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


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.

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


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.

Proc Natl Acad Sci U S A ; 116(10): 4307-4315, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30765529


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.

Proc Natl Acad Sci U S A ; 115(40): 10100-10105, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30224463


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.

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
MAbs ; 6(2): 460-73, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24492306


Viral entry targets with therapeutic neutralizing potential are subject to multiple escape mechanisms, including antigenic drift, immune dominance of functionally irrelevant epitopes, and subtle variations in host cell mechanisms. A surprising finding of recent years is that potent neutralizing antibodies to viral epitopes independent of strain exist, but are poorly represented across the diverse human population. Identifying these antibodies and understanding the biology mediating the specific immune response is thus difficult. An effective strategy for meeting this challenge is to incorporate multiplexed antigen screening into a high throughput survey of the memory B cell repertoire from immune individuals. We used this approach to discover suites of cross-clade antibodies directed to conformational epitopes in the stalk region of the influenza A hemagglutinin (HA) protein and to select high-affinity anti-peptide antibodies to the glycoprotein B (gB) of human cytomegalovirus. In each case, our screens revealed a restricted VH and VL germline usage, including published and previously unidentified gene families. The in vivo evolution of paratope specificity with optimal neutralizing activity was understandable after correlating biological activities with kinetic binding and epitope recognition. Iterative feedback between antigen probe design based on structure and function information with high throughput multiplexed screening demonstrated a generally applicable strategy for efficient identification of safe, native, finely tuned antibodies with the potential for high genetic barriers to viral escape.

Anticorpos Bloqueadores/metabolismo , Antígenos Virais/metabolismo , Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Epitopos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Proteínas do Envelope Viral/metabolismo , Anticorpos Bloqueadores/imunologia , Afinidade de Anticorpos , Antígenos Virais/imunologia , Linhagem Celular , Infecções por Citomegalovirus/terapia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Ensaios de Triagem em Larga Escala , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Imunidade Humoral , Imunidade Inata , Memória Imunológica , Influenza Humana/terapia , Conformação Proteica , Proteínas do Envelope Viral/imunologia