RESUMEN
Myelin basic protein (MBP) is a key component of myelin, the specialized lipid membrane that encases the axons of all neurons. Both plasminogen (Pg) and tissue-type plasminogen activator (t-PA) bind to MBP with high affinity. We investigated the kinetics and mechanisms involved in this process using immobilized MBP and found that Pg activation by t-PA is significantly stimulated by MBP. This mechanism involves the binding of t-PA via a lysine-dependent mechanism to the Lys91 residue of the MBP NH2-terminal region Asp82 -Pro99, and the binding of Pg via a lysine-dependent mechanism to the Lys122 residue of the MBP COOH-terminal region Leu109-Gly126. In this context, MBP mimics fibrin and because MBP is a plasmin substrate, our results suggest direct participation of the Pg activation system on MBP physiology.
Asunto(s)
Proteína Básica de Mielina/metabolismo , Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Sitios de Unión , Activación Enzimática , Humanos , Cinética , Lisina/análisis , Lisina/metabolismo , Proteína Básica de Mielina/química , Unión Proteica , Dominios Proteicos , ProteolisisRESUMEN
The glucose-regulated protein 78 (GRP78) is a plasminogen (Pg) receptor on the cell surface. In this study, we demonstrate that GRP78 also binds the tissue-type plasminogen activator (t-PA), which results in a decrease in K(m) and an increase in the V(max) for both its amidolytic activity and activation of its substrate, Pg. This results in accelerated Pg activation when GRP78, t-PA, and Pg are bound together. The increase in t-PA activity is the result of a mechanism involving a t-PA lysine-dependent binding site in the GRP78 amino acid sequence (98)LIGRTWNDPSVQQDIKFL(115). We found that GRP78 is expressed on the surface of neuroblastoma SK-N-SH cells where it is co-localized with the voltage-dependent anion channel (VDAC), which is also a t-PA-binding protein in these cells. We demonstrate that both Pg and t-PA serve as a bridge between GRP78 and VDAC bringing them together to facilitate Pg activation. t-PA induces SK-N-SH cell proliferation via binding to GRP78 on the cell surface. Furthermore, Pg binding to the COOH-terminal region of GRP78 stimulates cell proliferation via its microplasminogen domain. This study confirms previous findings from our laboratory showing that GRP78 acts as a growth factor-like receptor and that its association with t-PA, Pg, and VDAC on the cell surface may be part of a system controlling cell growth.
Asunto(s)
Proliferación Celular , Proteínas de Choque Térmico/metabolismo , Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Membrana Celular/metabolismo , Chaperón BiP del Retículo Endoplásmico , Activación Enzimática , Humanos , Immunoblotting , Cinética , Microscopía Fluorescente , Datos de Secuencia Molecular , Neuroblastoma/metabolismo , Neuroblastoma/patología , Unión Proteica , Especificidad por Sustrato , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
The voltage-dependent anion channel (VDAC), a major pore-forming protein in the outer membrane of mitochondria, is also found in the plasma membrane of a large number of cells where in addition to its role in regulating cellular ATP release and volume control it is important for maintaining redox homeostasis. Cell surface VDAC is a receptor for plasminogen kringle 5, which promotes partial closure of the channel. In this study, we demonstrate that VDAC binds tissue-type plasminogen activator (t-PA) on human neuroblastoma SK-N-SH cells. Binding of t-PA to VDAC induced a decrease in K(m) and an increase in the V(max) for activation of its substrate, plasminogen (Pg). This resulted in accelerated Pg activation when VDAC, t-PA, and Pg were bound together. VDAC is also a substrate for plasmin; hence, it mimics fibrin activity. Binding of t-PA to VDAC occurs between a t-PA fibronectin type I finger domain located between amino acids Ile(5) and Asn(37) and a VDAC region including amino acids (20)GYGFG(24). These VDAC residues correspond to a GXXXG repeat motif commonly found in amyloid ß peptides that is necessary for aggregation when these peptides form fibrillar deposits on the cell surface. Furthermore, we also show that Pg kringle 5 is a substrate for the NADH-dependent reductase activity of VDAC. This ternary complex is an efficient proteolytic complex that may facilitate removal of amyloid ß peptide deposits from the normal brain and cell debris from injured brain tissue.
Asunto(s)
Membrana Celular/metabolismo , Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Aminoácidos/química , Apoptosis , Encéfalo/metabolismo , Línea Celular Tumoral , Cromatografía/métodos , Fibrinolisina/metabolismo , Fibrinólisis , Calor , Humanos , Cinética , Modelos Genéticos , Unión ProteicaRESUMEN
GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH(2)-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78(low)) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH(2)-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78.
