR2163 was given in 5 dose-escalation cohorts ranging from 1 to 20 mg (n = 3 patients/cohort; n = 15 total patients). The second stage evaluated the maximum tested dose of 20 mg administered every 4 weeks for 3 doses (n = 13 patients). RESULTS: No dose limiting toxicities or ocular serious AEs were reported. The most frequently reported AEs in the study eye were conjunctival hemorrhage (n = 7), conjunctival hyperemia (n = 4), and eye pain (n = 2). No non-ocular or ocular AEs were assessed as drug related. There were no clinically significant changes in ocular exams. A sustained pharmacodynamic effect of anti-HtrA1 was observed in the aqueous humor, as measured by levels of cleaved DKK3. CONCLUSIONS: FHTR2163, a novel Fab directed against HtrA1, was well tolerated with no DLTs or significant ocular AEs. The molecule when injected intravitreally for 3 doses showed a sustained pharmacodynamic effect at the maximum tested dose of 20 mg.


Assuntos
Atrofia Geográfica , Degeneração Macular , Atrofia Geográfica/diagnóstico , Atrofia Geográfica/tratamento farmacológico , Atrofia Geográfica/etiologia , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Injeções Intravítreas , Degeneração Macular/complicações , Degeneração Macular/tratamento farmacológico , Acuidade Visual
4.
Sci Rep ; 10(1): 10951, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616769

RESUMO

The N-terminal domain (NTD) of the GluN1 subunit (GluN1-NTD) is important for NMDA receptor structure and function, but the interacting proteins of the GluN1-NTD are not well understood. Starting with an unbiased screen of ~ 1,500 transmembrane proteins using the purified GluN1-NTD protein as a bait, we identify Protocadherin 7 (PCDH7) as a potential interacting protein. PCDH7 is highly expressed in the brain and has been linked to CNS disorders, including epilepsy. Using primary neurons and brain slice cultures, we find that overexpression and knockdown of PCDH7 induce opposing morphological changes of dendritic structures. We also find that PCDH7 overexpression reduces synaptic NMDA receptor currents. These data show that PCDH7 can regulate dendritic spine morphology and synaptic function, possibly via interaction with the GluN1 subunit.


Assuntos
Caderinas/metabolismo , Espinhas Dendríticas/fisiologia , Hipocampo/citologia , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica , Animais , Caderinas/genética , Feminino , Hipocampo/fisiologia , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais
5.
Proc Natl Acad Sci U S A ; 117(18): 9952-9963, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32345717

RESUMO

Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in preclinical models to elucidate the in vivo repertoire of HtrA1-specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf-related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD who were treated with the HtrA1-blocking Fab fragment. This pharmacodynamic biomarker provides important information on HtrA1 activity and pharmacological inhibition within the ocular compartment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Anticorpos Anti-Idiotípicos/farmacologia , Atrofia Geográfica/tratamento farmacológico , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Degeneração Macular/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Idoso , Animais , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/imunologia , Biomarcadores/sangue , Progressão da Doença , Feminino , Predisposição Genética para Doença , Genótipo , Atrofia Geográfica/sangue , Atrofia Geográfica/genética , Atrofia Geográfica/imunologia , Serina Peptidase 1 de Requerimento de Alta Temperatura A/antagonistas & inibidores , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Degeneração Macular/sangue , Degeneração Macular/genética , Degeneração Macular/imunologia , Masculino , Polimorfismo de Nucleotídeo Único/genética , Proteoma/genética , Proteoma/imunologia , Ratos , Retina/efeitos dos fármacos , Retina/imunologia , Retina/patologia , Bibliotecas de Moléculas Pequenas/farmacologia
7.
Neuron ; 91(2): 328-40, 2016 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-27477018

