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1.
J Am Soc Nephrol ; 32(5): 1249-1261, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33833079

RESUMO

BACKGROUND: Membranous nephropathy (MN) results from deposition of antigen-antibody complexes along the glomerular basement membrane (GBM). PLA2R, THSD7A, NELL1, and SEMA3B account for 80%-90% of target antigens in MN. METHODS: We performed laser microdissection and mass spectrometry (MS/MS) in kidney biopsies from 135 individuals with PLA2R-negative MN, and used immunohistochemistry/immunofluorescence and confocal microscopy to confirm the MS/MS finding, detect additional cases, and localize the novel protein. We also performed MS/MS and immunohistochemistry on 116 controls and used immunofluorescence microscopy to screen biopsy samples from two validation cohorts. Western blot and elution studies were performed to detect antibodies in serum and biopsy tissue. RESULTS: MS/MS studies detected a unique protein, protocadherin 7 (PCDH7), in glomeruli of ten (5.7%) PLA2R-negative MN cases, which also were negative for PLA2R, THSD7A, EXT1/EXT2, NELL1, and SEMA3B. Spectral counts ranged from six to 24 (average 13.2 [SD 6.6]). MS/MS did not detect PCDH7 in controls (which included 28 PLA2R-positive cases). In all ten PCDH7-positive cases, immunohistochemistry showed bright granular staining along the GBM, which was absent in the remaining cases of PLA2R-negative MN and control cases. Four of 69 (5.8%) cases in the validation cohorts (all of which were negative for PLA2R, THSD7A, EXT1, NELL1, and SEMA3B) were PCDH7-positive MN. Kidney biopsy showed minimal complement deposition in 12 of the 14 PCDH7-associated cases. Confocal microscopy showed colocalization of PCDH7 and IgG along the GBM. Western blot analysis using sera from six patients showed antibodies to nonreduced PCDH7. Elution of IgG from frozen tissue of PCDH7-associated MN showed reactivity against PCDH7. CONCLUSIONS: MN associated with the protocadherin PCDH7 appears to be a distinct, previously unidentified type of MN.


Assuntos
Caderinas/metabolismo , Glomerulonefrite Membranosa/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Glomerulonefrite Membranosa/patologia , Humanos , Microdissecção e Captura a Laser , Masculino , Espectrometria de Massas , Microscopia Confocal , Pessoa de Meia-Idade , Protocaderinas
2.
Tissue Eng Part A ; 18(23-24): 2466-76, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22738340

RESUMO

Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and α-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies.


Assuntos
Células Cultivadas/citologia , Condrócitos/citologia , Cultura Primária de Células/instrumentação , Actinas/biossíntese , Actinas/genética , Animais , Apoptose , Materiais Biocompatíveis , Bovinos , Desdiferenciação Celular , Divisão Celular , Inibição de Contato , Elasticidade , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Feminino , Glicosaminoglicanos/biossíntese , Poliestirenos , Cultura Primária de Células/métodos , RNA Mensageiro/biossíntese , Elastômeros de Silicone , Propriedades de Superfície , Transcriptoma
3.
Integr Biol (Camb) ; 4(4): 410-21, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22410748

RESUMO

Fibroblasts differentiate into the highly synthetic and contractile myofibroblast phenotype when exposed to substrates with an elastic modulus corresponding to pathologically stiff fibrotic tissue. Cellular responses to changes in substrate stiffness are typically analyzed after hours or days, which does not enable the monitoring of myofibroblast persistence, a hallmark of fibrosis. To determine long-lasting effects on the fibrotic behavior of lung fibroblasts, we followed a novel approach of explanting and repeatedly passaging fibroblasts on silicone substrates with stiffness representing various states of lung health. Fibrotic activity was determined by assaying for myofibroblast proliferation, cell contractility, expression of α-smooth muscle actin, extracellular matrix and active TGFß1. As predicted, myofibroblast activity was low on healthy soft substrates and increased with increasing substrate stiffness. However, explanting and mechanically priming lung fibroblasts for 3 weeks on pathologically stiff substrates resulted in sustained myofibroblast activity even after the cells were returned to healthy soft cultures for 2 weeks. Such primed cells retained higher fibrotic activity than cells that had been exclusively cultured on soft substrates, and were not statistically different from cells continuously passaged on stiff surfaces. Inversely, priming lung fibroblasts for 3 weeks on soft substrates partially protected from myofibroblast activation after the shift to stiff substrates. Hence, mechano-sensed information relating to physical conditions of the local cellular environment could permanently induce fibrotic behavior of lung fibroblasts. This priming effect has important implications for the progression and persistence of aggressive fibrotic diseases such as idiopathic pulmonary fibrosis.


