RESUMEN
BACKGROUND: Pseudoxanthoma elasticum (PXE), a monogenic disorder resulting in calcification affecting the skin, eyes and peripheral arteries, is caused by mutations in the ABCC6 gene, and is associated with low plasma inorganic pyrophosphate (PPi). It is unknown how ABCC6 genotype affects plasma PPi. METHODS: We studied the association of ABCC6 genotype (192 patients with biallelic pathogenic ABCC6 mutations) and PPi levels, and its association with the severity of arterial and ophthalmological phenotypes. ABCC6 variants were classified as truncating or non-truncating, and three groups of the 192 patients were formed: those with truncating mutations on both chromosomes (n = 121), those with two non-truncating mutations (n = 10), and a group who had one truncating and one non-truncating ABCC6 mutation (n = 61). The hypothesis formulated before this study was that there was a negative association between PPi level and disease severity. RESULTS: Our findings confirm low PPi in PXE compared with healthy controls (0.53 ± 0.15 vs. 1.13 ± 0.29 µM, p < 0.01). The PPi of patients correlated with increasing age (ß: 0.05 µM, 95% CI: 0.03-0.06 per 10 years) and was higher in females (0.55 ± 0.17 vs. 0.51 ± 0.13 µM in males, p = 0.03). However, no association between PPi and PXE phenotypes was found. When adjusted for age and sex, no association between PPi and ABCC6 genotype was found. CONCLUSIONS: Our data suggest that the relationship between ABCC6 mutations and reduced plasma PPi may not be as direct as previously thought. PPi levels varied widely, even in patients with the same ABCC6 mutations, further suggesting a lack of direct correlation between them, even though the ABCC6 protein-mediated pathway is responsible for ~60% of this metabolite in the circulation. We discuss potential factors that may perturb the expected associations between ABCC6 genotype and PPi and between PPi and disease severity. Our findings support the argument that predictions of pathogenicity made on the basis of mutations (or on the structure of the mutated protein) could be misleading.
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Organic anion transporting polypeptide 3A1 (OATP3A1, encoded by the SLCO3A1 gene) is a prostaglandin, oligopeptide, and steroid/thyroid hormone transporter with wide tissue distribution, expressed, e.g., in the human brain and testis. Although the physiological importance of OATP3A1 has not yet been clarified, based on its expression pattern, substrate recognition, and evolutionary conservation, OATP3A1 is a potential pharmacological target. Previously, two isoforms of OATP3A1, termed as V1 and V2, have been characterized. Here, we describe the cloning and functional characterization of a third isoform, OATP3A1_V3. The mRNA of isoform V3 is formed by alternative splicing and results in an OATP3A1 protein with an altered C-terminus compared to isoforms V1 and V2. Based on quantitative PCR, we demonstrate the widespread expression of SLCO3A1_V3 mRNA in human organs, with the highest expression in the brain and testis. By generation of an isoform V3-specific antibody and immunostaining, we show that the encoded protein is expressed in the human choroid plexus, neurons, and both germ and Sertoli cells of the testis. Moreover, we demonstrate that in contrast to isoform V1, OATP3A1_V3 localizes to the apical membrane of polarized MDCKII cells. Using HEK-293 cells engineered to overexpress OATP3A1_V3, we verify the protein's functionality and identify dehydroepiandrosterone sulfate as a novel OATP3A1 substrate. Based on their distinct expression patterns but overlapping functions, OATP3A1 isoforms may contribute to transcellular (neuro)steroid transport in the central nervous system.
