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1.
J Lipid Res ; 61(3): 413-421, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31941672

RESUMO

Zinc metallopeptidase STE24 (ZMPSTE24) is essential for the conversion of farnesyl-prelamin A to mature lamin A, a key component of the nuclear lamina. In the absence of ZMPSTE24, farnesyl-prelamin A accumulates in the nucleus and exerts toxicity, causing a variety of disease phenotypes. By ∼4 months of age, both male and female Zmpste24 -/- mice manifest a near-complete loss of adipose tissue, but it has never been clear whether this phenotype is a direct consequence of farnesyl-prelamin A toxicity in adipocytes. To address this question, we generated a conditional knockout Zmpste24 allele and used it to create adipocyte-specific Zmpste24-knockout mice. To boost farnesyl-prelamin A levels, we bred in the "prelamin A-only" Lmna allele. Gene expression, immunoblotting, and immunohistochemistry experiments revealed that adipose tissue in these mice had decreased Zmpste24 expression along with strikingly increased accumulation of prelamin A. In male mice, Zmpste24 deficiency in adipocytes was accompanied by modest changes in adipose stores (an 11% decrease in body weight, a 23% decrease in body fat mass, and significantly smaller gonadal and inguinal white adipose depots). No changes in adipose stores were detected in female mice, likely because prelamin A expression in adipose tissue is lower in female mice. Zmpste24 deficiency in adipocytes did not alter the number of macrophages in adipose tissue, nor did it alter plasma levels of glucose, triglycerides, or fatty acids. We conclude that ZMPSTE24 deficiency in adipocytes, and the accompanying accumulation of farnesyl-prelamin A, reduces adipose tissue stores, but only modestly and only in male mice.

2.
Proc Natl Acad Sci U S A ; 116(51): 25870-25879, 2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31796586

RESUMO

Deficiencies in either lamin B1 or lamin B2 cause both defective migration of cortical neurons in the developing brain and reduced neuronal survival. The neuronal migration abnormality is explained by a weakened nuclear lamina that interferes with nucleokinesis, a nuclear translocation process required for neuronal migration. In contrast, the explanation for impaired neuronal survival is poorly understood. We hypothesized that the forces imparted on the nucleus during neuronal migration result in nuclear membrane (NM) ruptures, causing interspersion of nuclear and cytoplasmic contents-and ultimately cell death. To test this hypothesis, we bred Lmnb1-deficient mice that express a nuclear-localized fluorescent Cre reporter. Migrating neurons within the cortical plate of E18.5 Lmnb1-deficient embryos exhibited NM ruptures, evident by the escape of the nuclear-localized reporter into the cytoplasm and NM discontinuities by electron microscopy. The NM ruptures were accompanied by DNA damage and cell death. The NM ruptures were not observed in nonmigrating cells within the ventricular zone. NM ruptures, DNA damage, and cell death were also observed in cultured Lmnb1 -/- and Lmnb2 -/- neurons as they migrated away from neurospheres. To test whether mechanical forces on the cell nucleus are relevant to NM ruptures in migrating neurons, we examined cultured Lmnb1 -/- neurons when exposed to external constrictive forces (migration into a field of tightly spaced silicon pillars). As the cells entered the field of pillars, there were frequent NM ruptures, accompanied by DNA damage and cell death.

3.
Elife ; 82019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31486771

RESUMO

Cultured mouse peritoneal macrophages release large numbers of ~30-nm cholesterol-rich particles. Here, we show that those particles represent fragments of the plasma membrane that are pulled away and left behind during the projection and retraction of filopodia and lamellipodia. Consistent with this finding, the particles are enriched in proteins found in focal adhesions, which attach macrophages to the substrate. The release of particles is abolished by blocking cell movement (either by depolymerizing actin with latrunculin A or by inhibiting myosin II with blebbistatin). Confocal microscopy and NanoSIMS imaging studies revealed that the plasma membrane-derived particles are enriched in 'accessible cholesterol' (a mobile pool of cholesterol detectable with the modified cytolysin ALO-D4) but not in sphingolipid-sequestered cholesterol [a pool detectable with ostreolysin A (OlyA)]. The discovery that macrophages release cholesterol-rich particles during cellular locomotion is likely relevant to cholesterol efflux and could contribute to extracellular cholesterol deposition in atherosclerotic plaques.


