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1.
Science ; 384(6701): 1196-1202, 2024 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-38870301

RESUMEN

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Edición Génica , Liposomas , Pulmón , Nanopartículas , Células Madre , Animales , Humanos , Ratones , Sistemas CRISPR-Cas , Fibrosis Quística/terapia , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Terapia Genética/métodos , Pulmón/metabolismo , Organoides , Células Madre/metabolismo
2.
J Neurosci ; 32(15): 5223-36, 2012 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-22496568

RESUMEN

Alterations in the lipid composition of endosomal-lysosomal membranes may constitute an early event in Alzheimer's disease (AD) pathogenesis. In this study, we investigated the possibility that GM2 ganglioside accumulation in a mouse model of Sandhoff disease might be associated with the accumulation of intraneuronal and extracellular proteins commonly observed in AD. Our results show intraneuronal accumulation of amyloid-ß peptide (Aß)-like, α-synuclein-like, and phospho-tau-like immunoreactivity in the brains of ß-hexosaminidase knock-out (HEXB KO) mice. Biochemical and immunohistochemical analyses confirmed that at least some of the intraneuronal Aß-like immunoreactivity (iAß-LIR) represents amyloid precursor protein C-terminal fragments (APP-CTFs) and/or Aß. In addition, we observed increased levels of Aß40 and Aß42 peptides in the lipid-associated fraction of HEXB KO mouse brains, and intraneuronal accumulation of ganglioside-bound Aß (GAß) immunoreactivity in a brain region-specific manner. Furthermore, α-synuclein and APP-CTFs and/or Aß were found to accumulate in different regions of the substantia nigra, indicating different mechanisms of accumulation or turnover pathways. Based on the localization of the accumulated iAß-LIR to endosomes, lysosomes, and autophagosomes, we conclude that a significant accumulation of iAß-LIR may be associated with the lysosomal-autophagic turnover of Aß and fragments of APP-containing Aß epitopes. Importantly, intraneuronal GAß immunoreactivity, a proposed prefibrillar aggregate found in AD, was found to accumulate throughout the frontal cortices of postmortem human GM1 gangliosidosis, Sandhoff disease, and Tay-Sachs disease brains. Together, these results establish an association between the accumulation of gangliosides, autophagic vacuoles, and the intraneuronal accumulation of proteins associated with AD.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Gangliósidos/metabolismo , Hexosaminidasa B/genética , Lisosomas/fisiología , Enfermedad de Sandhoff/patología , Adulto , Animales , Western Blotting , Química Encefálica/genética , Química Encefálica/fisiología , Preescolar , Gangliósido G(M2)/metabolismo , Humanos , Inmunohistoquímica , Lactante , Metabolismo de los Lípidos , Bulbo Raquídeo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Médula Espinal/metabolismo , Sustancia Negra/metabolismo , Adulto Joven , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo
3.
Virchows Arch ; 451(4): 823-34, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17674039

RESUMEN

The pathogenesis of Fabry disease is poorly understood. We used a variety of immunohistological techniques to localize globotriaosylceramide, the main glycolipid that accumulates in Fabry disease. Globotriaosylceramide immunoreactivity in a heterogenous pattern was present in all organs examined of a patient on long-term enzyme replacement therapy. In the brain, immmunopositivity was found only in the parahippocampal region. Globotriaosylceramide immunostaining was present in the cell membrane and cytoplasm of endothelial cells, even in the absence of lysosomal inclusions. In kidney tissue, globotriaosylceramide colocalized with lysosomal, endoplasmic reticulum, and nuclear markers. Pre- and postembedding immunogold electron microscopy of skin biopsies and untreated patient cultured skin fibroblasts confirmed the presence of globotriaosylceramide in the cell membrane, in various cytoplasmic structures, and in the nucleus. Control organ tissues and cultured fibroblasts from five unaffected subjects were uniformly negative for globotriaosylceramide by immunohistochemistry and immunogold electron microscopy. We conclude that a substantial amount of lysosomal and extralysosomal globotriaosylceramide immunoreactivity remains in cells and tissues even after years of enzyme replacement therapy in Fabry disease. These findings are crucial for the understanding of the disease mechanism and suggest the usefulness of immunostaining for globotriaosylceramide as a means to assess response to novel, specific therapies.