Asunto(s)
Proteínas de Escherichia coli/farmacología , Escherichia coli/enzimología , Proteínas de Choque Térmico/metabolismo , Melanoma/metabolismo , Neoplasias de la Próstata/metabolismo , Proteolisis/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Subtilisinas/farmacología , Animales , Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/metabolismo , Autoanticuerpos/genética , Autoanticuerpos/metabolismo , Dominio Catalítico , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/genética , Células Hep G2 , Humanos , Masculino , Melanoma/genética , Ratones , Neoplasias de la Próstata/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Previous data indicate that dopamine neurotransmission is differently regulated in male and female rats. The purpose of the present study was to investigate the dopamine transporter and autoreceptor as potential loci responsible for this sex difference. Fast cyclic voltammetry at carbon-fiber microelectrodes was used to monitor changes in electrically evoked levels of extracellular dopamine in the striata of anesthetized male and female rats before and after administration of an uptake inhibitor, a dopamine D2 antagonist, or a D3/D2 agonist. Administration of 40 mg/kg cocaine ip increased electrically-evoked extracellular dopamine concentrations in both sexes, but to a significantly greater extent in female striatum at the higher stimulation frequencies. The typical antipsychotic, haloperidol, increased dopamine efflux in both sexes but the effect was twice as large in the female striatum. The D3/D2 agonist quinpirole induced an unexpected, transient increase in dopamine efflux following high-frequency stimulation only in females, and evoked dopamine was higher in females across this entire time course. More detailed analysis of cocaine effects revealed no fundamental sex differences in the interaction of cocaine with DAT in vivo or in synaptosomes. These results indicate that nigrostriatal dopamine neurotransmission in the female rat is more tightly regulated by autoreceptor and transporter mechanisms, perhaps related by greater autoreceptor control of DAT activity. Thus, baseline sex differences in striatal dopamine regulation induce different pharmacologic responses. These results contribute to understanding sex differences in stimulant-induced locomotor activity in rats and may have broader implications for neurologic disorders and their pharmacotherapies in humans.
Asunto(s)
Química Encefálica/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Dopaminérgicos/farmacología , Caracteres Sexuales , Análisis de Varianza , Animales , Química Encefálica/efectos de la radiación , Cocaína/farmacología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/efectos de la radiación , Dopamina/metabolismo , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Electroquímica/métodos , Femenino , Haloperidol/farmacología , Masculino , Quinpirol/farmacología , Ratas , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Sinaptosomas/efectos de la radiación , Factores de TiempoRESUMEN
Autoantibodies from autistic spectrum disorder (ASD) patients react with multiple proteins expressed in the brain. One such autoantibody targets myelin basic protein (MBP). ASD patients have autoantibodies to MBP of both the IgG and IgA classes in high titers, but no autoantibodies of the IgM class. IgA autoantibodies act as serine proteinases and degrade MBP in vitro. They also induce a decrease in long-term potentiation in the hippocampi of rats either perfused with or previously inoculated with this IgA. Because this class of autoantibody causes myelin sheath destruction in multiple sclerosis (MS), we hypothesized a similar pathological role for them in ASD.
Asunto(s)
Trastorno Autístico/sangre , Hipocampo/citología , Inmunoglobulina A/sangre , Inmunoglobulina A/farmacología , Proteína Básica de Mielina/inmunología , Transmisión Sináptica/efectos de los fármacos , Adolescente , Animales , Trastorno Autístico/inmunología , Trastorno Autístico/fisiopatología , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Encéfalo/metabolismo , Niño , Preescolar , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunoglobulina A/efectos de los fármacos , Técnicas In Vitro , Leupeptinas/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Técnicas de Placa-Clamp , Proteolisis/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
The HSP70 family member GRP78 is a selective tumor marker upregulated on the surface of many tumor cell types, including melanoma, where it acts as a growth factor receptor-like protein. Receptor-recognized forms of the proteinase inhibitor α2-macroglobulin (α2M*) are the best-characterized ligands for GRP78, but in melanoma and other cancer patients, autoantibodies arise against the NH2-terminal domain of GRP78 that react with tumor cell-surface GRP78. This causes the activation of signaling cascades that are proproliferative and antiapoptotic. Antibodies directed against the COOH-terminal domain of GRP78, however, upregulate p53-mediated proapoptotic signaling, leading to cell death. Here, we describe the binding characteristics, cell signaling properties, and downstream cellular effects of three novel murine monoclonal antibodies. The NH2-terminal domain-reactive antibody, N88, mimics α2M* as a ligand and drives PI 3-kinase-dependent activation of Akt and the subsequent stimulation of cellular proliferation in vitro. The COOH-terminal domain-reactive antibody, C38, acts as an antagonist of both α2M* and N88, whereas another, C107, directly induces apoptosis in vitro. In a murine B16F1 melanoma flank tumor model, we demonstrate the acceleration of tumor growth by treatment with N88, whereas C107 significantly slowed tumor growth whether administered before (P<0.005) or after (P<0.05) tumor implantation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Proteínas de Choque Térmico/inmunología , Melanoma Experimental/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/biosíntesis , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Chaperón BiP del Retículo Endoplásmico , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos , Femenino , Citometría de Flujo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Hibridomas , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Carga TumoralRESUMEN