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Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes and the cardiovascular system. Skin findings often lead to early diagnosis of PXE, but currently, no specific treatment exists to counteract the progression of symptoms. PXE belongs to a group of Mendelian calcification disorders linked to pyrophosphate metabolism, which also includes generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Inactivating mutations in ABCC6, ENPP1 and NT5E are the genetic cause of these diseases, respectively, and all of them result in reduced inorganic pyrophosphate (PPi ) concentration in the circulation. Although PPi is a strong inhibitor of ectopic calcification, oral supplementation therapy was initially not considered because of its low bioavailability. Our earlier work however demonstrated that orally administered pyrophosphate inhibits ectopic calcification in the animal models of PXE and GACI, and that orally given Na4 P2 O7 is absorbed in humans. Here, we report that gelatin-encapsulated Na2 H2 P2 O7 has similar absorption properties in healthy volunteers and people affected by PXE. The sodium-free K2 H2 P2 O7 form resulted in similar uptake in healthy volunteers and inhibited calcification in Abcc6-/- mice as effectively as its sodium counterpart. Novel pyrophosphate compounds showing higher bioavailability in mice were also identified. Our results provide an important step towards testing oral PPi in clinical trials in PXE, or potentially any condition accompanied by ectopic calcification including diabetes, chronic kidney disease or ageing.
Asunto(s)
Seudoxantoma Elástico , Calcificación Vascular , Animales , Suplementos Dietéticos , Difosfatos , Humanos , Ratones , Mutación , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/uso terapéutico , Seudoxantoma Elástico/tratamiento farmacológico , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Pirofosfatasas/uso terapéutico , Calcificación Vascular/tratamiento farmacológico , Calcificación Vascular/genéticaRESUMEN
Mutations in the ABCC6 gene result in calcification diseases such as pseudoxanthoma elasticum or Generalized Arterial Calcification of Infancy. Generation of antibodies recognizing an extracellular (EC) epitope of ABCC6 has been hampered by the short EC segments of the protein. To overcome this limitation, we immunized bovine FcRn transgenic mice exhibiting an augmented humoral immune response with Human Embryonic Kidney 293 cells cells expressing human ABCC6 (hABCC6). We obtained a monoclonal antibody recognizing an EC epitope of hABCC6 that we named mEChC6. Limited proteolysis revealed that the epitope is within a loop in the N-terminal half of ABCC6 and probably spans amino acids 338-347. mEChC6 recognizes hABCC6 in the liver of hABCC6 transgenic mice, verifying both specificity and EC binding to intact hepatocytes.
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Anticuerpos Monoclonales de Origen Murino/inmunología , Epítopos/inmunología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/inmunología , Animales , Epítopos/genética , Humanos , Ratones , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genéticaRESUMEN
Trauma-induced calcification is the pathological consequence of complex injuries which often affect the central nervous system and other parts of the body simultaneously. We demonstrated by an animal model recapitulating the calcification of the above condition that adrenaline transmits the stress signal of brain injury to the calcifying tissues. We have also found that although the level of plasma pyrophosphate, the endogenous inhibitor of calcification, was normal in calcifying animals, it could not counteract the acute calcification. However, externally added pyrophosphate inhibited calcification even when it was administered after the complex injuries. Our finding suggests a potentially powerful clinical intervention of calcification triggered by polytrauma injuries which has no effective treatment.
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Lesiones Traumáticas del Encéfalo/complicaciones , Difosfatos/uso terapéutico , Osificación Heterotópica/complicaciones , Calcificación Vascular/etiología , Antagonistas Adrenérgicos/farmacología , Animales , Lesiones Traumáticas del Encéfalo/patología , Cardiotoxinas , Difosfatos/sangre , Modelos Animales de Enfermedad , Epinefrina , Femenino , Regulación de la Expresión Génica , Ratones Endogámicos C57BL , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/patología , Osificación Heterotópica/sangre , Osificación Heterotópica/diagnóstico por imagen , Receptores Adrenérgicos/metabolismo , Calcificación Vascular/sangre , Calcificación Vascular/diagnóstico por imagen , Calcificación Vascular/genética , Microtomografía por Rayos XRESUMEN
Pseudoxanthoma elasticum is a heritable disease caused by ABCC6 deficiency. Patients develop ectopic calcification in skin, eyes, and vascular tissues. ABCC6, primarily found in liver and kidneys, mediates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi), a potent inhibitor of calcification. Pseudoxanthoma elasticum patients and Abcc6-/- mice display reduced PPi levels in plasma and peripheral tissues. Pseudoxanthoma elasticum is currently incurable, although some palliative treatments exist. In recent years, we have successfully developed therapeutic methodologies to compensate the PPi deficit in animal models and humans. Here, we inadvertently discovered that modulating dietary PPi can also be an effective approach to reducing calcification in Abcc6-/- mice. Our findings were prompted by a change in institutional rodent diet. The new chow was enriched in PPi, which increased plasma PPi, and significantly reduced mineralization in Abcc6-/- mice. We also found that dietary PPi is readily absorbed in humans. Our results suggest that the consumption of food naturally or artificially enriched in PPi represents a possible intervention to mitigate calcification progression in pseudoxanthoma elasticum, that dietary preferences of patients may explain pseudoxanthoma elasticum heterogeneous manifestations, and that animal chow has the potential to influence data reproducibility.
