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1.
Biochemistry ; 56(33): 4405-4418, 2017 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-28715204

RESUMO

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor ß (TGF-ß) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60-114, PDP60-114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP60-114 had no impact on activity. The specific activity of the GDF11/PDP60-114 complex (EC50 = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP60-114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP60-114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP60-114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC50 = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC50 = 2 nM) by protease treatment. Complex formation with PDP60-114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-ß family that form latent pro/mature domain complexes.


Assuntos
Proteínas Morfogenéticas Ósseas , Fatores de Diferenciação de Crescimento , Multimerização Proteica , Proteólise , Animais , Proteínas Morfogenéticas Ósseas/biossíntese , Proteínas Morfogenéticas Ósseas/química , Proteínas Morfogenéticas Ósseas/genética , Células CHO , Cricetinae , Cricetulus , Fatores de Diferenciação de Crescimento/biossíntese , Fatores de Diferenciação de Crescimento/química , Fatores de Diferenciação de Crescimento/genética , Humanos , Concentração de Íons de Hidrogênio , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Domínios Proteicos , Solubilidade
2.
Anal Chem ; 89(7): 4021-4030, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28245108

RESUMO

Meteorin and Cometin (Meteorin-like) are secreted proteins belonging to a newly discovered growth factor family. Both proteins play important roles in neural development and may have potential as therapeutic targets or agents. Meteorin and Cometin are homologues and contain ten evolutionarily conserved Cys residues across a wide variety of species. However, the status of the Cys residues has remained unknown. Here, we have successfully determined the disulfide structure for murine Meteorin by LC-MS analysis of fragments generated by trypsin plus endoprotease-Asp-N. For proteolytic fragments linked by more than one disulfide bond, we used electron transfer dissociation (ETD) to partially dissociate disulfide bonds followed by high-energy collisional dissociation (HCD) to determine disulfide linkages. Our analysis revealed that the ten Cys residues in murine Meteorin form five disulfide bonds with Cys7 (C1) linked to Cys28 (C2), Cys59 (C3) to Cys95 (C4), Cys148 (C5) to Cys219 (C8), Cys151 (C6) to Cys243 (C9), and Cys161 (C7) to Cys266 (C10). Since the ten Cys residues are highly conserved in Meteorin and Cometin, it is likely that the disulfide linkages are also conserved. This disulfide structure information should facilitate structure-function relationship studies on this new class of neurotrophic factors and also assist in evaluation of their therapeutic potentials.


Assuntos
Dissulfetos/análise , Fatores de Crescimento Neural/química , Fragmentos de Peptídeos/química , Proteólise , Animais , Cromatografia Líquida , Transporte de Elétrons , Transferência de Energia , Camundongos , Estrutura Molecular , Espectrometria de Massas em Tandem
3.
MAbs ; 12(1): 1713648, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31928294

RESUMO

LINGO-1 is a membrane protein of the central nervous system (CNS) that suppresses myelination of axons. Preclinical studies have revealed that blockade of LINGO-1 function leads to CNS repair in demyelinating animal models. The anti-LINGO-1 antibody Li81 (opicinumab), which blocks LINGO-1 function and shows robust remyelinating activity in animal models, is currently being investigated in a Phase 2 clinical trial as a potential treatment for individuals with relapsing forms of multiple sclerosis (AFFINITY: clinical trial.gov number NCT03222973). Li81 has the unusual feature that it contains two LINGO-1 binding sites: a classical site utilizing its complementarity-determining regions and a cryptic secondary site involving Li81 light chain framework residues that recruits a second LINGO-1 molecule only after engagement of the primary binding site. Concurrent binding at both sites leads to formation of a 2:2 complex of LINGO-1 with the Li81 antigen-binding fragment, and higher order complexes with intact Li81 antibody. To elucidate the role of the secondary binding site, we designed a series of Li81 variant constructs that eliminate it while retaining the classic site contacts. These Li81 mutants retained the high affinity binding to LINGO-1, but lost the antibody-induced oligodendrocyte progenitor cell (OPC) differentiation activity and myelination activity in OPC- dorsal root ganglion neuron cocultures seen with Li81. The mutations also attenuate antibody-induced internalization of LINGO-1 on cultured cortical neurons, OPCs, and cells over-expressing LINGO-1. Together these studies reveal that engagement at both LINGO-1 binding sites of Li81 is critical for robust functional activity of the antibody.


