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1.
J Am Soc Nephrol ; 27(10): 3204-3219, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27005919

RESUMO

Two common missense variants in APOL1 (G1 and G2) have been definitively linked to CKD in black Americans. However, not all individuals with the renal-risk genotype develop CKD, and little is known about how APOL1 variants drive disease. Given the association of APOL1 with HDL particles, which are cleared by the kidney, differences in the level or quality of mutant APOL1­HDL particles could be causal for disease and might serve as a useful risk stratification marker. We measured plasma levels of G0 (low risk), G1, and G2 APOL1 in 3450 individuals in the Dallas Heart Study using a liquid chromatography-MS method that enabled quantitation of the different variants. Additionally, we characterized native APOL1­HDL from donors with no or two APOL1 risk alleles by size-exclusion chromatography and analysis of immunopurified APOL1­HDL particles. Finally, we identified genetic loci associated with plasma APOL1 levels and tested for APOL1-dependent association with renal function. Although we replicated the previous association between APOL1 variant status and renal function in nondiabetic individuals, levels of circulating APOL1 did not associate with microalbuminuria or GFR. Furthermore, the size or known components of APOL1­HDL did not consistently differ in subjects with the renal-risk genotype. Genetic association studies implicated variants in loci harboring haptoglobin-related protein (HPR), APOL1, and ubiquitin D (UBD) in the regulation of plasma APOL1 levels, but these variants did not associate with renal function. Collectively, these data demonstrate that the risk of renal disease associated with APOL1 is probably not related to circulating levels of the mutant protein.


Assuntos
Apolipoproteínas/sangue , Lipoproteínas HDL/sangue , Insuficiência Renal Crônica/sangue , Adulto , Apolipoproteína L1 , Apolipoproteínas/genética , Estudos de Coortes , Estudos Transversais , Feminino , Variação Genética , Genótipo , Humanos , Lipoproteínas HDL/genética , Masculino , Insuficiência Renal Crônica/epidemiologia , Insuficiência Renal Crônica/genética , Fatores de Risco
2.
Rapid Commun Mass Spectrom ; 27(23): 2639-47, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24591025

RESUMO

RATIONALE: Human genetics studies in African Americans have shown a strong correlation between polymorphisms in the ApoL1 gene and chronic kidney disease (CKD). To gain further insight into the etiology of ApoL1-associated kidney diseases, the determination of circulating levels of both wild type as well as ApoL1 variants could be of significant use. To date, antibodies that discriminate between all three ApoL1 variant forms (wild type, G1 and G2) are not available. We aimed to develop a rapid method for detecting and quantifying ApoL1 variants and total levels in plasma. METHODS: Ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) in multiple-reaction monitoring acquisition mode was used to quantify ApoL1. RESULTS: We demonstrated that it is feasible to detect and quantify ApoL1 variants (wild type, G1 and G2), and total ApoL1 concentrations in plasma. ApoL1 genotypes determined by LC/MS agreed perfectly with the traditional method DNA sequencing for 74 human subjects. The method exhibited at least three orders of linearity with a lower limit of quantification of 10 nM. Moreover, the method can readily be multiplexed for the quantification of a panel of protein markers in a single sample. CONCLUSIONS: The method reported herein obviates the need to perform DNA genotyping of ApoL1 variants, which is of significant value in cases where stored samples are unsuitable for DNA analysis. More importantly, the method could potentially be of use in the early identification of individuals at risk of developing CKD, and for the stratification of patients for treatment with future ApoL1-modifying therapies.