Asunto(s)
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Enfermedad de Fabry/metabolismo , Lisosomas/metabolismo , Trihexosilceramidas/metabolismo , Adulto , Encéfalo/metabolismo , Encéfalo/patología , Membrana Celular/patología , Membrana Celular/ultraestructura , Núcleo Celular/patología , Núcleo Celular/ultraestructura , Células Cultivadas , Retículo Endoplásmico/patología , Retículo Endoplásmico/ultraestructura , Enfermedad de Fabry/etiología , Enfermedad de Fabry/patología , Humanos , Riñón/metabolismo , Riñón/patología , Lisosomas/patología , Lisosomas/ultraestructura , Persona de Mediana Edad , Piel/metabolismo , Piel/patología
4.
PLoS One ; 10(8): e0134341, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26252393

RESUMEN

UNLABELLED: Migalastat HCl (AT1001, 1-Deoxygalactonojirimycin) is an investigational pharmacological chaperone for the treatment of α-galactosidase A (α-Gal A) deficiency, which leads to Fabry disease, an X-linked, lysosomal storage disorder. The currently approved, biologics-based therapy for Fabry disease is enzyme replacement therapy (ERT) with either agalsidase alfa (Replagal) or agalsidase beta (Fabrazyme). Based on preclinical data, migalastat HCl in combination with agalsidase is expected to result in the pharmacokinetic (PK) enhancement of agalsidase in plasma by increasing the systemic exposure of active agalsidase, thereby leading to increased cellular levels in disease-relevant tissues. This Phase 2a study design consisted of an open-label, fixed-treatment sequence that evaluated the effects of single oral doses of 150 mg or 450 mg migalastat HCl on the PK and tissue levels of intravenously infused agalsidase (0.2, 0.5, or 1.0 mg/kg) in male Fabry patients. As expected, intravenous administration of agalsidase alone resulted in increased α-Gal A activity in plasma, skin, and peripheral blood mononuclear cells (PBMCs) compared to baseline. Following co-administration of migalastat HCl and agalsidase, α-Gal A activity in plasma was further significantly increased 1.2- to 5.1-fold compared to agalsidase administration alone, in 22 of 23 patients (95.6%). Importantly, similar increases in skin and PBMC α-Gal A activity were seen following co-administration of migalastat HCl and agalsidase. The effects were not related to the administered migalastat HCl dose, as the 150 mg dose of migalastat HCl increased α-Gal A activity to the same extent as the 450 mg dose. Conversely, agalsidase had no effect on the plasma PK of migalastat. No migalastat HCl-related adverse events or drug-related tolerability issues were identified. TRIAL REGISTRATION: ClinicalTrials.gov NCT01196871.


Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/enzimología , Isoenzimas/uso terapéutico , alfa-Galactosidasa/metabolismo , 1-Desoxinojirimicina/administración & dosificación , 1-Desoxinojirimicina/sangre , 1-Desoxinojirimicina/farmacocinética , 1-Desoxinojirimicina/uso terapéutico , Administración Oral , Adulto , Área Bajo la Curva , Demografía , Enfermedad de Fabry/sangre , Humanos , Bombas de Infusión , Isoenzimas/administración & dosificación , Masculino , Persona de Mediana Edad , Proteínas Recombinantes , Piel/enzimología , alfa-Galactosidasa/administración & dosificación , alfa-Galactosidasa/sangre , alfa-Galactosidasa/uso terapéutico
5.
PLoS One ; 9(7): e102092, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25036864

RESUMEN

Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA) activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER)-retained P545L mutant form of human GAA (P545L GAA). AT2220 increased the specific activity of P545L GAA toward both natural (glycogen) and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO) and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As such, AT2220 may warrant further evaluation as a treatment for Pompe disease.