RESUMO

Genetic variants of TREM2, a protein expressed selectively by microglia in the brain, are associated with Alzheimer's disease (AD). Starting from an unbiased protein microarray screen, we identified a set of lipoprotein particles (including LDL) and apolipoproteins (including CLU/APOJ and APOE) as ligands of TREM2. Binding of these ligands by TREM2 was abolished or reduced by disease-associated mutations. Overexpression of wild-type TREM2 was sufficient to enhance uptake of LDL, CLU, and APOE in heterologous cells, whereas TREM2 disease variants were impaired in this activity. Trem2 knockout microglia showed reduced internalization of LDL and CLU. ß-amyloid (Aß) binds to lipoproteins and this complex is efficiently taken up by microglia in a TREM2-dependent fashion. Uptake of Aß-lipoprotein complexes was reduced in macrophages from human subjects carrying a TREM2 AD variant. These data link three genetic risk factors for AD and reveal a possible mechanism by which mutant TREM2 increases risk of AD. VIDEO ABSTRACT.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas/metabolismo , Encéfalo/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Receptores Imunológicos/metabolismo , Linhagem Celular , Humanos , Glicoproteínas de Membrana/genética , Ligação Proteica , Receptores Imunológicos/genética
8.
Nat Commun ; 7: 11473, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-27145901

RESUMO

Viruses encode secreted and cell-surface expressed proteins essential to modulate host immune defenses and establish productive infections. However, to date there has been no systematic study of the extracellular interactome of any human virus. Here we utilize the E3 proteins, diverse and rapidly evolving transmembrane-containing proteins encoded by human adenoviruses, as a model system to survey the extracellular immunomodulatory landscape. From a large-scale protein interaction screen against a microarray of more than 1,500 human proteins, we find and validate 51 previously unidentified virus-host interactions. Our results uncover conserved strategies as well as substantial diversity and multifunctionality in host targeting within and between viral species. Prominent modulation of the leukocyte immunoglobulin-like and signalling lymphocyte activation molecule families and a number of inhibitory receptors were identified as hubs for viral perturbation, suggesting unrecognized immunoregulatory strategies. We describe a virus-host extracellular interaction map of unprecedented scale that provides new insights into viral immunomodulation.


Assuntos
Adenovírus Humanos/imunologia , Imunomodulação/imunologia , Mapas de Interação de Proteínas/imunologia , Proteínas Virais/imunologia , Células A549 , Adenovírus Humanos/metabolismo , Adenovírus Humanos/fisiologia , Animais , Células CHO , Linhagem Celular Tumoral , Células Cultivadas , Cricetulus , Espaço Extracelular/imunologia , Espaço Extracelular/metabolismo , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/imunologia , Humanos , Células Jurkat , Células K562 , Ligação Proteica , Proteoma/imunologia , Proteoma/metabolismo , Proteínas Virais/metabolismo
9.
Science ; 350(6263): 961-5, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26586761

RESUMO

Axon pathfinding is orchestrated by numerous guidance cues, including Slits and their Robo receptors, but it remains unclear how information from multiple cues is integrated or filtered. Robo3, a Robo family member, allows commissural axons to reach and cross the spinal cord midline by antagonizing Robo1/2-mediated repulsion from midline-expressed Slits and potentiating deleted in colorectal cancer (DCC)-mediated midline attraction to Netrin-1, but without binding either Slits or Netrins. We identified a secreted Robo3 ligand, neural epidermal growth factor-like-like 2 (NELL2), which repels mouse commissural axons through Robo3 and helps steer them to the midline. These findings identify NELL2 as an axon guidance cue and establish Robo3 as a multifunctional regulator of pathfinding that simultaneously mediates NELL2 repulsion, inhibits Slit repulsion, and facilitates Netrin attraction to achieve a common guidance purpose.


Assuntos
Axônios/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Medula Espinal/embriologia , Animais , Axônios/metabolismo , Ligantes , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Netrina-1 , Neurogênese/genética , Receptores de Superfície Celular , Receptores Imunológicos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Roundabout
10.
Anal Biochem ; 479: 1-5, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25797350