Autoantibodies that react with GRP78 expressed on the cell-surface of many tumor cell lines occur in the sera of patients with prostate cancer, melanoma, and ovarian cancer. These autoantibodies are a negative prognostic factor in prostate cancer and, when purified, stimulate tumor cell proliferation in vitro. It is unclear, however, whether these immunoglobulin Gs are merely a biomarker, or whether they actually promote the tumor growth in vivo. We immunized C57Bl/6 mice with recombinant GRP78 and then implanted the B16F1 murine melanoma cell line as flank tumors. We used the antisera from these mice for in-vitro cell signaling and proliferation assays. The immunodominant epitope in patients with cancer was well represented in the antibody repertoire of these immunized mice. We observed significantly accelerated tumor growth, and shortened survival in GRP78-immunized mice compared with controls. Furthermore, antisera from these mice, and purified anti-GRP78 immunoglobulin G from similarly immunized mice, stimulate Akt phosphorylation and proliferation in B16F1 and human DM6 melanoma cells in culture. These studies show a causal link between a humoral response to GRP78 and the progression of cancer in a murine melanoma model. They support the hypothesis that such autoantibodies are involved in the progression of human cancers and are not simply a biomarker. As GRP78 is present on the surface of many types of cancer cells, this hypothesis has broad clinical and therapeutic implications.
Asunto(s)
Autoanticuerpos/inmunología , Proteínas de Choque Térmico/inmunología , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Proliferación Celular , Chaperón BiP del Retículo Endoplásmico , Epítopos/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Péptidos/inmunología , Fosforilación , Proteínas Recombinantes/inmunología , alfa-Macroglobulinas/metabolismoRESUMEN
The unfolded protein response (UPR) is an adaptive survival mechanism through which cells can weather the stress of misfolded protein accumulation induced by a wide variety of pathophysiologic and pharmacologic insults. The ER chaperone GRP78 is a central modulator of the UPR both through its protein-binding capacity and its direct regulation of the UPR signaling molecules IRE1α, PERK, and ATF6. Recent reports have revealed the presence of GRP78 on the surface of cancer cells. Biological roles for cell-surface GRP78 include competing NH(2)-domain and COOH-domain agonist receptor activities that induce opposite effects on proliferation and apoptosis. Modulation of the UPR impacts both of these processes directly and indirectly. Here, we outline methods that we use to investigate UPR modulation via direct ligation of cell-surface GRP78. Specifically, we review methods of cell culture, cell-signaling analysis with emphasis on UPR components, and ultimately, the impact that these have on cell proliferation, survival, and apoptosis.
Asunto(s)
Proteínas de Choque Térmico/fisiología , Chaperonas Moleculares/fisiología , Neoplasias de la Próstata/fisiopatología , Respuesta de Proteína Desplegada , Factor de Transcripción Activador 6/fisiología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Endorribonucleasas/fisiología , Proteínas de Choque Térmico/inmunología , Humanos , Masculino , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal , eIF-2 Quinasa/fisiologíaAsunto(s)
Algoritmos , Péptidos/química , Proteínas/química , Secuencia de Aminoácidos , Electroforesis en Gel Bidimensional/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Indicadores y Reactivos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Proteoma , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , TripsinaAsunto(s)
Proteínas Fúngicas/química , Fosfoproteínas/química , Proteoma/química , Saccharomyces cerevisiae/química , Sitios de Unión , Eliminación de Gen , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteína Fosfatasa 1 , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The quinolines have been used in the treatment of malaria, arthritis, and lupus for many years, yet the precise mechanism of their action remains unclear. In this study, we used a functional proteomics approach that exploited the structural similarities between the quinoline compounds and the purine ring of ATP to identify quinoline-binding proteins. Several quinoline drugs were screened by displacement affinity chromatography against the purine binding proteome captured with gamma-phosphate-linked ATP-Sepharose. Screening of the human red blood cell purine binding proteome identified two human proteins, aldehyde dehydrogenase 1 (ALDH1) and quinone reductase 2 (QR2). In contrast, no proteins were detected upon screening of the Plasmodium falciparum purine binding proteome with the quinolines. In a complementary approach, we passed cell lysates from mice, red blood cells, or P. falciparum over hydroxychloroquine- or primaquine-Sepharose. Consistent with the displacement affinity chromatography screen, ALDH and QR2 were the only proteins recovered from mice and human red blood cell lysate and no proteins were recovered from P. falciparum. Furthermore, the activity of QR2 was potently inhibited by several of the quinolines in vitro. Our results show that ALDH1 and QR2 are selective targets of the quinolines and may provide new insights into the mechanism of action of these drugs.