RESUMO
Although both protein tyrosine phosphatases and kinases are constitutively active in healthy human red blood cells (RBCs), the preponderance of phosphatase activities maintains the membrane proteins in a predominantly unphosphorylated state. We report here that unlike healthy RBCs, proteins in sickle cells are heavily tyrosine phosphorylated, raising the question regarding the mechanism underpinning this tyrosine phosphorylation. Upon investigating possible causes, we observe that protein tyrosine phosphatase 1B (PTP1B), the major erythrocyte tyrosine phosphatase, is largely digested to a lower molecular weight fragment in sickle cells. We further find that the resulting truncated form of PTP1B is significantly less active than its intact counterpart, probably accounting for the intense tyrosine phosphorylation of Band 3 in sickle erythrocytes. Because this tyrosine phosphorylation of Band 3 promotes erythrocyte membrane weakening that causes release of both membrane vesicles and cell free hemoglobin that in turn initiates vaso-occlusive events, we conclude that cleavage of PTP1B could contribute to the symptoms of sickle cell disease. We further posit that methods to inhibit proteolysis of PTP1B could mitigate symptoms of the disease.
Assuntos
Anemia Falciforme , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Anemia Falciforme/metabolismo , Membrana Eritrocítica/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Tirosina/metabolismoRESUMO
Many hypotheses have been proposed to explain how a glutamate to valine substitution in sickle haemoglobin (HbS) can cause sickle cell disease (SCD). We propose and document a new mechanism in which elevated tyrosine phosphorylation of Band 3 initiates sequelae that cause vaso-occlusion and the symptoms of SCD. In this mechanism, denaturation of HbS and release of heme generate intracellular oxidants which cause inhibition of erythrocyte tyrosine phosphatases, thus permitting constitutive tyrosine phosphorylation of Band 3. This phosphorylation in turn induces dissociation of the spectrin-actin cytoskeleton from the membrane, leading to membrane weakening, discharge of membrane-derived microparticles (which initiate the coagulation cascade) and release of cell-free HbS (which consumes nitric oxide) and activates the endothelium to express adhesion receptors). These processes promote vaso-occlusive events which cause SCD. We further show that inhibitors of Syk tyrosine kinase block Band 3 tyrosine phosphorylation, prevent release of cell-free Hb, inhibit discharge of membrane-derived microparticles, increase sickle cell deformability, reduce sickle cell adhesion to human endothelial cells, and enhance sickle cell flow through microcapillaries. In view of reports that imatinib (a Syk inhibitor) successfully treats symptoms of sickle cell disease, we suggest that Syk tyrosine kinase inhibitors warrant repurposing as potential treatments for SCD.
Assuntos
Anemia Falciforme/tratamento farmacológico , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Anemia Falciforme/sangue , Adesão Celular/efeitos dos fármacos , Micropartículas Derivadas de Células/química , Avaliação Pré-Clínica de Medicamentos , Endotélio Vascular/metabolismo , Deformação Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos Anormais/efeitos dos fármacos , Eritrócitos Anormais/metabolismo , Hemoglobina Falciforme/análise , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Estresse Oxidativo , Oxigênio/sangue , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Plasma , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Traço Falciforme/sangue , Talassemia beta/sangueRESUMO
To egress from its erythrocyte host, the malaria parasite, Plasmodium falciparum, must destabilize the erythrocyte membrane by activating an erythrocyte tyrosine kinase. Because imatinib inhibits erythrocyte tyrosine kinases and because imatinib has a good safety profile, we elected to determine whether coadministration of imatinib with standard of care (SOC) might be both well tolerated and therapeutically efficacious in malaria patients. Patients with uncomplicated P. falciparum malaria from a region in Vietnam where one third of patients experience delayed parasite clearance (DPC; continued parasitemia after 3 d of therapy) were treated for 3 d with either the region's SOC (40 mg dihydroartemisinin + 320 mg piperaquine/d) or imatinib (400 mg/d) + SOC. Imatinib + SOC-treated participants exhibited no increase in number or severity of adverse events, a significantly accelerated decline in parasite density and pyrexia, and no DPC. Surprisingly, these improvements were most pronounced in patients with the highest parasite density, where serious complications and death are most frequent. Imatinib therefore appears to improve SOC therapy, with no obvious drug-related toxicities.
Assuntos
Antimaláricos/efeitos adversos , Antimaláricos/uso terapêutico , Mesilato de Imatinib/uso terapêutico , Malária Falciparum/tratamento farmacológico , Adolescente , Adulto , Artemisininas/uso terapêutico , Quimioterapia Combinada , Febre/tratamento farmacológico , Febre/microbiologia , Humanos , Mesilato de Imatinib/efeitos adversos , Malária Falciparum/parasitologia , Pessoa de Meia-Idade , Quinolinas/uso terapêutico , Resultado do Tratamento , Vietnã , Adulto JovemRESUMO
Although current malaria therapies inhibit pathways encoded in the parasite's genome, we have looked for anti-malaria drugs that can target an erythrocyte component because development of drug resistance might be suppressed if the parasite cannot mutate the drug's target. In search for such erythrocyte targets, we noted that human erythrocytes express tyrosine kinases, whereas the Plasmodium falciparum genome encodes no obvious tyrosine kinases. We therefore screened a library of tyrosine kinase inhibitors from Eli Lilly and Co. in a search for inhibitors with possible antimalarial activity. We report that although most tyrosine kinase inhibitors exerted no effect on parasite survival, a subset of tyrosine kinase inhibitors displayed potent anti-malarial activity. Moreover, all inhibitors found to block tyrosine phosphorylation of band 3 specifically suppressed P. falciparum survival at the parasite egress stage of its intra-erythrocyte life cycle. Conversely, tyrosine kinase inhibitors that failed to block band 3 tyrosine phosphorylation but still terminated the parasitemia were observed to halt parasite proliferation at other stages of the parasite's life cycle. Taken together these results suggest that certain erythrocyte tyrosine kinases may be important to P. falciparum maturation and that inhibitors that block these kinases may contribute to novel therapies for P. falciparum malaria.