Assuntos
Membrana Celular/metabolismo , Movimento Celular , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/metabolismo , Colesterol/análise , Macrófagos Peritoneais/metabolismo , Pseudópodes/metabolismo , Animais , Células Cultivadas , Camundongos , Proteínas/análise
4.
Elife ; 82019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31169500

RESUMO

GPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) within the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1-bound LPL is essential for the margination of triglyceride-rich lipoproteins (TRLs) along capillaries, allowing the lipolytic processing of TRLs to proceed. In peripheral tissues, the intravascular processing of TRLs by the GPIHBP1-LPL complex is crucial for the generation of lipid nutrients for adjacent parenchymal cells. GPIHBP1 is absent from the capillaries of the brain, which uses glucose for fuel; however, GPIHBP1 is expressed in the capillaries of mouse and human gliomas. Importantly, the GPIHBP1 in glioma capillaries captures locally produced LPL. We use NanoSIMS imaging to show that TRLs marginate along glioma capillaries and that there is uptake of TRL-derived lipid nutrients by surrounding glioma cells. Thus, GPIHBP1 expression in gliomas facilitates TRL processing and provides a source of lipid nutrients for glioma cells.


Assuntos
Glioma/metabolismo , Lipoproteínas/metabolismo , Receptores de Lipoproteínas/metabolismo , Animais , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Capilares/metabolismo , Isótopos de Carbono/metabolismo , Células Endoteliais/metabolismo , Ácidos Graxos/metabolismo , Glioma/irrigação sanguínea , Glioma/patologia , Glioma/ultraestrutura , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Humanos , Lipase Lipoproteica/metabolismo , Camundongos Endogâmicos C57BL , Triglicerídeos/metabolismo
5.
Proc Natl Acad Sci U S A ; 116(10): 4307-4315, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30765529

RESUMO

The nuclear lamina is an intermediate filament meshwork adjacent to the inner nuclear membrane (INM) that plays a critical role in maintaining nuclear shape and regulating gene expression through chromatin interactions. Studies have demonstrated that A- and B-type lamins, the filamentous proteins that make up the nuclear lamina, form independent but interacting networks. However, whether these lamin subtypes exhibit a distinct spatial organization or whether their organization has any functional consequences is unknown. Using stochastic optical reconstruction microscopy (STORM) our studies reveal that lamin B1 and lamin A/C form concentric but overlapping networks, with lamin B1 forming the outer concentric ring located adjacent to the INM. The more peripheral localization of lamin B1 is mediated by its carboxyl-terminal farnesyl group. Lamin B1 localization is also curvature- and strain-dependent, while the localization of lamin A/C is not. We also show that lamin B1's outer-facing localization stabilizes nuclear shape by restraining outward protrusions of the lamin A/C network. These two findings, that lamin B1 forms an outer concentric ring and that its localization is energy-dependent, are significant as they suggest a distinct model for the nuclear lamina-one that is able to predict its behavior and clarifies the distinct roles of individual nuclear lamin proteins and the consequences of their perturbation.

6.
J Lipid Res ; 60(4): 869-879, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30598475

RESUMO

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1 Enh/Enh). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia-even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1 Enh/- mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.

7.
Prev Vet Med ; 159: 51-56, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30314790

RESUMO

Pseudorabies (PR), also known as Aujeszky's disease, is a highly contagious disease affecting pigs and a wide range of animals. Pseudorabies is enzootic in many countries. In China, it is a priority animal disease for control and eradication, however the data on disease frequency in intensive pig farms and the information on associated risk factors is inadequate. A cross-sectional study of intensive pig farms (≥350 sows) in Shanghai was conducted to determine herd-level prevalence of PRV and associated risk factors. Following a two-stage random sampling design, a total of 1349 sow serum samples were tested by gpI-ELISA from a total of 91 intensive pig farms in Shanghai. A herd was classified as positive if at least one PRV test-positive sow was present. Information on putative risk/protective factors was collected using questionnaires to pig farm owners or veterinarians. A logistic regression model was built to identify risk/protective factors for herd positivity. The results indicated that the herd-level true prevalence was 67.6% (95% CI:57.0-77.0). In the multivariable logistic regression model using backward stepwise procedure, two risk factors were found to be significantly associated with herd positivity: 'Breeding with introduced sows in the last 12 months' (OR = 3.5, 95%CI:1.2, 10.3) and 'Presence of stray dogs or cats' (OR = 4.0, 95%CI: 1.2, 12.6). The multivariable logistic model fitted the data well. Hosmer-Lemeshow goodness of fit test showed χ2 = 10.86 (df = 8, p = 0.21 > 0.05) and the predictability (area under the ROC curve) was 0.86. This study suggested that PR was highly endemic in intensive pig farms in Shanghai. The risk and protective factors identified in this study could be useful to improve the prevention policy of PR in Shanghai and other areas of China.