Asunto(s)
1-Desoxinojirimicina/farmacología , Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/metabolismo , Glucógeno/metabolismo , Lisosomas/efectos de los fármacos , Mutación , 1-Desoxinojirimicina/administración & dosificación , 1-Desoxinojirimicina/farmacocinética , Administración Oral , Animales , Biocatálisis/efectos de los fármacos , Disponibilidad Biológica , Células COS , Chlorocebus aethiops , Modelos Animales de Enfermedad , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Estabilidad de Enzimas/efectos de los fármacos , Técnicas de Inactivación de Genes , Glucano 1,4-alfa-Glucosidasa/biosíntesis , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo II/patología , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/metabolismo , Lisosomas/metabolismo , Ratones , Ratones Transgénicos , Proteínas Mutantes/biosíntesis , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos
6.
Neurotherapeutics ; 11(4): 840-56, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25037721

RESUMEN

Mutation of the lysosomal hydrolase acid-ß-glucosidase (GCase), which leads to reduced GCase activity, is one of the most frequent genetic risk factors for Parkinson's disease (PD) and promotes α-synuclein accumulation in the brain, a hallmark of PD and other synucleinopathies. Whether targeting GCase pharmacologically is a valid therapeutic strategy for sporadic PD in the absence of GCase mutation is unknown. We have investigated whether increasing the stability, trafficking, and activity of wild-type GCase could be beneficial in synucleinopathies by administering the pharmacological chaperone AT2101 (afegostat-tartrate, isofagomine) to mice that overexpress human wild-type α-synuclein (Thy1-aSyn mice). AT2101 administered orally for 4 months to Thy1-aSyn mice improved motor and nonmotor function, abolished microglial inflammatory response in the substantia nigra, reduced α-synuclein immunoreactivity in nigral dopaminergic neurons, and reduced the number of small α-synuclein aggregates, while increasing the number of large α-synuclein aggregates. These data support the further investigation of pharmacological chaperones that target GCase as a therapeutic approach for sporadic PD and other synucleinopathies, even in the absence of glucocerebrosidase mutations.


Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/prevención & control , alfa-Sinucleína/metabolismo , beta-Glucosidasa/antagonistas & inhibidores , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Humanos , Iminopiranosas/farmacología , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Agregado de Proteínas/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Tartratos
7.
J Med Chem ; 56(7): 2705-25, 2013 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-23363020

RESUMEN

Lysosomal enzymes are responsible for the degradation of a wide variety of glycolipids, oligosaccharides, proteins, and glycoproteins. Inherited mutations in the genes that encode these proteins can lead to reduced stability of newly synthesized lysosomal enzymes. While often catalytically competent, the mutated enzymes are unable to efficiently pass the quality control mechanisms of the endoplasmic reticulum, resulting in reduced lysosomal trafficking, substrate accumulation, and cellular dysfunction. Pharmacological chaperones (PCs) are small molecules that bind and stabilize mutant lysosomal enzymes, thereby allowing proper cellular translocation. Such compounds have been shown to increase enzyme activity and reduce substrate burden in a number of preclinical models and clinical studies. In this Perspective, we review several of the lysosomal diseases for which PCs have been studied and the SAR of the various classes of molecules.


Asunto(s)
Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Chaperonas Moleculares/uso terapéutico , Línea Celular , Glucolípidos/metabolismo , Humanos , Relación Estructura-Actividad
8.
FEBS J ; 277(7): 1618-38, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20148966