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Calcinosis/tratamiento farmacológico , Suplementos Dietéticos , Seudoxantoma Elástico/tratamiento farmacológico , Seudoxantoma Elástico/patología , Pirofosfatasas/administración & dosificación , Animales , Biopsia con Aguja , Calcinosis/patología , Modelos Animales de Enfermedad , Femenino , Voluntarios Sanos , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Medición de Riesgo , Especificidad de la Especie , Resultado del TratamientoRESUMEN
Various disorders including pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI), which are caused by inactivating mutations in ABCC6 and ENPP1, respectively, present with extensive tissue calcification due to reduced plasma pyrophosphate (PPi). However, it has always been assumed that the bioavailability of orally administered PPi is negligible. Here, we demonstrate increased PPi concentration in the circulation of humans after oral PPi administration. Furthermore, in mouse models of PXE and GACI, oral PPi provided via drinking water attenuated their ectopic calcification phenotype. Noticeably, provision of drinking water with 0.3 mM PPi to mice heterozygous for inactivating mutations in Enpp1 during pregnancy robustly inhibited ectopic calcification in their Enpp1-/- offspring. Our work shows that orally administered PPi is readily absorbed in humans and mice and inhibits connective tissue calcification in mouse models of PXE and GACI PPi, which is recognized as safe by the FDA, therefore not only has great potential as an effective and extremely low-cost treatment for these currently intractable genetic disorders, but also in other conditions involving connective tissue calcification.
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Difosfatos/uso terapéutico , Seudoxantoma Elástico/tratamiento farmacológico , Calcificación Vascular/tratamiento farmacológico , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Administración Oral , Adulto , Anciano , Animales , Calcio/análisis , Tejido Conectivo/metabolismo , Difosfatos/sangre , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Miocardio/química , Miocardio/metabolismo , Hidrolasas Diéster Fosfóricas/deficiencia , Hidrolasas Diéster Fosfóricas/genética , Embarazo , Seudoxantoma Elástico/patología , Pirofosfatasas/deficiencia , Pirofosfatasas/genética , Calcificación Vascular/patología , Adulto JovenRESUMEN
BACKGROUND: Cells deploy quality control mechanisms to remove damaged or misfolded proteins. Recently, we have reported that a mutation (R43W) in the Frank-ter Haar syndrome protein Tks4 resulted in aberrant intracellular localization. RESULTS: Here we demonstrate that the accumulation of Tks4(R43W) depends on the intact microtubule network. Detergent-insoluble Tks4 mutant colocalizes with the centrosome and its aggregate is encaged by the intermediate filament protein vimentin. Both the microtubule inhibitor nocodazole and the histone deacetylase inhibitor Trichostatin A inhibit markedly the aggresome formation in cells expressing Tks4(R43W). Finally, pretreatment of cells with the proteasome inhibitor MG132 markedly increases the level of aggresomes formed by Tks4(R43W). Furthermore, two additional mutant Tks4 proteins (Tks4(1-48) or Tks4(1-341)) have been investigated. Whereas the shorter Tks4 mutant, Tks4(1-48), shows no expression at all, the longer Tks4 truncation mutant accumulates in the nuclei of the cells. CONCLUSIONS: Our results suggest that misfolded Frank-ter Haar syndrome protein Tks4(R43W) is transported via the microtubule system to the aggresomes. Lack of expression of Tks4(1-48) or aberrant intracellular expressions of Tks4(R43W) and Tks4(1-341) strongly suggest that these mutations result in dysfunctional proteins which are not capable of operating properly, leading to the development of FTHS.