Assuntos
Criação de Animais Domésticos/métodos , Pseudorraiva/epidemiologia , Doenças dos Suínos/epidemiologia , Animais , China/epidemiologia , Estudos Transversais , Feminino , Prevalência , Fatores de Risco , Suínos
8.
Biochem Biophys Res Commun ; 504(4): 899-902, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30224066

RESUMO

Heterogeneity in the metabolic properties of adipocytes in white adipose tissue has been well documented. We sought to investigate metabolic heterogeneity in adipocytes of brown adipose tissue (BAT), focusing on heterogeneity in nutrient uptake. To explore the possibility of metabolic heterogeneity in brown adipocytes, we used nanoscale secondary ion mass spectrometry (NanoSIMS) to quantify uptake of lipids in adipocytes interscapular BAT and perivascular adipose tissue (PVAT) after an intravenous injection of triglyceride-rich lipoproteins (TRLs) containing [2H]triglycerides (2H-TRLs). The uptake of deuterated lipids into brown adipocytes was quantified by NanoSIMS. We also examined 13C enrichment in brown adipocytes after administering [13C]glucose or 13C-labeled mixed fatty acids by gastric gavage. The uptake of 2H-TRLs-derived lipids into brown adipocytes was heterogeneous, with 2H enrichment in adjacent adipocytes varying by more than fourfold. We also observed substantial heterogeneity in 13C enrichment in adjacent brown adipocytes after administering [13C]glucose or [13C]fatty acids by gastric gavage. The uptake of nutrients by adjacent brown adipocytes within a single depot is variable, suggesting that there is heterogeneity in the metabolic properties of brown adipocytes.


Assuntos
Adipócitos Marrons/metabolismo , Nutrientes/farmacocinética , Espectrometria de Massa de Íon Secundário/métodos , Animais , Isótopos de Carbono/análise , Ácidos Graxos/farmacocinética , Glucose/farmacocinética , Lipídeos/farmacocinética , Lipoproteínas/administração & dosagem , Lipoproteínas/farmacocinética , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptores de Lipoproteínas/genética
9.
Sci Transl Med ; 10(460)2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30257952

RESUMO

Hutchinson-Gilford progeria syndrome is a disorder of premature aging in children caused by de novo mutations in LMNA that lead to the synthesis of an internally truncated form of prelamin A (commonly called progerin). The production of progerin causes multiple disease phenotypes, including an unusual vascular phenotype characterized by the loss of smooth muscle cells in the arterial media and fibrosis of the adventitia. We show that progerin expression, combined with mechanical stress, promotes smooth muscle cell death. Disrupting the linker of the nucleoskeleton and cytoskeleton (LINC) complex in smooth muscle cells ameliorates the toxic effects of progerin on smooth muscle cells and limits the accompanying adventitial fibrosis.


Assuntos
Doenças da Aorta/complicações , Complexos Multiproteicos/metabolismo , Miócitos de Músculo Liso/metabolismo , Progéria/complicações , Progéria/metabolismo , Túnica Adventícia/metabolismo , Túnica Adventícia/patologia , Animais , Aorta/metabolismo , Aorta/patologia , Morte Celular , Células Cultivadas , Colágeno Tipo VIII/biossíntese , Modelos Animais de Doenças , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Camundongos , Miócitos de Músculo Liso/ultraestrutura , Fenótipo
10.
Proc Natl Acad Sci U S A ; 115(40): 10100-10105, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30224463