RESUMO

When used in conjunction with multivalent protein probes, protein microarrays offer a robust technology for discovery of low-affinity extracellular protein-protein interactions. Probes for receptor-matching screens generally consist of purified extracellular domains fused to affinity tags. Given that approximately two-thirds of extracellular proteins are transmembrane domain-containing proteins, it would be desirable to develop a system to express and display probe receptors in a native-like membrane environment. Toward this end, we evaluated baculovirus display as a platform for generating multivalent probes for protein microarray screens. Virion particles were generated displaying single-transmembrane domain receptors BTLA, CD200, and EFNB2, representing a range of affinities for their interacting partners. Virions directly labeled with Cy5 fluorophore were screened against a microarray containing more than 600 extracellular proteins, and the results were compared with data derived from soluble Fc protein or probe-coated protein A microbeads. An optimized protocol employing a blocking step with a nonrelated probe-expressing control baculovirus allowed identification of the expected interactions with a signal-to-noise ratio similar to or higher than those obtained with the other formats. Our results demonstrate that baculovirus display is suitable for detection of high- and low-affinity extracellular protein-protein interactions on protein microarrays. This platform eliminates the need for protein purification and provides a native-like lipid environment for membrane-associated receptors.


Assuntos
Antígenos CD/metabolismo , Baculoviridae/genética , Efrina-B2/metabolismo , Análise Serial de Proteínas/métodos , Receptores Imunológicos/metabolismo , Animais , Antígenos CD/genética , Baculoviridae/metabolismo , Linhagem Celular , Efrina-B2/genética , Humanos , Ligantes , Ligação Proteica , Receptores Imunológicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Razão Sinal-Ruído , Vírion/genética , Vírion/metabolismo
11.
PLoS One ; 9(1): e84823, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24400119

RESUMO

The growth and guidance of many axons in the developing nervous system require Netrin-mediated activation of Deleted in Colorectal Cancer (DCC) and other still unknown signaling cues. Commissural axon guidance defects are more severe in DCC mutant mice than Netrin-1 mutant mice, suggesting additional DCC activating signals besides Netrin-1 are involved in proper axon growth. Here we report that interaction screens on extracellular protein microarrays representing over 1,000 proteins uniquely identified Cerebellin 4 (CBLN4), a member of the C1q-tumor necrosis factor (TNF) family, and Netrin-1 as extracellular DCC-binding partners. Immunofluorescence and radio-ligand binding studies demonstrate that Netrin-1 competes with CBLN4 binding at an overlapping site within the membrane-proximal fibronectin domains (FN) 4-6 of DCC and binds with ∼5-fold higher affinity. CBLN4 also binds to the DCC homolog, Neogenin-1 (NEO1), but with a lower affinity compared to DCC. CBLN4-null mice did not show a defect in commissural axons of the developing spinal cord but did display a transient increase in the number of wandering axons in the brachial plexus, consistent with a role in axon guidance. Overall, the data solidifies CBLN4 as a bona fide DCC ligand and strengthens its implication in axon guidance.


Assuntos
Receptores de Superfície Celular/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Axônios/metabolismo , Proteínas de Transporte , Receptor DCC , Desenvolvimento Embrionário/genética , Humanos , Cinética , Ligantes , Camundongos , Mutação , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Netrina-1 , Neurogênese/genética , Neurônios/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Precursores de Proteínas/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
12.
Curr Protoc Protein Sci ; Chapter 27: Unit 27.3, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23546621

RESUMO

Functional protein microarrays offer the capability for high-throughput protein interaction analysis and have long promised to be a powerful tool for understanding protein interactions at the proteome scale. Although popular techniques for protein-protein interaction mapping like yeast-two-hybrid and affinity-purification mass spectrometry have performed well for identifying intracellular protein-protein interactions, the study of interactions between extracellular proteins has remained challenging for these methods. Instead, the use of protein microarrays appears to be a robust and efficient method for the identification of interactions among the members of this class of protein. This unit describes methods for extracellular protein microarray production, screening, and analysis. A protocol is described for enhanced detection of low-affinity interactions by generating multivalent complexes using Fc-fusion bait proteins and protein A microbeads, along with a statistical method for hit scoring and identification of nonspecific interactions.