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Proteínas Adaptadoras Transductoras de Señales/genética , Anomalías Craneofaciales/genética , Cardiopatías Congénitas/genética , Microtúbulos/patología , Osteocondrodisplasias/congénito , Mutación Puntual , Agregación Patológica de Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células COS , Chlorocebus aethiops , Anomalías Craneofaciales/metabolismo , Anomalías Craneofaciales/patología , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Cardiopatías Congénitas/metabolismo , Cardiopatías Congénitas/patología , Humanos , Microtúbulos/genética , Microtúbulos/metabolismo , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Pliegue de Proteína , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
The disordered Tubulin Polymerization Promoting Protein (TPPP/p25), a prototype of neomorphic moonlighting proteins, displays physiological and pathological functions by interacting with distinct partners. Here the role of the disordered N- and C-termini straddling a middle flexible segment in the distinct functions of TPPP/p25 was established, and the binding motives responsible for its heteroassociations with tubulin and α-synuclein, its physiological and pathological interacting partner, respectively, were identified. We showed that the truncation of the disordered termini altered the folding state of the middle segment and has functional consequences concerning its physiological function. Double truncation diminished its binding to tubulin/microtubules, consequently the tubulin polymerization/microtubule bundling activities of TPPP/p25 were lost highlighting the role of the disordered termini in its physiological function. In contrast, interaction of TPPP/p25 with α-synuclein was not affected by the truncations and its α-synuclein aggregation promoting activity was preserved, showing that the α-synuclein binding motif is localized within the middle segment. The distinct tubulin and α-synuclein binding motives of TPPP/p25 were also demonstrated at the cellular level: the double truncated TPPP/p25 did not align along the microtubules in contrast to the full length form, while it induced α-synuclein aggregation. The localization of the binding motives on TPPP/p25 were established by specific ELISA experiments performed with designed and synthesized peptides: motives at the 178-187 and 147-156 segments are involved in the binding of tubulin and α-synuclein, respectively. The dissimilarity of these binding motives responsible for the neomorphic moonlighting feature of TPPP/p25 has significant innovative impact in anti-Parkinson drug research.
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Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Secuencia de Aminoácidos , Animales , Células CHO , Cricetulus , Células HeLa , Humanos , Datos de Secuencia Molecular , Tubulina (Proteína)/química , alfa-Sinucleína/químicaRESUMEN
The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with α-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of α-synuclein with ß-amyloid (Aß) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Aß oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aß with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Aß(42) tightly bound to TPPP/p25 (K(d) = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aß(42), α-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Aß was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Aß can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aß and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease.
