RESUMEN
Chromatin fibers must fold or coil in the process of chromosome condensation. Patterns of coiling have been demonstrated for reconstituted chromatin, but the actual trajectories of fibers in condensed states of chromosomes could not be visualized because of the high density of the material. We have exploited partial decondensation of mitotic chromosomes to reveal their internal structure at sub-nucleosomal resolution by cryo-electron tomography, without the use of stains, fixatives, milling, or sectioning. DNA gyres around nucleosomes were visible, allowing the nucleosomes to be identified and their orientations to be determined. Linker DNA regions were traced, revealing the trajectories of the chromatin fibers. The trajectories were irregular, with almost no evidence of coiling and no short- or long-range order of the chromosomal material. The 146-bp core particle, long known as a product of nuclease digestion, is identified as the native state of the nucleosome, with no regular spacing along the chromatin fibers.
Asunto(s)
Cromosomas/ultraestructura , ADN/química , Mitosis , Nucleosomas/metabolismo , Secuencias de Aminoácidos , Cromatina/química , Microscopía por Crioelectrón , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Histonas/química , Humanos , Microscopía Fluorescente , Nucleosomas/química , Espermidina/química , TomografíaRESUMEN
We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Evolución Biológica , Cromosomas/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Eucariontes/genética , Genoma , Complejos Multiproteicos/genética , Complejos Multiproteicos/fisiología , Adenosina Trifosfatasas/química , Algoritmos , Animales , Nucléolo Celular/ultraestructura , Núcleo Celular/ultraestructura , Centrómero/ultraestructura , Cromosomas/química , Cromosomas Humanos/química , Cromosomas Humanos/ultraestructura , Proteínas de Unión al ADN/química , Genoma Humano , Genómica , Heterocromatina/ultraestructura , Humanos , Interfase , Mitosis , Modelos Biológicos , Complejos Multiproteicos/química , Telómero/ultraestructuraRESUMEN
BACKGROUND: Dermatomyositis, an inflammatory myopathy with cutaneous involvement, is associated with malignancy and often manifests paraneoplastically. While co-occurrence with small cell carcinoma is well attested, primary lung adenocarcinoma, which may present as focal ground-glass opacification on computed tomography of the thorax, is less frequently coincident. CASE PRESENTATION: We report the case of a 72-year-old female patient with dermatomyositis - treated with a combination of prednisone, methotrexate, and intravenous immunoglobulin - and an indolent, subsolid, non-hypermetabolic pulmonary lesion, which was determined to be invasive primary lung adenocarcinoma. Supporting a paraneoplastic basis, immunosuppressive therapy was discontinued following tumor excision without relapse of signs or symptoms of dermatomyositis. CONCLUSIONS: While dermatomyositis prodromal to lung adenocarcinoma is not without precedent, association with an indolent, subsolid lesion has, to the best of our knowledge, not been reported. The case described herein illustrates the importance of maintaining a high index of suspicion for malignancy in the setting of dermatomyositis.
Asunto(s)
Adenocarcinoma/diagnóstico por imagen , Dermatomiositis/etiología , Neoplasias Pulmonares/diagnóstico por imagen , Síndromes Paraneoplásicos/etiología , Tomografía Computarizada por Rayos X , Adenocarcinoma/complicaciones , Adenocarcinoma del Pulmón , Anciano , Femenino , Humanos , Pulmón/diagnóstico por imagen , Neoplasias Pulmonares/complicaciones , Recurrencia Local de NeoplasiaRESUMEN
C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-ß polypeptides, which are associated with Alzheimer's disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded "N-helix" followed by a short "N-loop" connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer's therapeutics.
Asunto(s)
Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Colesterol/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Micelas , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
It is generally believed that cholesterol homoeostasis in the brain is both linked to and impacted by Alzheimer's disease (AD). For example, elevated levels of cholesterol in neuronal plasma and endosome membranes appear to be a pro-amyloidogenic factor. The recent observation that the C-terminal transmembrane domain (C99, also known as the beta-C-terminal fragment, or beta-CTF) of the amyloid precursor protein (APP) specifically binds cholesterol helps to tie together previously loose ends in the web of our understanding of Alzheimer's-cholesterol relationships. In particular, binding of cholesterol to C99 appears to favor the amyloidogenic pathway in cells by promoting localization of C99 in lipid rafts. In turn, the products of this pathway-amyloid-beta and the intracellular domain of the APP (AICD)-may down-regulate ApoE-mediated cholesterol uptake and cholesterol biosynthesis. If confirmed, this negative-feedback loop for membrane cholesterol levels has implications for understanding the function of the APP and for devising anti-amyloidogenic preventive strategies for AD.
Asunto(s)
Enfermedad de Alzheimer/etiología , Precursor de Proteína beta-Amiloide/metabolismo , Colesterol/metabolismo , Lípidos/fisiología , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Metabolismo de los Lípidos/fisiología , Modelos Biológicos , Datos de Secuencia Molecular , Unión Proteica/fisiologíaRESUMEN
Though challenging, solution NMR spectroscopy allows fundamental interrogation of the structure and dynamics of membrane proteins. One major technical hurdle in studies of helical membrane proteins by NMR is the difficulty of obtaining sufficient long range NOEs to determine tertiary structure. For this reason, long range distance information is sometimes sought through measurement of paramagnetic relaxation enhancements (PRE) of NMR nuclei as a function of distance from an introduced paramagnetic probe. Current PRE interpretation is based on the assumption of Lorentzian resonance lineshapes. However, in order to optimize spectral resolution, modern multidimensional NMR spectra are almost always subjected to resolution-enhancement, leading to distortions in the Lorentizian peak shape. Here it is shown that when PREs are derived using peak intensities (i.e., peak height) and linewidths from both real and simulated spectra that were produced using a wide range of apodization/window functions, that there is little variation in the distances determined (<1 A at the extremes). This indicates that the high degree of resolution enhancement required to obtain well-resolved spectra from helical membrane proteins is compatible with the use of PRE data as a source of distance restraints. While these conclusions are particularly important for helical membrane proteins, they are generally applicable to all PRE measurements made using resolution-enhanced data.
Asunto(s)
Antígenos CD/química , Moléculas de Adhesión Celular/química , Resonancia Magnética Nuclear Biomolecular/métodos , Canales de Potasio con Entrada de Voltaje/química , Antígeno 12E7 , Animales , Antígenos CD/genética , Moléculas de Adhesión Celular/genética , Humanos , Canales de Potasio con Entrada de Voltaje/genética , Estructura Secundaria de Proteína/fisiologíaRESUMEN
Evidence that certain gamma-secretase modulators (GSMs) target the 99-residue C-terminal domain (C99) of the amyloid precursor protein, a substrate of gamma-secretase, but not the protease complex itself has been presented [Kukar, T. L., et al. (2008) Nature 453, 925-929]. Here, NMR results demonstrate a lack of specific binding of these GSMs to monodisperse C99 in LMPG micelles. In addition, results indicate that C99 was likely to have been aggregated in some of the key experiments of the previous work and that binding of GSMs to these C99 aggregates is also of a nonspecific nature.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/química , Precursor de Proteína beta-Amiloide/química , Humanos , Micelas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Unión Proteica , Especificidad por SustratoRESUMEN
The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.