RESUMO

The nuclear lamina, an intermediate filament meshwork lining the inner nuclear membrane, is formed by the nuclear lamins (lamins A, C, B1, and B2). Defects or deficiencies in individual nuclear lamin proteins have been reported to elicit nuclear blebs (protrusions or outpouchings of the nuclear envelope) and increase susceptibility for nuclear membrane ruptures. It is unclear, however, how a complete absence of nuclear lamins would affect nuclear envelope morphology and nuclear membrane integrity (i.e., whether nuclear membrane blebs or protrusions would occur and, if not, whether cells would be susceptible to nuclear membrane ruptures). To address these issues, we generated mouse embryonic fibroblasts (MEFs) lacking all nuclear lamins. The nuclear lamin-deficient MEFs had irregular nuclear shapes but no nuclear blebs or protrusions. Despite a virtual absence of nuclear blebs, MEFs lacking nuclear lamins had frequent, prolonged, and occasionally nonhealing nuclear membrane ruptures. By transmission electron microscopy, the inner nuclear membrane in nuclear lamin-deficient MEFs have a "wavy" appearance, and there were discrete discontinuities in the inner and outer nuclear membranes. Nuclear membrane ruptures were accompanied by a large increase in DNA damage, as judged by γ-H2AX foci. Mechanical stress increased both nuclear membrane ruptures and DNA damage, whereas minimizing transmission of cytoskeletal forces to the nucleus had the opposite effects.


Assuntos
Dano ao DNA , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Laminas/deficiência , Membrana Nuclear/metabolismo , Estresse Mecânico , Animais , Embrião de Mamíferos/ultraestrutura , Fibroblastos/ultraestrutura , Camundongos , Camundongos Knockout , Membrana Nuclear/genética , Membrana Nuclear/ultraestrutura
11.
J Lipid Res ; 59(4): 706-713, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29449313

RESUMO

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), an endothelial cell protein, binds LPL in the subendothelial spaces and transports it to the capillary lumen. In Gpihbp1-/- mice, LPL remains stranded in the subendothelial spaces, causing hypertriglyceridemia, but how Gpihbp1-/- mice respond to metabolic stress (e.g., cold exposure) has never been studied. In wild-type mice, cold exposure increases LPL-mediated processing of triglyceride-rich lipoproteins (TRLs) in brown adipose tissue (BAT), providing fuel for thermogenesis and leading to lower plasma triglyceride levels. We suspected that defective TRL processing in Gpihbp1-/- mice might impair thermogenesis and blunt the fall in plasma triglyceride levels. Indeed, Gpihbp1-/- mice exhibited cold intolerance, but the effects on plasma triglyceride levels were paradoxical. Rather than falling, the plasma triglyceride levels increased sharply (from ∼4,000 to ∼15,000 mg/dl), likely because fatty acid release by peripheral tissues drives hepatic production of TRLs that cannot be processed. We predicted that the sharp increase in plasma triglyceride levels would not occur in Gpihbp1-/-Angptl4-/- mice, where LPL activity is higher and baseline plasma triglyceride levels are lower. Indeed, the plasma triglyceride levels in Gpihbp1-/-Angptl4-/- mice fell during cold exposure. Metabolic studies revealed increased levels of TRL processing in the BAT of Gpihbp1-/-Angptl4-/- mice.


Assuntos
Temperatura Baixa , Receptores de Lipoproteínas/sangue , Receptores de Lipoproteínas/deficiência , Termogênese , Triglicerídeos/sangue , Animais , Apolipoproteínas B/sangue , Camundongos , Camundongos Knockout
13.
JCI Insight ; 2(20)2017 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-29046479

RESUMO

In mammals, GPIHBP1 is absolutely essential for transporting lipoprotein lipase (LPL) to the lumen of capillaries, where it hydrolyzes the triglycerides in triglyceride-rich lipoproteins. In all lower vertebrate species (e.g., birds, amphibians, reptiles, fish), a gene for LPL can be found easily, but a gene for GPIHBP1 has never been found. The obvious question is whether the LPL in lower vertebrates is able to reach the capillary lumen. Using purified antibodies against chicken LPL, we showed that LPL is present on capillary endothelial cells of chicken heart and adipose tissue, colocalizing with von Willebrand factor. When the antibodies against chicken LPL were injected intravenously into chickens, they bound to LPL on the luminal surface of capillaries in heart and adipose tissue. LPL was released rapidly from chicken hearts with an infusion of heparin, consistent with LPL being located inside blood vessels. Remarkably, chicken LPL bound in a specific fashion to mammalian GPIHBP1. However, we could not identify a gene for GPIHBP1 in the chicken genome, nor could we identify a transcript for GPIHBP1 in a large chicken RNA-seq data set. We conclude that LPL reaches the capillary lumen in chickens - as it does in mammals - despite an apparent absence of GPIHBP1.