Assuntos
Anticorpos Monoclonais/imunologia , Sítios de Ligação de Anticorpos/imunologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/imunologia , Humanos
4.
Exp Neurol ; 237(2): 260-6, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22766205

RESUMO

Neuropathic pain is caused by a lesion or disease to the somatosensory nervous system and current treatment merely reduces symptoms. Here, we investigate the potential therapeutic effect of the neurotrophic factor Meteorin on multiple signs of neuropathic pain in two distinct rat models. In a first study, two weeks of intermittent systemic administration of recombinant Meteorin led to a dose-dependent reversal of established mechanical and cold hypersensitivity in rats after photochemically-induced sciatic nerve injury. Moreover, analgesic efficacy lasted for at least one week after treatment cessation. In rats with a chronic constriction injury (CCI) of the sciatic nerve, five systemic injections of Meteorin over 9 days dose-dependently reversed established mechanical and thermal hypersensitivity as well as weight bearing deficits taken as a surrogate marker of spontaneous pain. The beneficial effects of systemic Meteorin were sustained for at least three weeks after treatment ended and no adverse side effects were observed. Pharmacokinetic analysis indicated that plasma Meteorin exposure correlated well with dosing and was no longer detectable after 24 hours. This pharmacokinetic profile combined with a delayed time of onset and prolonged duration of analgesic efficacy on multiple parameters suggests a disease-modifying mechanism rather than symptomatic pain relief. In sciatic nerve lesioned rats, delivery of recombinant Meteorin by intrathecal injection was also efficacious in reversing mechanical and cold hypersensitivity. Together, these data demonstrate that Meteorin represents a novel treatment strategy for the effective and long lasting relief from the debilitating consequences of neuropathic pain.


Assuntos
Analgésicos/farmacologia , Hiperalgesia/tratamento farmacológico , Proteínas do Tecido Nervoso/farmacologia , Neuralgia/tratamento farmacológico , Animais , Modelos Animais de Doenças , Hiperalgesia/etiologia , Masculino , Neuralgia/complicações , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Nervo Isquiático/lesões
5.
Nat Med ; 17(7): 816-21, 2011 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-21725297

RESUMO

Survival and differentiation of oligodendrocytes are important for the myelination of central nervous system (CNS) axons during development and crucial for myelin repair in CNS demyelinating diseases such as multiple sclerosis. Here we show that death receptor 6 (DR6) is a negative regulator of oligodendrocyte maturation. DR6 is expressed strongly in immature oligodendrocytes and weakly in mature myelin basic protein (MBP)-positive oligodendrocytes. Overexpression of DR6 in oligodendrocytes leads to caspase 3 (casp3) activation and cell death. Attenuation of DR6 function leads to enhanced oligodendrocyte maturation, myelination and downregulation of casp3. Treatment with a DR6 antagonist antibody promotes remyelination in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis (EAE) models. Consistent with the DR6 antagoinst antibody studies, DR6-null mice show enhanced remyelination in both demyelination models. These studies reveal a pivotal role for DR6 signaling in immature oligodendrocyte maturation and myelination that may provide new therapeutic avenues for the treatment of demyelination disorders such as multiple sclerosis.


Assuntos
Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Animais , Western Blotting , Caspase 3/metabolismo , Caspase 3/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/fisiopatologia , Encefalomielite Autoimune Experimental/terapia , Ativação Enzimática , Regulação da Expressão Gênica , Camundongos , Esclerose Múltipla/metabolismo , Esclerose Múltipla/terapia , Bainha de Mielina/fisiologia , Oligodendroglia/fisiologia , Ratos , Receptores do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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