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Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Células CHO , Proteínas Portadoras/genética , Cricetinae , Cricetulus , Humanos , Cuerpos de Lewy/genética , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Atrofia de Múltiples Sistemas/genética , Atrofia de Múltiples Sistemas/metabolismo , Atrofia de Múltiples Sistemas/patología , Proteínas del Tejido Nervioso/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Análisis por Matrices de Proteínas , Unión Proteica , Ratas , Ratas Wistar , Tubulina (Proteína)/genética , alfa-Sinucleína/genéticaRESUMEN
Vinblastine is a widely used anticancer drug with undesired side effects. Its conjugation with carrier molecules could be an efficient strategy to reduce these side effects. Besides this, the conjugate could exhibit increased efficiency against resistant cells, e.g., due to the altered internalization pathway. Oligoarginines, as cell-penetrating peptides, can transport covalently attached compounds into different kinds of cells and enhance the efficiency of those compounds. We report here the coupling of vinblastine through its carboxyl group at position 16 with the N-terminal amino function of L-Trp methyl ester. After hydrolysis of the ester group, 17-desacetylvinblastineTrp was conjugated to the N-terminal amino group of oligoarginine via the C-terminal carboxyl group of the Trp moiety in solution. The antitumor effect of conjugates was studied on sensitive and resistant human leukemia (HL-60) cells in vitro. Our data suggest that all conjugates investigated possess an antiproliferative effect against the studied cells. However, the effect was dependent on the number of Arg residues in the conjugates: Arg8 > Arg6 â« Arg4. The conjugate with Arg8 exhibited similar efficicacy as compared with free 17-desacetylvinblastineTrp. The in vitro studies also showed that the tubulin binding ability of vinblastine was essentially preserved even in the octaarginine conjugate. We also observed that two isomers were formed during conjugation. These isomers showed different levels of activity against tubulin polymerization in vitro and in vivo. The 17-desacetylvinblastineTrp-Arg8-1 isomer conjugate possessed high selectivity against the mitotic spindles. HRMS and NMR data suggest that 17-desacetylvinblastineTrp-Arg8-1 and 17-desacetylvinblastineTrp-Arg8-2 are epimers at the tryptophan α carbon atom.
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Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Arginina/análogos & derivados , Arginina/farmacología , Vinblastina/química , Antineoplásicos/química , Arginina/química , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Células HeLa , Humanos , Estructura Molecular , EstereoisomerismoRESUMEN
TPPP/p25 (tubulin polymerization-promoting protein/p25) is an unstructured protein that induces microtubule polymerization in vitro and is aligned along the microtubule network in transfected mammalian cells. In normal human brain, TPPP/p25 is expressed predominantly in oligodendrocytes, where its expression is proved to be crucial for their differentiation process. Here we demonstrated that the expression of TPPP/p25 in HeLa cells, in doxycycline-inducible CHO10 cells, and in the oligodendrocyte CG-4 cells promoted the acetylation of alpha-tubulin at residue Lys-40, whereas its down-regulation by specific small interfering RNA in CG-4 cells or by the withdrawal of doxycycline from CHO10 cells decreased the acetylation level of alpha-tubulin. Our results indicate that TPPP/p25 binds to HDAC6 (histone deacetylase 6), an enzyme responsible for tubulin deacetylation. Moreover, we demonstrated that the direct interaction of these two proteins resulted in the inhibition of the deacetylase activity of HDAC6. The measurement of HDAC6 activity showed that TPPP/p25 is able to induce almost complete (90%) inhibition at 3 microM concentration. In addition, treatment of the cells with nocodazole, vinblastine, or cold exposure revealed that microtubule acetylation induced by trichostatin A, a well known HDAC6 inhibitor, does not cause microtubule stabilization. In contrast, the microtubule bundling activity of TPPP/p25 was able to protect the microtubules from depolymerization. Finally, we demonstrated that, similarly to other HDAC6 inhibitors, TPPP/p25 influences the microtubule dynamics by decreasing the growth velocity of the microtubule plus ends and also affects cell motility as demonstrated by time lapse video experiments. Thus, we suggest that TPPP/p25 is a multiple effector of the microtubule organization.
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Histona Desacetilasas/metabolismo , Proteínas del Tejido Nervioso/fisiología , Tubulina (Proteína)/química , Animales , Línea Celular , Movimiento Celular , Cricetinae , Cricetulus , Células HeLa , Histona Desacetilasa 6 , Humanos , Ácidos Hidroxámicos/farmacología , Microtúbulos/metabolismo , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
TPPP/p25, a recently identified tubulin polymerization-promoting protein (TPPP), is expressed mainly in myelinating oligodendrocytes of the CNS. Here, we show that TPPP/p25 is strongly upregulated during the differentiation of primary oligodendrocyte cells as well as the CG-4 cell line. The microRNA expression profile of CG-4 cells before and after induction of differentiation was established and revealed differential regulation of a limited subset of microRNAs. miR-206, a microRNA predicted to target TPPP/p25, was not detected in oligodendrocytes. Overexpression of miR-206 led to downregulation of TPPP/p25 resulting in inhibition of differentiation. Transfection of siRNAs against TPPP/p25 also inhibited cell differentiation and promoted cell proliferation, providing evidence for an important role of TPPP/p25 during oligodendrogenesis. These results support an essential role for TPPP/p25 in oligodendrocyte differentiation likely via rearrangement of the microtubule system during the process elongation prior to the onset of myelination.