Assuntos
Apolipoproteínas/sangue , Apolipoproteínas/genética , Cromatografia Líquida de Alta Pressão/métodos , Variação Genética , Nefropatias/sangue , Lipoproteínas HDL/sangue , Lipoproteínas HDL/genética , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Apolipoproteína L1 , Genótipo , Humanos , Nefropatias/diagnóstico , Nefropatias/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular
3.
FASEB Bioadv ; 4(1): 76-89, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35024574

RESUMO

Tumor innervation has recently been documented and characterized in various settings and tumor types. However, the role that nerves innervating tumors play in the pathogenesis of cancer has not been clarified. In this study, we searched for neural signaling from bulk RNA sequencing from The Cancer Genome Atlas (TCGA) dataset and looked for patterns of interactions between different cell types within the tumor environment. Using a presynapse signature (PSS) as a probe, we showed that multiple stromal cell types crosstalk and/or contribute to neural signals. Based on the correlation and linear regression, we hypothesized that neural signals contribute to an immune-suppressive tumor microenvironment (TME). To test this hypothesis, we performed in vitro dorsal root ganglion (DRG)/macrophage coculture experiments. Compared to the M2 macrophage monoculture, the DRG/M2 macrophage coculture prevented anti-inflammatory M2 to pro-inflammatory M1 polarization by LPS stimulation. Finally, a survey of different TCGA tumor types indicated that higher RNA neural signature is predictive of poor patient outcomes in multiple tumor types.

4.
Biochim Biophys Acta ; 1801(12): 1349-60, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20817122

RESUMO

The purinergic receptor P2Y(13) has been shown to play a role in the uptake of holo-HDL particles in in vitro hepatocyte experiments. In order to determine the role of P2Y(13) in lipoprotein metabolism in vivo, we ablated the expression of this gene in mice. Here we show that P2Y(13) knockout mice have lower fecal concentrations of neutral sterols (-27%±2.1% in males) as well as small decreases in plasma HDL (-13.1%±3.2% in males; -17.5%±4.0% in females) levels. In addition, significant decreases were detected in serum levels of fatty acids and glycerol in female P2Y(13) knockout mice. Hepatic mRNA profiling analyses showed increased expression of SREBP-regulated cholesterol and fatty acid biosynthesis genes, while fatty acid ß-oxidation genes were significantly decreased. Liver gene signatures also identified changes in PPARα-regulated transcript levels. With the exception of a small increase in bone area, P2Y(13) knockout mice do not show any additional major abnormalities, and display normal body weight, fat mass and lean body mass. No changes in insulin sensitivity and oral glucose tolerance could be detected. Taken together, our experiments assess a role for the purinergic receptor P2Y(13) in the regulation of lipoprotein metabolism and demonstrate that modulating its activity could be of benefit to the treatment of dyslipidemia in people.


Assuntos
Lipoproteínas/metabolismo , Receptores Purinérgicos P2/fisiologia , Animais , Feminino , Perfilação da Expressão Gênica , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Receptores Purinérgicos P2/genética
5.
Bioorg Med Chem Lett ; 20(11): 3426-30, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20444602

RESUMO

Niacin is an effective drug for raising HDL cholesterol. However, niacin must be taken in large doses and significant side effects are often observed, including facial flushing, loss of glucose tolerance, and liver toxicity. An anthranilic acid was identified as an agonist of the niacin receptor. In order to improve efficacy and provide structural diversity, replacements for the anthranilic acid were investigated and several compounds with improved properties were identified.


Assuntos
Niacina/metabolismo , Receptores de Droga/metabolismo , ortoaminobenzoatos/química , Disponibilidade Biológica
6.
Bioorg Med Chem Lett ; 19(16): 4768-72, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19592242

RESUMO

Niacin is an effective drug for raising HDL cholesterol and reducing coronary risks, but patients show low compliance with treatment due to severe facial flushing upon taking the drug. A series of bicyclic pyrazole carboxylic acids were synthesized and tested for their ability to activate the niacin receptor. One analog, 23, showed improved potency and lacked flushing at doses that effectively altered the lipid profile of rats.