Assuntos
Análise Serial de Proteínas/instrumentação , Análise Serial de Proteínas/métodos , Mapeamento de Interação de Proteínas/instrumentação , Mapeamento de Interação de Proteínas/métodos , Proteínas/análise , Animais , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Humanos , Ligação Proteica , Proteínas/metabolismo
13.
J Biol Chem ; 287(19): 15837-50, 2012 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-22396535

RESUMO

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.


Assuntos
Evolução Molecular , Glicoproteínas de Membrana/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Receptores Imunológicos/metabolismo , Antígeno 12E7 , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Arginina/química , Arginina/genética , Arginina/metabolismo , Sítios de Ligação/genética , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Colectinas/química , Colectinas/genética , Colectinas/metabolismo , Sequência Conservada/genética , Células HEK293 , Humanos , Ligantes , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Depuradores/química , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Homologia de Sequência de Aminoácidos , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Células Vero
14.
Anal Biochem ; 420(2): 127-38, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21982860

RESUMO

Characterization of the extracellular protein interactome has lagged far behind that of intracellular proteins, where mass spectrometry and yeast two-hybrid technologies have excelled. Improved methods for identifying receptor-ligand and extracellular matrix protein interactions will greatly accelerate biological discovery in cell signaling and cellular communication. These technologies must be able to identify low-affinity binding events that are often observed between membrane-bound coreceptor molecules during cell-cell or cell-extracellular matrix contact. Here we demonstrate that functional protein microarrays are particularly well-suited for high-throughput screening of extracellular protein interactions. To evaluate the performance of the platform, we screened a set of 89 immunoglobulin (Ig)-type receptors against a highly diverse extracellular protein microarray with 686 genes represented. To enhance detection of low-affinity interactions, we developed a rapid method to assemble bait Fc fusion proteins into multivalent complexes using protein A microbeads. Based on these screens, we developed a statistical methodology for hit calling and identification of nonspecific interactions on protein microarrays. We found that the Ig receptor interactions identified using our methodology are highly specific and display minimal off-target binding, resulting in a 70% true-positive to false-positive hit ratio. We anticipate that these methods will be useful for a wide variety of functional protein microarray users.


Assuntos
Espaço Extracelular/metabolismo , Análise Serial de Proteínas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Reações Falso-Positivas , Biblioteca Gênica , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Microesferas , Proteínas/química , Receptores Imunológicos/metabolismo , Reprodutibilidade dos Testes , Especificidade por Substrato
15.
J Biol Chem ; 286(21): 18969-81, 2011 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-21454693

RESUMO

Psoriasis is a human skin condition characterized by epidermal hyperproliferation and infiltration of multiple leukocyte populations. In characterizing a novel insulin growth factor (IGF)-like (IGFL) gene in mice (mIGFL), we found transcripts of this gene to be most highly expressed in skin with enhanced expression in models of skin wounding and psoriatic-like inflammation. A possible functional ortholog in humans, IGFL1, was uniquely and significantly induced in psoriatic skin samples. In vitro IGFL1 expression was up-regulated in cultured primary keratinocytes stimulated with tumor necrosis factor α but not by other psoriasis-associated cytokines. Finally, using a secreted and transmembrane protein library, we discovered high affinity interactions between human IGFL1 and mIGFL and the TMEM149 ectodomain. TMEM149 (renamed here as IGFLR1) is an uncharacterized gene with structural similarity to the tumor necrosis factor receptor family. Our studies demonstrate that IGFLR1 is expressed primarily on the surface of mouse T cells. The connection between mIGFL and IGFLR1 receptor suggests mIGFL may influence T cell biology within inflammatory skin conditions.


Assuntos
Fator de Crescimento Insulin-Like I/biossíntese , Psoríase/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Pele/metabolismo , Regulação para Cima , Ferimentos e Lesões/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Fator de Crescimento Insulin-Like I/genética , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Terciária de Proteína , Psoríase/genética , Psoríase/patologia , Receptores do Fator de Necrose Tumoral/genética , Pele/patologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Ferimentos e Lesões/genética , Ferimentos e Lesões/patologia
16.
Nat Immunol ; 10(1): 48-57, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19011627