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Proteínas Portadoras/metabolismo , Diferenciación Celular/fisiología , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/fisiología , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Línea Celular , Células HeLa , Humanos , Ratones , MicroARNs/metabolismo , Microtúbulos/fisiología , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Oligodendroglía/citología , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Ácido Nucleico , Células Madre/citología , Células Madre/fisiología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Bis-indols are a large group of the anti-cancer agents, which effectively arrest the uncontrolled division of the cancerous cells. Their use in clinical chemotherapy is still limited because of: (i) the non-specific targeting of the mitotic cells; (ii) low bioavailability of the drugs. KAR-2 has been identified as a tubulin binding agent which displays significantly lower cytotoxicity but favourable anti-cancer potency than its mother molecule, vinblastine. The objective of this paper, on one hand, was to show that the human intestinal epithelial Caco-2 cells, used for pharmacokinetic studies display distinct sensitivity against KAR-2 and vinblastine due to their distinct targeting of mitotic and interphase microtubular systems. We showed that KAR-2 impeded specifically the cell division. On the other hand, we elucidated the transport mechanisms of KAR-2 as compared to vinblastine, applying pharmacokinetic modelling based upon the combination of the data obtained from in situ animal and in vitro cell level experiments. The Caco-2 cell line and in in situ intestinal perfusion experiments in rat small intestine showed that vinblastine and KAR-2 are substrates of secretion transporters but with different affinity and KAR-2 presents a higher passive diffusion permeability. The information obtained from the kinetic modelling of each type of experiments rendered it possible to apply a single mathematical model to the whole (in vitro and in situ) data set including the adequate scale-up parameters. The model was validated by means of a model selection procedure based on the goodness of fit indexes to the experimental data. This unified quantitative model which characterizes the absorption/efflux features of KAR-2 as compared to vinblastine revealed its more favourable intestinal permeability. In addition, our pharmacokinetic analyses evaluated in this study showed the advantages of the modelling approach in maximizing of the information obtained from experimental data.
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Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/farmacocinética , Huso Acromático/efectos de los fármacos , Vinblastina/análogos & derivados , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Algoritmos , Animales , Transporte Biológico Activo , Células CACO-2 , Cromatografía Líquida de Alta Presión , Humanos , Inyecciones , Absorción Intestinal , Masculino , Modelos Estadísticos , Permeabilidad , Ratas , Ratas Wistar , Espectrometría de Fluorescencia , Vinblastina/farmacocinética , Vinblastina/farmacologíaRESUMEN
Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Delta3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 +/- 0.3, 2.2 +/- 0.3, and 0.9 +/- 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr(14), Ser(18), Ser(160) for Cdk5; Ser(18), Ser(160) for ERK2, and Ser(32) for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (K(d) = 2.5-2.7 microM) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr(14) and/or Ser(18) in its unfolded N-terminal tail.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/química , Proteínas del Tejido Nervioso/química , Proteínas Quinasas/química , Animales , Sitios de Unión , Dicroismo Circular , Quinasa 5 Dependiente de la Ciclina/metabolismo , Humanos , Cinética , Microscopía Electrónica de Transmisión , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/fisiología , Fosforilación , Unión Proteica , Proteínas Recombinantes/química , Serina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Chromosomal rearrangements apparently account for the presence of a primate-specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72-residue leader peptide on Isoform A, and a CUG site that codes for a 28-residue leader peptide on Isoform B. We report studies that provide evidence for the N-terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28-amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.