Assuntos
Diferenciação Celular/fisiologia , Matriz Extracelular/fisiologia , Pulmão/citologia , Miofibroblastos/citologia , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Separação Celular , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/química , Meios de Cultivo Condicionados/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Elasticidade , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Dureza , Antígeno Ki-67/metabolismo , Masculino , Miofibroblastos/metabolismo , Ratos , Ratos Sprague-Dawley , Silicones/química , Silicones/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
4.
Nat Biotechnol ; 27(7): 667-70, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19561594

RESUMO

Metazoan genomes encode hundreds of RNA-binding proteins (RBPs) but RNA-binding preferences for relatively few RBPs have been well defined. Current techniques for determining RNA targets, including in vitro selection and RNA co-immunoprecipitation, require significant time and labor investment. Here we introduce RNAcompete, a method for the systematic analysis of RNA binding specificities that uses a single binding reaction to determine the relative preferences of RBPs for short RNAs that contain a complete range of k-mers in structured and unstructured RNA contexts. We tested RNAcompete by analyzing nine diverse RBPs (HuR, Vts1, FUSIP1, PTB, U1A, SF2/ASF, SLM2, RBM4 and YB1). RNAcompete identified expected and previously unknown RNA binding preferences. Using in vitro and in vivo binding data, we demonstrate that preferences for individual 7-mers identified by RNAcompete are a more accurate representation of binding activity than are conventional motif models. We anticipate that RNAcompete will be a valuable tool for the study of RNA-protein interactions.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Bases de Dados de Ácidos Nucleicos , Genoma , Dados de Sequência Molecular , RNA/química , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Curva ROC , Especificidade por Substrato
5.
Mol Cell Proteomics ; 8(1): 157-71, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18782753

RESUMO

The serine/threonine protein phosphatases are targeted to specific subcellular locations and substrates in part via interactions with a wide variety of regulatory proteins. Understanding these interactions is thus critical to understanding phosphatase function. Using an iterative affinity purification/mass spectrometry approach, we generated a high density interaction map surrounding the protein phosphatase 2A catalytic subunit. This approach recapitulated the assembly of the PP2A catalytic subunit into many different trimeric complexes but also revealed several new protein-protein interactions. Here we define a novel large multiprotein assembly, referred to as the striatin-interacting phosphatase and kinase (STRIPAK) complex. STRIPAK contains the PP2A catalytic (PP2Ac) and scaffolding (PP2A A) subunits, the striatins (PP2A regulatory B''' subunits), the striatin-associated protein Mob3, the novel proteins STRIP1 and STRIP2 (formerly FAM40A and FAM40B), the cerebral cavernous malformation 3 (CCM3) protein, and members of the germinal center kinase III family of Ste20 kinases. Although the function of the CCM3 protein is unknown, the CCM3 gene is mutated in familial cerebral cavernous malformations, a condition associated with seizures and strokes. Our proteomics survey indicates that a large portion of the CCM3 protein resides within the STRIPAK complex, opening the way for further studies of CCM3 biology. The STRIPAK assembly establishes mutually exclusive interactions with either the CTTNBP2 proteins (which interact with the cytoskeletal protein cortactin) or a second subcomplex consisting of the sarcolemmal membrane-associated protein (SLMAP) and the related coiled-coil proteins suppressor of IKKepsilon (SIKE) and FGFR1OP2. We have thus identified several novel PP2A-containing protein complexes, including a large assembly linking kinases and phosphatases to a gene mutated in human disease.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfotransferases/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas do Citoesqueleto , Células HeLa , Humanos , Proteínas de Ligação a Fosfato , Ligação Proteica
7.
J Cell Sci ; 120(Pt 7): 1189-99, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17341581

RESUMO

Caspases are crucial activators of apoptosis and NF-kappaB signaling in vertebrates and invertebrates. In Drosophila, the caspase-9 counterpart Dronc is essential for most apoptotic death, whereas the caspase-8 homolog Dredd activates NF-kappaB signaling in response to gram-negative bacterial infection. The mechanics of caspase regulation are conserved and include the activities of a family of inhibitor of apoptosis (IAP) proteins. The RING-domain-bearing protein Defense repressor 1 (Dnr1), blocks ectopic Dredd-mediated induction of an NF-kappaB reporter in the Drosophila S2 cell line. In this study, we present novel data indicating that Dnr1 impacts on Dronc-dependent regulation of the apoptotic program. We show that depletion of Dnr1 results in elevated Dronc protein levels, which translates to increased caspase activation and activity upon induction of apoptosis. Conversely, we demonstrate that overexpression of Dnr1 blocks apoptotic caspase activity and prevents induction of apoptosis in tissue culture assays. Furthermore, we show that Dnr1 overexpression significantly reduces Dronc protein levels and identify the domains of Dnr1 necessary for these effects. From these data, we propose that Dnr1 inhibits initiator caspases in S2 cells.


Assuntos
Apoptose , Caspases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Repressoras/metabolismo , Animais , Caspases/análise , Linhagem Celular , Proteínas de Drosophila/análise , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Técnica Direta de Fluorescência para Anticorpo , Genes Reporter , Proteínas Inibidoras de Apoptose/análise , Microscopia de Fluorescência , Microscopia de Vídeo , NF-kappa B/genética , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Proteínas Repressoras/análise , Proteínas Repressoras/química , Proteínas Repressoras/genética
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