Assuntos
Capilares/metabolismo , Galinhas/metabolismo , Lipase Lipoproteica/metabolismo , Receptores de Lipoproteínas/metabolismo , Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/metabolismo , Animais , Anticorpos , Células Endoteliais/metabolismo , Feminino , Cabras , Coração , Heparina , Humanos , Imunoglobulina G , Metabolismo dos Lipídeos , Lipase Lipoproteica/genética , Lipoproteínas/metabolismo , Masculino , Camundongos , Receptores de Lipoproteínas/análise , Receptores de Lipoproteínas/genética , Triglicerídeos/metabolismo
14.
J Lipid Res ; 58(7): 1453-1461, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28476858

RESUMO

Mutation of conserved cysteines in proteins of the Ly6 family cause human disease-chylomicronemia in the case of glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) and paroxysmal nocturnal hemoglobinuria in the case of CD59. A mutation in a conserved cysteine in CD59 prevented the protein from reaching the surface of blood cells. In contrast, mutation of conserved cysteines in human GPIHBP1 had little effect on GPIHBP1 trafficking to the surface of cultured CHO cells. The latter findings were somewhat surprising and raised questions about whether CHO cell studies accurately model the fate of mutant GPIHBP1 proteins in vivo. To explore this concern, we created mice harboring a GPIHBP1 cysteine mutation (p.C63Y). The p.C63Y mutation abolished the ability of mouse GPIHBP1 to bind LPL, resulting in severe chylomicronemia. The mutant GPIHBP1 was detectable by immunohistochemistry on the surface of endothelial cells, but the level of expression was ∼70% lower than in WT mice. The mutant GPIHBP1 protein in mouse tissues was predominantly monomeric. We conclude that mutation of a conserved cysteine in GPIHBP1 abolishes the ability of GPIHBP1 to bind LPL, resulting in mislocalization of LPL and severe chylomicronemia. The mutation reduced but did not eliminate GPIHBP1 on the surface of endothelial cells in vivo.


Assuntos
Sequência Conservada , Cisteína , Lipase Lipoproteica/metabolismo , Mutação , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Feminino , Humanos , Lipase Lipoproteica/genética , Camundongos , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Lipoproteínas/genética , Triglicerídeos/sangue
15.
Exp Dermatol ; 26(11): 1134-1136, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28418591

RESUMO

Mutations in SLURP1, a secreted protein of keratinocytes, cause a palmoplantar keratoderma (PPK) known as mal de Meleda. Slurp1 deficiency in mice faithfully recapitulates the human disease, with increased keratinocyte proliferation and thickening of the epidermis on the volar surface of the paws. There has long been speculation that SLURP1 serves as a ligand for a receptor that regulates keratinocyte growth and differentiation. We were intrigued that mutations leading to increased signalling through the epidermal growth factor receptor (EGFR) cause PPK. Here, we sought to determine whether reducing EGFR signalling would ameliorate the PPK associated with SLURP1 deficiency. To address this issue, we bred Slurp1-deficient mice that were homozygous for a hypomorphic Egfr allele. The hypomorphic Egfr allele, which leads to reduced EGFR signalling in keratinocytes, did not ameliorate the PPK elicited by SLURP1 deficiency, suggesting that SLURP1 deficiency causes PPK independently (or downstream) from the EGFR pathway.