RESUMEN

Gaucher disease is caused by mutations in the gene that encodes the lysosomal enzyme acid beta-glucosidase (GCase). We have shown previously that the small molecule pharmacological chaperone isofagomine (IFG) binds and stabilizes N370S GCase, resulting in increased lysosomal trafficking and cellular activity. In this study, we investigated the effect of IFG on L444P GCase. Incubation of Gaucher patient-derived lymphoblastoid cell lines (LCLs) or fibroblasts with IFG led to approximately 3.5- and 1.3-fold increases in L444P GCase activity, respectively, as measured in cell lysates. The effect in fibroblasts was increased approximately 2-fold using glycoprotein-enrichment, GCase-immunocapture, or by incubating cells overnight in IFG-free media prior to assay, methods designed to maximize GCase activity by reducing IFG carryover and inhibition in the enzymatic assay. IFG incubation also increased the lysosomal trafficking and in situ activity of L444P GCase in intact cells, as measured by reduction in endogenous glucosylceramide levels. Importantly, this reduction was seen only following three-day incubation in IFG-free media, underscoring the importance of IFG removal to restore lysosomal GCase activity. In mice expressing murine L444P GCase, oral administration of IFG resulted in significant increases (2- to 5-fold) in GCase activity in disease-relevant tissues, including brain. Additionally, eight-week IFG administration significantly lowered plasma chitin III and IgG levels, and 24-week administration significantly reduced spleen and liver weights. Taken together, these data suggest that IFG can increase the lysosomal activity of L444P GCase in cells and tissues. Moreover, IFG is orally available and distributes into multiple tissues, including brain, and may thus merit therapeutic evaluation for patients with neuronopathic and non-neuronopathic Gaucher disease.


Asunto(s)
Enfermedad de Gaucher/genética , Iminopiranosas/química , Enfermedades por Almacenamiento Lisosomal/genética , Mutación , beta-Glucosidasa/genética , Animales , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Glucosilceramidasa/metabolismo , Humanos , Masculino , Ratones , Microscopía Confocal/métodos , Chaperonas Moleculares/metabolismo , Ratas , Ratas Sprague-Dawley
9.
Nat Chem Biol ; 3(2): 101-7, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17187079

RESUMEN

Gaucher disease results from mutations in the lysosomal enzyme acid beta-glucosidase (GCase). Although enzyme replacement therapy has improved the health of some affected individuals, such as those with the prevalent N370S mutation, oral treatment with pharmacological chaperones may be therapeutic in a wider range of tissue compartments by restoring sufficient activity of endogenous mutant GCase. Here we demonstrate that isofagomine (IFG, 1) binds to the GCase active site, and both increases GCase activity in cell lysates and restores lysosomal trafficking in cells containing N370S mutant GCase. We also compare the crystal structures of IFG-bound GCase at low pH with those of glycerol-bound GCase at low pH and apo-GCase at neutral pH. Our data indicate that IFG induces active GCase, which is secured by interactions with Asn370. The design of small molecules that stabilize substrate-bound conformations of mutant proteins may be a general therapeutic strategy for diseases caused by protein misfolding and mistrafficking.


Asunto(s)
Enfermedad de Gaucher/enzimología , Glucosilceramidasa/química , Piperidinas/química , Dominio Catalítico , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/metabolismo , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/metabolismo , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Iminopiranosas/química , Iminopiranosas/farmacología , Modelos Moleculares , Piperidinas/farmacología , Unión Proteica , Conformación Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos
10.
Biopolymers ; 74(5): 363-76, 2004 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-15222016

RESUMEN

The AP7 and AP24 proteins represent a class of mineral-interaction polypeptides that are found in the aragonite-containing nacre layer of mollusk shell (H. rufescens). These proteins have been shown to preferentially interfere with calcium carbonate mineral growth in vitro. It is believed that both proteins play an important role in aragonite polymorph selection in the mollusk shell. Previously, we demonstrated the 1-30 amino acid (AA) N-terminal sequences of AP7 and AP24 represent mineral interaction/modification domains in both proteins, as evidenced by their ability to frustrate calcium carbonate crystal growth at step edge regions. In this present report, using free N-terminal, C(alpha)-amide "capped" synthetic polypeptides representing the 1-30 AA regions of AP7 (AP7-1 polypeptide) and AP24 (AP24-1 polypeptide) and NMR spectroscopy, we confirm that both N-terminal sequences possess putative Ca (II) interaction polyanionic sequence regions (2 x -DD- in AP7-1, -DDDED- in AP24-1) that are random coil-like in structure. However, with regard to the remaining sequences regions, each polypeptide features unique structural differences. AP7-1 possesses an extended beta-strand or polyproline type II-like structure within the A11-M10, S12-V13, and S28-I27 sequence regions, with the remaining sequence regions adopting a random-coil-like structure, a trait common to other polyelectrolyte mineral-associated polypeptide sequences. Conversely, AP24-1 possesses random coil-like structure within A1-S9 and Q14-N16 sequence regions, and evidence for turn-like, bend, or loop conformation within the G10-N13, Q17-N24, and M29-F30 sequence regions, similar to the structures identified within the putative elastomeric proteins Lustrin A and sea urchin spicule matrix proteins. The similarities and differences in AP7 and AP24 N-terminal domain structure are discussed with regard to joint AP7-AP24 protein modification of calcium carbonate growth.