Assuntos
Dislipidemias/tratamento farmacológico , Hipolipemiantes/farmacologia , Niacina/farmacologia , Agonistas Nicotínicos/farmacologia , Pirazóis/farmacologia , Animais , HDL-Colesterol/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Hipolipemiantes/síntese química , Hipolipemiantes/química , Camundongos , Niacina/química , Agonistas Nicotínicos/síntese química , Agonistas Nicotínicos/química , Pirazóis/síntese química , Pirazóis/química , Ratos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo
7.
Chem Biol ; 11(12): 1677-87, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15610852

RESUMO

Peptide:N-glycanase (PNGase) is ostensibly the sole enzyme responsible for deglycosylation of unfolded N-linked glycoproteins dislocated from the ER to the cytosol. Here we show the pan-caspase inhibitor, Z-VAD-fmk, to be an active site-directed irreversible inhibitor of yeast and mammalian PNGase at concentrations below those used to inhibit caspases in vivo. Through chemical synthesis we determined that the P1 residue, electrophile position, and leaving group are important structural parameters for PNGase inhibition. We show that Z-VAD-fmk inhibits PNGase in living cells and that degradation of class I MHC heavy chains and TCRalpha, in an identical cellular setting, is markedly different. Remarkably, proteasome-mediated turnover of class I MHC heavy chains proceeds even when PNGase is completely inhibited, suggesting that the function of PNGase may be to facilitate more efficient proteasomal proteolysis of N-linked glycoproteins through glycan removal.


Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Glicoproteínas/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/antagonistas & inibidores , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/fisiologia , Animais , Sítios de Ligação , Inibidores de Caspase , Linhagem Celular Tumoral , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/efeitos dos fármacos , Humanos , Camundongos , Estrutura Molecular , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Receptores de Antígenos de Linfócitos T alfa-beta/efeitos dos fármacos , Relação Estrutura-Atividade
8.
Int J Biol Sci ; 8(3): 310-27, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22355267

RESUMO

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticolesterolemiantes/uso terapêutico , LDL-Colesterol/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Imunização Passiva , Síndrome Metabólica/terapia , Pró-Proteína Convertases/antagonistas & inibidores , Pró-Proteína Convertases/imunologia , Serina Endopeptidases/imunologia , Sinvastatina/uso terapêutico , Proteínas de Ligação a Elemento Regulador de Esterol/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Anticolesterolemiantes/administração & dosagem , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Perfilação da Expressão Gênica , Células Hep G2/efeitos dos fármacos , Células Hep G2/metabolismo , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macaca mulatta , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/genética , Camundongos , Camundongos Transgênicos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/biossíntese , Pró-Proteína Convertases/genética , RNA Mensageiro/metabolismo , Receptores de LDL/biossíntese , Receptores de LDL/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Sinvastatina/administração & dosagem
9.
PLoS One ; 2(7): e679, 2007 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-17653289

RESUMO

BACKGROUND: The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15. RESULTS: We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome. CONCLUSIONS: By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice.


Assuntos
Citocinas/imunologia , Ubiquitinas/imunologia , Animais , Conjugação Genética , Reações Cruzadas , Citocinas/genética , Citocinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Evolução Molecular , Cinética , Camundongos , Filogenia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Transcrição Gênica , Ubiquitinas/genética , Ubiquitinas/metabolismo
10.
EMBO J ; 22(5): 1036-46, 2003 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-12606569

RESUMO

Successful maturation determines the intracellular fate of secretory and membrane proteins in the endoplasmic reticulum (ER). Failure of proteins to fold or assemble properly can lead to their retention in the ER and redirects them to the cytosol for degradation by the proteasome. Proteasome inhibitors can yield deglycosylated cytoplasmic intermediates that are the result of an N-glycanase activity, believed to act prior to destruction of these substrates by the proteasome. A gene encoding a yeast peptide:N-glycanase, PNG1, has been cloned, but this N-glycanase and its mammalian homolog were reported to be incapable of deglycosylating full-length glycoproteins. We show that both the yeast PNG1 enzyme and its mammalian homolog display N-glycanase activity towards intact glycoproteins. As substrates, cytosolic PNGase activity prefers proteins containing high-mannose over those bearing complex type oligosaccharides. Importantly, PNG1 discriminates between non-native and folded glycoproteins, consistent with a role for N-glycanase in cytoplasmic turnover of glycoproteins.