Assuntos
Antígenos Ly/genética , Antígenos Ly/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ceratodermia Palmar e Plantar/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Alelos , Animais , Genótipo , Ceratodermia Palmar e Plantar/patologia , Masculino , Camundongos Knockout , Fenótipo , Transdução de Sinais/genética , Ativador de Plasminogênio Tipo Uroquinase/deficiência
16.
J Lipid Res ; 58(1): 208-215, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27875259

RESUMO

GPIHBP1, an endothelial cell protein, binds LPL in the interstitial spaces and shuttles it to its site of action inside blood vessels. For years, studies of human GPIHBP1 have been hampered by an absence of useful antibodies. We reasoned that monoclonal antibodies (mAbs) against human GPIHBP1 would be useful for 1) defining the functional relevance of GPIHBP1's Ly6 and acidic domains to the binding of LPL; 2) ascertaining whether human GPIHBP1 is expressed exclusively in capillary endothelial cells; and 3) testing whether GPIHBP1 is detectable in human plasma. Here, we report the development of a panel of human GPIHBP1-specific mAbs. Two mAbs against GPIHBP1's Ly6 domain, RE3 and RG3, abolished LPL binding, whereas an antibody against the acidic domain, RF4, did not. Also, mAbs RE3 and RG3 bound with reduced affinity to a mutant GPIHBP1 containing an Ly6 domain mutation (W109S) that abolishes LPL binding. Immunohistochemistry studies with the GPIHBP1 mAbs revealed that human GPIHBP1 is expressed only in capillary endothelial cells. Finally, we created an ELISA that detects GPIHBP1 in human plasma. That ELISA should make it possible for clinical lipidologists to determine whether plasma GPIHBP1 levels are a useful biomarker of metabolic or vascular disease.


Assuntos
Anticorpos Monoclonais/imunologia , Lipase Lipoproteica/imunologia , Receptores de Lipoproteínas/imunologia , Triglicerídeos/metabolismo , Animais , Sítios de Ligação/imunologia , Linhagem Celular , Drosophila , Células Endoteliais/enzimologia , Células Endoteliais/imunologia , Humanos , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/isolamento & purificação , Camundongos , Receptores de Lipoproteínas/genética , Triglicerídeos/imunologia
17.
Nucleus ; 7(5): 512-521, 2016 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-27841971

RESUMO

A variety of missense mutations in LMNA (the gene for lamin C and prelamin A) cause familial partial lipodystrophy (FPLD), a disease associated with reduced adipose tissue, particularly in the limbs. Several studies have reported that fibroblasts from FPLD subjects have an accumulation of prelamin A. Those findings were intriguing but also perplexing because many of the LMNA missense mutations associated with lipodystrophy are located in sequences distant from the sequences required for the farnesylation of prelamin A and ZMPSTE24-mediated conversion of prelamin A to mature lamin A. Here, we revisited the issue of prelamin A accumulation in the setting of FPLD mutations. We used western blots with lamin A/C antibodies and prelamin A-specific monoclonal antibodies to assess prelamin A levels in wild-type fibroblasts and fibroblasts carrying LMNA mutations associated with lipodystrophy (R482W, I299V, C591F, T528M). None of the mutant fibroblasts exhibited an accumulation of prelamin A. Also, the amount of prelamin A accumulation in response to lopinavir (an inhibitor of ZMPSTE24) was similar in wild-type and mutant fibroblasts. Thus, the LMNA lipodystrophy mutations that we examined did not lead to prelamin A accumulation, nor did they render those cells more susceptible to prelamin A accumulation when ZMPSTE24 was inhibited by lopinavir.


Assuntos
Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Mutação de Sentido Incorreto , Sequência de Bases , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Lipodistrofia Parcial Familiar/genética , Lipodistrofia Parcial Familiar/patologia , Lopinavir/farmacologia
18.
J Clin Invest ; 126(4): 1592-602, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26999604

RESUMO

The alternatively spliced products of LMNA, lamin C and prelamin A (the precursor to lamin A), are produced in similar amounts in most tissues and have largely redundant functions. This redundancy suggests that diseases, such as Hutchinson-Gilford progeria syndrome (HGPS), that are caused by prelamin A-specific mutations could be treated by shifting the output of LMNA more toward lamin C. Here, we investigated mechanisms that regulate LMNA mRNA alternative splicing and assessed the feasibility of reducing prelamin A expression in vivo. We identified an exon 11 antisense oligonucleotide (ASO) that increased lamin C production at the expense of prelamin A when transfected into mouse and human fibroblasts. The same ASO also reduced the expression of progerin, the mutant prelamin A protein in HGPS, in fibroblasts derived from patients with HGPS. Mechanistic studies revealed that the exon 11 sequences contain binding sites for serine/arginine-rich splicing factor 2 (SRSF2), and SRSF2 knockdown lowered lamin A production in cells and in murine tissues. Moreover, administration of the exon 11 ASO reduced lamin A expression in wild-type mice and progerin expression in an HGPS mouse model. Together, these studies identify ASO-mediated reduction of prelamin A as a potential strategy to treat prelamin A-specific diseases.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Lamina Tipo A/biossíntese , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Progéria/tratamento farmacológico , Progéria/metabolismo , RNA Mensageiro/metabolismo , Animais , Modelos Animais de Doenças , Éxons , Técnicas de Silenciamento de Genes , Humanos , Lamina Tipo A/genética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos Antissenso/genética , Progéria/genética , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Fatores de Processamento de Serina-Arginina
19.
J Invest Dermatol ; 136(2): 436-443, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26967477