Asunto(s)
Moluscos/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Calcificación Fisiológica , Carbonato de Calcio/química , Carbonato de Calcio/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Proteínas/química , Proteínas/metabolismo
11.
Biopolymers ; 63(6): 358-69, 2002 May.
Artículo en Inglés | MEDLINE | ID: mdl-11920437

RESUMEN

The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layer) in mollusk shell. Recent atomic force microscopy (AFM) pulling studies have demonstrated that the lustrin-containing organic nacre layer in the abalone, Haliotis rufescens, exhibits a typical sawtooth force-extension curve with hysteretic recovery. This force extension behavior is reminiscent of reversible unfolding and refolding in elastomeric proteins such as titin and tenascin. Since secondary structure plays an important role in force-induced protein unfolding and refolding, the question is, What secondary structure(s) exist within the major domains of Lustrin A? Using a model peptide (FPGKNVNCTSGE) representing the 12-residue consensus sequence found near the N-termini of the first eight cysteine-rich domains (C-domains) within the Lustrin A protein, we employed CD, NMR spectroscopy, and simulated annealing/minimization to determine the secondary structure preferences for this sequence. At pH 7.4, we find that the 12-mer sequence adopts a loop conformation, consisting of a "bend" or "turn" involving residues G3-K4 and N7-C8-T9, with extended conformations arising at F1-G3; K4-V6; T9-S10-G11 in the sequence. Minor pH-dependent conformational effects were noted for this peptide; however, there is no evidence for a salt-bridge interaction between the K4 and E12 side chains. The presence of a loop conformation within the highly conserved -PG-, -NVNCT- sequence of C1-C8 domains may have important structural and mechanistic implications for the Lustrin A protein with regard to elastic behavior.


Asunto(s)
Proteínas de la Matriz Extracelular/química , Secuencia de Aminoácidos , Dicroismo Circular , Secuencia de Consenso , Proteínas de la Matriz Extracelular/síntesis química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación Proteica , Termodinámica
12.
Connect Tissue Res ; 44 Suppl 1: 10-5, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-12952167

RESUMEN

The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layers) in mollusk shell. These proteins not only exhibit elastomeric behavior within the mineralized matrix, but also adhesion to the aragonite-containing composite layer. One member of the lustrin superfamily, Lustrin A, has been sequenced; the protein is organized into defined, modular sequence domains that are hypothesized to perform separate functions (i.e., force unfolding, mineral adhesion, intermolecular binding) within the Lustrin A protein. Using nuclear magnetic resonance (NMR) and in vitro mineralization assays, we investigated structure-function relationships for two Lustrin A putative mineral binding domains, the 30 AA Arg, Lys, Tyr, Ser-rich (RKSY) and the 24 AA Asp-rich (D4) sequence regions domain of the Lustrin A protein. The results indicate that both sequences adopt open, unfolded structures that represent either extended or random coil states. Using geologic calcite overgrowth assays and scanning electron microscopic analyses, we observe that the RKSY polypeptide does not significantly perturb calcium carbonate growth. However, the D4 domain does influence crystal growth in a concentration-dependent manner. Collectively, our data indicate that D4, and not the RKSY domain, exhibits structure-function activity consistent with a mineral binding region.