Asunto(s)
Codón , Regulación de la Expresión Génica , Leucina/química , Biosíntesis de Proteínas , Tripsinógeno/genética , Secuencia de Aminoácidos , Secuencia de Bases , Encéfalo/enzimología , ADN , Células HeLa , Humanos , Datos de Secuencia Molecular , ARN Mensajero/genética , Tripsinógeno/químicaRESUMEN
TPPP/p25 is a brain-specific protein, which induces tubulin polymerization and microtubule (MT) bundling and is enriched in Lewy bodies characteristic of Parkinson's disease [Tirián et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 13976-13981]. We identified two human gene sequences, CG1-38 and p25beta, which encoded homologous proteins, that we termed p20 and p18, respectively. These homologous proteins display 60% identity with tubulin polymerization promoting protein/p25 (TPPP/p25); however, the N-terminal segment of TPPP/p25 is missing. They could be clustered into three subfamilies present in mammals and other vertebrates. We cloned, isolated, and characterized the structural and functional properties of the recombinant human proteins at molecular, ultrastructural, and cellular levels using a number of tools. These data revealed that, while p20 behaved as a disorganized protein similarly to TPPP/p25, which was described as a flexible and inherently dynamic protein with a long unstructured N-terminal tail, p18 was featured in more ordered fashion. TPPP/p25 and p20 specifically attached to MTs causing MT bundling both in vitro and in vivo; p18 protein did not cross-link MTs, and it distributed homogeneously within the cytosol of the transfected HeLa cells. These data indicate that the two shorter homologues display distinct structural features that determine their associations to MTs. The properties of p20 resemble TPPP/p25. The bundling activity of these two proteins results in the stabilization of the microtubular network, which is likely related to their physiological functions.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Dicroismo Circular , Cartilla de ADN , Células HeLa , Humanos , Hidrólisis , Microscopía Electrónica , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/ultraestructura , Filogenia , Conformación Proteica , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
TPPP/p25, a flexible unstructured protein, binds to tubulin and induces aberrant microtubule assemblies. We identified hereby glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a new interacting partner of TPPP/p25. The immunoprecipitation and affinity chromatographic experiments with bovine brain cell-free extract revealed that the interaction was salt and NAD(+) sensitive while ELISA showed resistant and firm association of the two isolated proteins. In transfected HeLa cells at low expression level of EGFP-TPPP/p25, while the green fusion protein aligned at the microtubular network, GAPDH distributed uniformly in the cytosol. However, at high expression level, GAPDH co-localized with TPPP/p25 in the aggresome-like aggregate. Immunohistochemistry showed enrichment of TPPP/p25 and GAPDH within the alpha-synuclein positive Lewy body.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Cuerpos de Lewy/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Anciano , Animales , Encéfalo/metabolismo , Bovinos , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Inmunohistoquímica , Proteínas del Tejido Nervioso/genética , Enfermedad de Parkinson/metabolismo , Unión Proteica , Conejos , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Recently we identified TPPP/p25 (tubulin polymerization promoting protein/p25) as a brain-specific unstructured protein that induced aberrant microtubule assemblies and ultrastructure in vitro and as a new marker for Parkinson's disease and other synucleopathies. In this paper the structural and functional consequences of TPPP/p25 are characterized to elucidate the relationship between the in vitro and the pathological phenomena. We show that at low expression levels EGFP-TPPP/p25 specifically colocalizes with the microtubule network of HeLa and NRK cells. We found that the colocalization was dynamic (tg=5 seconds by fluorescence recovery after photobleaching) and changed during the phases of mitosis. Time-lapse and immunofluorescence experiments revealed that high levels of EGFP-TPPP/p25 inhibited cell division and promoted cell death. At high expression levels or in the presence of proteosome inhibitor, green fusion protein accumulated around centrosomes forming an aggresome-like structure protruding into the nucleus or a filamentous cage of microtubules surrounding the nucleus. These structures showed high resistance to vinblastin. We propose that a potential function of TPPP/p25 is the stabilization of physiological microtubular ultrastructures, however, its upregulation may directly or indirectly initiate the formation of aberrant protein aggregates such as pathological inclusions.