RESUMO

Here we have identified a surface protein, TIGIT, containing an immunoglobulin variable domain, a transmembrane domain and an immunoreceptor tyrosine-based inhibitory motif that was expressed on regulatory, memory and activated T cells. Poliovirus receptor, which is expressed on dendritic cells, bound TIGIT with high affinity. A TIGIT-Fc fusion protein inhibited T cell activation in vitro, and this was dependent on the presence of dendritic cells. The binding of poliovirus receptor to TIGIT on human dendritic cells enhanced the production of interleukin 10 and diminished the production of interleukin 12p40. Knockdown of TIGIT with small interfering RNA in human memory T cells did not affect T cell responses. TIGIT-Fc inhibited delayed-type hypersensitivity reactions in wild-type but not interleukin 10-deficient mice. Our data suggest that TIGIT exerts immunosuppressive effects by binding to poliovirus receptor and modulating cytokine production by dendritic cells.


Assuntos
Células Dendríticas/imunologia , Tolerância Imunológica , Proteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Células CHO , Comunicação Celular , Diferenciação Celular , Células Cultivadas , Cricetinae , Cricetulus , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Regulação para Baixo , Humanos , Memória Imunológica , Interleucina-10/biossíntese , Subunidade p40 da Interleucina-12/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ligação Proteica , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Virais/genética , Receptores Virais/metabolismo , Alinhamento de Sequência , Linfócitos T/metabolismo
17.
Peptides ; 29(3): 479-86, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17942192

RESUMO

Vasoactive intestinal peptide (VIP) binds to two receptors, VPAC1 and VPAC2. Non-selective VIP antagonists have been shown to inhibit human cancer cell proliferation and reduce tumor growth in mice. Many human cancers over-express VPAC1 but not VPAC2. We show that VPAC1-selective antagonists can inhibit human cancer cell proliferation and identify five positions in the VPAC1-selective antagonist PG 97-269 that may be responsible for VPAC1 selectivity. Position 16 appears to be particularly critical for selectivity, as demonstrated in the replacement of Arg16 of PG 97-269 with the native VIP amino acid; this single change results in greatly reduced VPAC1 binding and selectivity. Finally, we show that site-specific conjugation with a 22kDa polyethylene glycol (PEG) at the C-terminus of VPAC1-selective antagonists further improves VPAC1-selective binding and has minimal effect on antagonistic activity. Our studies have further solidified VPAC1 as a cancer target and offer the possibility of generating highly potent VPAC1-selective antagonists with minimal number of mutations to reduce the risk of immunogenicity and potentially prolonged duration of action to allow more efficient treatment regimen.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Polietilenoglicóis/química , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Radioimunoensaio , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Peptídeo Intestinal Vasoativo/química , Peptídeo Intestinal Vasoativo/farmacologia
18.
AAPS J ; 9(2): E227-34, 2007 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-17907763

RESUMO

A PEGylated glucagon-like peptide-1 (GLP-1) agonist and glucagon antagonist hybrid peptide was engineered as a potential treatment for type 2 diabetes. To support preclinical development of this PEGylated dual-acting peptide for diabetes (DAPD), we developed a reproducible method for PEGylation, purification, and analysis. Optimal conditions for site-specific PEGylation with 22 and 43 kDa maleimide-polyethylene glycol (maleimide-PEG) polymers were identified by evaluating pH, reaction time, and reactant molar ratio parameters. A 3-step purification process was developed and successfully implemented to purify PEGylated DAPD and remove excess uncoupled PEG and free peptide. Five lots of 43 kDa PEGylated DAPD with starting peptide amounts of 100 mg were produced with overall yields of 53% to 71%. Analytical characterization by N-terminal sequencing, amino acid analysis, matrix-assisted laser desorption/ionization mass spectrometry, and GLP-1 receptor activation assay confirmed site-specific attachment of PEG at the engineered cysteine residue, expected molecular weight, correct amino acid sequence and composition, and consistent functional activity. Purity and safety analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), analytical ion-exchange chromatography, reversed-phase high-performance liquid chromatography, and limulus amebocyte lysate test showed that the final products contained <1% free peptide, <5% uncoupled PEG, and <0.2 endotoxin units per milligram of peptide. These results demonstrate that the PEGylation and purification process we developed was consistent and effective in producing PEGylated DAPD preclinical materials at the 100 mg (peptide weight basis) or 1.2 g (drug substance weight basis) scale.