Assuntos
Amidoidrolases/metabolismo , Citoplasma/enzimologia , Proteínas Fúngicas/metabolismo , Glicoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Amidoidrolases/genética , Animais , Sítios de Ligação , Linhagem Celular , Cisteína/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Glicosilação , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Complexo de Endopeptidases do Proteassoma , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia
11.
EMBO J ; 23(3): 650-8, 2004 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-14749736

RESUMO

The human cytomegalovirus-encoded glycoprotein US2 catalyzes proteasomal degradation of Class I major histocompatibility complex (MHC) heavy chains (HCs) through dislocation of the latter from the endoplasmic reticulum (ER) to the cytosol. During this process, the Class I MHC HCs are deglycosylated by an N-glycanase-type activity. siRNA molecules designed to inhibit the expression of the light chain, beta(2)-microglobulin, block the dislocation of Class I MHC molecules, which implies that US2-dependent dislocation utilizes correctly folded Class I MHC molecules as a substrate. Here we demonstrate it is peptide: N-glycanase (PNGase or PNG1) that deglycosylates dislocated Class I MHC HCs. Reduction of PNGase activity by siRNA expression in US2-expressing cells inhibits deglycosylation of Class I MHC HC molecules. In PNGase siRNA-treated cells, glycosylated HCs appear in the cytosol, providing the first evidence for the presence of an intact N-linked type I membrane glycoprotein in the cytosol. N-glycanase activity is therefore not required for dislocation of glycosylated Class I MHC molecules from the ER.


Assuntos
Retículo Endoplasmático/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Proteínas do Envelope Viral/metabolismo , Microglobulina beta-2/metabolismo , Linhagem Celular Tumoral , Glicosilação , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Microglobulina beta-2/genética
12.
EMBO Rep ; 5(2): 201-6, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14726951

RESUMO

N-glycanase from Saccharomyces cerevisiae (Png1) preferentially removes N-glycans from misfolded proteins. The ability of Png1 to distinguish between folded and misfolded glycoproteins is reminiscent of substrate recognition by UDP-glucose glycoprotein glucosyl transferase, an enzyme that possesses this trait. The only known in vivo substrates of Png1 are aberrant glycoproteins that originate in the endoplasmic reticulum, and arrive in the cytoplasm for proteasomal degradation. The substrate specificity of Png1 is admirably suited for this task.


Assuntos
Glicoproteínas/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Linhagem Celular , Glicoproteínas/química , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Glicosilação , Humanos , Mutação/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/isolamento & purificação , Conformação Proteica , Dobramento de Proteína , Ribonucleases/química , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Especificidade por Substrato , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
13.
Traffic ; 4(12): 824-37, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14617346

RESUMO

Like all members of the herpesvirus family, human herpesvirus-7 has evolved mechanisms to evade immune detection. The human herpesvirus-7 gene product U21 encodes an immunoevasin that binds to class I major histocompatibility complex molecules and diverts them to a lysosomal compartment. Here we show that the cytoplasmic tail of U21, although sufficient to sequester a heterologous membrane protein (CD4 chimera), has no effect on U21's ability to redirect class I major histocompatibility complex molecules to lysosomes. Instead, the ER-lumenal domain of U21 is sufficient to redirect class I major histocompatibility complex molecules to the lysosomal compartment. These observations demonstrate a novel viral immunoevasive mechanism for U21, and implicate the ER-lumenal domain of a type I transmembrane protein in lysosomal sorting.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/farmacologia , Retículo Endoplasmático/metabolismo , Herpesvirus Humano 7/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Lisossomos/metabolismo , Proteínas Virais/química , Proteínas Virais/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Antígenos CD4/biossíntese , Antígenos CD4/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Citometria de Fluxo , Humanos , Leucina/química , Manose/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Retroviridae/genética
14.
Am J Hum Genet ; 72(1): 23-31, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12471562