RESUMO

SLURP1, a member of the lymphocyte antigen 6 protein family, is secreted by suprabasal keratinocytes. Mutations in SLURP1 cause a palmoplantar keratoderma (PPK) known as mal de Meleda. SLURP2, another secreted lymphocyte antigen 6 protein, is encoded by a gene located ?20 kb downstream from SLURP1. SLURP2 is produced by suprabasal keratinocytes. To investigate the importance of SLURP2, we first examined Slurp2 knockout mice in which exon 2-3 sequences had been replaced with lacZ and neo cassettes. Slurp2(-/-) mice exhibited hyperkeratosis on the volar surface of the paws (i.e., palmoplantar keratoderma), increased keratinocyte proliferation, and an accumulation of lipid droplets in the stratum corneum. They also exhibited reduced body weight and hind limb clasping. These phenotypes are similar to those of Slurp1(-/-) mice. To solidify a link between Slurp2 deficiency and palmoplantar keratoderma and to be confident that the disease phenotypes in Slurp2(-/-) mice were not secondary to the effects of the lacZ and neo cassettes on Slurp1 expression, we created a new line of Slurp2 knockout mice (Slurp2X(-/-)) in which Slurp2 was inactivated with a simple nonsense mutation. Slurp2X(-/-) mice exhibited the same disease phenotypes. Thus, Slurp2 deficiency and Slurp1 deficiencies cause the same disease phenotypes.


Assuntos
Antígenos Ly/genética , Códon sem Sentido , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica , Ceratodermia Palmar e Plantar/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Proteínas Ligadas por GPI/deficiência , Imuno-Histoquímica , Queratinócitos/citologia , Queratinócitos/metabolismo , Ceratodermia Palmar e Plantar/patologia , Camundongos , Camundongos Knockout , Fenótipo , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/métodos
20.
Hum Mol Genet ; 24(10): 2826-40, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25652409

RESUMO

Neurons in the brain produce lamin C but almost no lamin A, a consequence of the removal of prelamin A transcripts by miR-9, a brain-specific microRNA. We have proposed that miR-9-mediated regulation of prelamin A in the brain could explain the absence of primary neurological disease in Hutchinson-Gilford progeria syndrome, a genetic disease caused by the synthesis of an internally truncated form of farnesyl-prelamin A (progerin). This explanation makes sense, but it is not entirely satisfying because it is unclear whether progerin-even if were expressed in neurons-would be capable of eliciting neuropathology. To address that issue, we created a new Lmna knock-in allele, Lmna(HG-C), which produces progerin transcripts lacking an miR-9 binding site. Mice harboring the Lmna(HG-C) allele produced progerin in neurons, but they had no pathology in the central nervous system. However, these mice invariably developed esophageal achalasia, and the enteric neurons and nerve fibers in gastrointestinal tract were markedly abnormal. The same disorder, achalasia, was observed in genetically modified mice that express full-length farnesyl-prelamin A in neurons (Zmpste24-deficient mice carrying two copies of a Lmna knock-in allele yielding full-length prelamin A transcripts lacking a miR-9 binding site). Our findings indicate that progerin and full-length farnesyl-prelamin A are toxic to neurons of the enteric nervous system.


Assuntos
Sistema Nervoso Entérico/patologia , Acalasia Esofágica/genética , Lamina Tipo A/genética , Neurônios/metabolismo , Prenilação de Proteína , Animais , Acalasia Esofágica/patologia , Feminino , Técnicas de Introdução de Genes , Lamina Tipo A/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/metabolismo , Mutação , Neurônios/patologia , Interferência de RNA
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