Assuntos
Desenho de Fármacos , Peptídeo 1 Semelhante ao Glucagon/agonistas , Hipoglicemiantes/síntese química , Peptídeos/síntese química , Polietilenoglicóis/síntese química , Animais , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Polietilenoglicóis/farmacologia , Polietilenoglicóis/uso terapêutico , Ratos
19.
J Pharmacol Exp Ther ; 320(2): 900-6, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17110523

RESUMO

A previously described VPAC2-selective agonist, BAY 55-9837 (peptide HSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY), had several limitations with respect to its potential as an insulin secretagogue for the treatment of type 2 diabetes. These limitations were primarily poor stability in aqueous buffer and short duration of action in vivo. In this report, we describe a series of novel analogs of BAY 55-9837 that were designed around the likely degradation mechanisms and structure-activity relationship of this peptide with a view to overcoming its limitations. These analogs were tested for improved liquid stability and retention of VPAC2-selective binding and activation, as well as prolonged activity in vivo. Although several degradation mechanisms were possible based on the degradation pattern, it was determined that deamidation at the two asparagines (N9 and N28) was the major instability determinant. Changing these two asparagines to glutamines did not negatively affect VPAC2-selective binding and activation. The double glutamine mutein analog, BAY(Q9Q28), retained full VPAC2 activity and selectivity while displaying no significant degradation when stored at 40 degrees C for 4 weeks. This is in contrast to BAY 55-9837, which showed greater than 80% degradation when stored at 40 degrees C for 2 weeks. A cysteine was added to the C terminus of BAY(Q9Q28), followed by site-specific cysteine conjugation with a 22- or 43-kDa polyethylene glycol (PEG) to yield BAY(Q9Q28C32)PEG22 or BAY(Q9Q28C32)PEG43, respectively. These PEGylated peptides retain the ability to selectively bind and activate the VPAC2 receptor and have prolonged glucose-lowering activity in vivo.


Assuntos
Hipoglicemiantes/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/agonistas , Sequência de Aminoácidos , Animais , Glicemia/análise , Células CHO , Cricetinae , Cricetulus , Estabilidade de Medicamentos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Polietilenoglicóis , Ratos , Ratos Wistar , Peptídeo Intestinal Vasoativo/farmacologia
20.
J Biol Chem ; 281(18): 12506-15, 2006 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-16505481

RESUMO

The closely related peptides glucagon-like peptide (GLP-1) and glucagon have opposing effects on blood glucose. GLP-1 induces glucose-dependent insulin secretion in the pancreas, whereas glucagon stimulates gluconeogenesis and glycogenolysis in the liver. The identification of a hybrid peptide acting as both a GLP-1 agonist and a glucagon antagonist would provide a novel approach for the treatment of type 2 diabetes. Toward this end a series of hybrid peptides made up of glucagon and either GLP-1 or exendin-4, a GLP-1 agonist, was engineered. Several peptides that bind to both the GLP-1 and glucagon receptors were identified. The presence of glucagon sequence at the N terminus removed the dipeptidylpeptidase IV cleavage site and increased plasma stability compared with GLP-1. Targeted mutations were incorporated into the optimal dual-receptor binding peptide to identify a peptide with the highly novel property of functioning as both a GLP-1 receptor agonist and a glucagon receptor antagonist. To overcome the short half-life of this mutant peptide in vivo, while retaining dual GLP-1 agonist and glucagon antagonist activities, site-specific attachment of long chained polyethylene glycol (PEGylation) was pursued. PEGylation at the C terminus retained the in vitro activities of the peptide while dramatically prolonging the duration of action in vivo. Thus, we have generated a novel dual-acting peptide with potential for development as a therapeutic for type 2 diabetes.


Assuntos
Peptídeos/química , Receptores de Glucagon/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Desenho de Fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Glucagon/agonistas , Homologia de Sequência de Aminoácidos
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