RESUMO

For more than a decade, protein-replacement therapy has been employed successfully for the treatment of Gaucher disease. Recently, a comparable therapy has become available for the related lipid-storage disorder Fabry disease. Two differently produced recombinant alpha-galactosidase A (alpha-gal A) preparations are used independently for this purpose. Agalsidase alpha is obtained from human fibroblasts that have been modified by gene activation; agalsidase beta is obtained from Chinese hamster ovary cells that are transduced with human alpha-gal A cDNA. It has previously been claimed that alpha-gal A mRNA undergoes editing, which may result in coproduction of an edited protein (Phe 396 Tyr) that might have a relevant physiological function. We therefore analyzed the occurrence of alpha-gal A editing, as well as the precise nature, in this respect, of the therapeutic enzymes. No indications were obtained for the existence of editing at the protein or RNA level. Both recombinant enzymes used in therapy are unedited and are capable of functionally correcting cultured fibroblasts from Fabry patients in their excessive globotriaosylceramide accumulation. Although RNA editing is apparently not relevant in the case of alpha-gal A, a thorough analysis of the potential occurrence of editing of transcripts is nevertheless advisable in connection with newly developed protein-replacement therapies.


Assuntos
Doença de Fabry/enzimologia , Doença de Fabry/genética , Terapia Genética , Isoenzimas/genética , Edição de RNA/genética , alfa-Galactosidase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Fibroblastos , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Macrófagos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triexosilceramidas/metabolismo , alfa-Galactosidase/química , alfa-Galactosidase/metabolismo
15.
Kidney Int ; 65(1): 310-22, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14675065

RESUMO

BACKGROUND: Dialysis-related amyloidosis (DRA) is a frequent complication of end-stage renal disease (ESRD) that has been associated with the accumulation of beta2-microglobulin (beta2-m). Removal of beta2-m results in the loss of important proteins due to the nonspecific nature of current therapies. Although whole antibodies can potentially be used to confer specificity to beta2-m removal from blood, single-chain variable region (scFv) antibody fragments could potentially offer several advantages as immunoadsorption ligands due to their size, genetic definition, ability to be expressed by microbes, and amenability for in vitro evolution. METHODS: An antihuman beta2-m scFv was constructed from the BBM.1 hybridoma and expressed by a yeast display vector. The binding affinity of the wild-type scFv fragment was quantified by flow cytometry analysis. Soluble scFv was expressed by a yeast secretion vector, purified, and immobilized onto agarose beads. The binding capacity of the immunoadsorbent was measured by equilibrating samples with saturating quantities of fluorescent beta2-m in serum. RESULTS: The displayed scFv possessed a nanomolar affinity (KD= 0.008 +/- 0.004 mg-beta2-m/L). The immunoadsorbent exhibited an adsorption site density of 0.41 +/- 0.01 mg beta2-m/mL settled gel. Under saturating conditions, the mass ratio of adsorbed beta2-m to immobilized antibody is 70% greater than any previous literature report for whole antibodies. Preliminary specificity experiments suggest that the scFv-based immunoadsorbent is specific toward human beta2-m. CONCLUSION: Recombinant DNA technology was successfully used to engineer an scFv-based immunoadsorbent. Use of immobilized scFvs during hemodialysis may minimize loss of valuable proteins and facilitate the removal of macromolecules that are significantly larger than the molecular weight cut-off of the membrane.


Assuntos
Amiloidose/terapia , Técnicas de Imunoadsorção , Falência Renal Crônica/terapia , Microglobulina beta-2/imunologia , Microglobulina beta-2/isolamento & purificação , Sequência de Aminoácidos , Amiloidose/sangue , Sequência de Bases , Clonagem Molecular , Circulação Extracorpórea , Humanos , Hibridomas , Região Variável de Imunoglobulina/genética , Falência Renal Crônica/sangue , Dados de Sequência Molecular , Plasmídeos , Leveduras/genética
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