Asunto(s)
Biopolímeros/química , Calcificación Fisiológica/fisiología , Proteínas de la Matriz Extracelular/química , Secuencia de Aminoácidos , Animales , Electrólitos/química , Proteínas de la Matriz Extracelular/síntesis química , Datos de Secuencia Molecular , Moluscos , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad
13.
Biopolymers ; 65(5): 362-72, 2002 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-12389216

RESUMEN

Fracture resistance in biomineralized structures has been linked to the presence of proteins, some of which possess sequences that are associated with elastic behavior. One such protein superfamily, the Pro,Gly-rich sea urchin intracrystalline spicule matrix proteins, form protein-protein supramolecular assemblies that modify the microstructure and fracture-resistant properties of the calcium carbonate mineral phase within embryonic sea urchin spicules and adult sea urchin spines. In this report, we detail the identification of a repetitive keratin-like "glycine-loop"- or coil-like structure within the 34-AA (AA: amino acid) N-terminal domain, (PGMG)(8)PG, of the spicule matrix protein, PM27. The identification of this repetitive structural motif was accomplished using two capped model peptides: a 9-AA sequence, GPGMGPGMG, and a 34-AA peptide representing the entire motif. Using CD, NMR spectrometry, and molecular dynamics simulated annealing/minimization simulations, we have determined that the 9-AA model peptide adopts a loop-like structure at pH 7.4. The structure of the 34-AA polypeptide resembles a coil structure consisting of repeating loop motifs that do not exhibit long-range ordering. Given that loop structures have been associated with protein elastic behavior and protein motion, it is plausible that the 34-AA Pro,Gly,Met repeat sequence motif in PM27 represents a putative elastic or mobile domain.


Asunto(s)
Proteínas/química , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Secuencias Repetitivas de Aminoácido , Erizos de Mar
14.
Biopolymers ; 70(4): 522-33, 2003 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-14648763

RESUMEN

Ethylenediamine-tetraacetic acid extracted water-soluble matrix proteins in molluscan shells secreted from the mantle epithelia are believed to control crystal nucleation, morphology, orientation, and phase of the deposited mineral. Previously, atomic force microscopy demonstrated that abalone nacre proteins bind to growing step edges and to specific crystallographic faces of calcite, suggesting that inhibition of calcite growth may be one of the molecular processes required for growth of the less thermodynamically stable aragonite phase. Previous experiments were done with protein mixtures. To elucidate the role of single proteins, we have characterized two proteins isolated from the aragonitic component of nacre of the red abalone, Haliotis rufescens. These proteins, purified by hydrophobic interaction chromatography, are designated AP7 and AP24 (aragonitic protein of molecular weight 7 kDa and 24 kDa, respectively). Degenerate oligonucleotide primers corresponding to N-terminal and internal peptide sequences were used to amplify cDNA clones by a polymerase chain reaction from a mantle cDNA library; the deduced primary amino acid sequences are presented. Preliminary crystal growth experiments demonstrate that protein fractions enriched in AP7 and AP24 produced CaCO(3) crystals with morphology distinct from crystals grown in the presence of the total mixture of soluble aragonite-specific proteins. Peptides corresponding to the first 30 residues of the N-terminal sequences of both AP7 and AP24 were generated. The synthetic peptides frustrate the progression of step edges of a growing calcite surface, indicating that sequence features within the N-termini of AP7 and AP24 include domains that interact with CaCO(3). CD analyses demonstrate that the N-terminal peptide sequences do not possess significant percentages of alpha-helix or beta-strand secondary structure in solution. Instead, in both the presence and absence of Ca(II), the peptides retain unfolded conformations that may facilitate protein-mineral interaction.


Asunto(s)
Carbonato de Calcio/química , Moluscos/química , Proteínas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Carbonato de Calcio/metabolismo , Dicroismo Circular , Modelos Moleculares , Datos de Secuencia Molecular , Moluscos/metabolismo , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Proteínas/aislamiento & purificación , Proteínas/metabolismo
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