RESUMEN
In patients infected with the human immunodeficiency virus (HIV), the HIV-Tat protein may be continually produced despite adequate antiretroviral therapy. As the HIV-infected population is aging, it is becoming increasingly important to understand how HIV-Tat may interact with proteins such as amyloid ß and Tau which accumulate in the aging brain and eventually result in Alzheimer's disease. In this review, we examine the in vivo data from HIV-infected patients and animal models and the in vitro experiments that show how protein complexes between HIV-Tat and amyloid ß occur through novel protein-protein interactions and how HIV-Tat may influence the pathways for amyloid ß production, degradation, phagocytosis, and transport. HIV-Tat may also induce Tau phosphorylation through a cascade of cellular processes that lead to the formation of neurofibrillary tangles, another hallmark of Alzheimer's disease. We also identify gaps in knowledge and future directions for research. Available evidence suggests that HIV-Tat may accelerate Alzheimer-like pathology in patients with HIV infection which cannot be impacted by current antiretroviral therapy.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Infecciones por VIH/metabolismo , Fragmentos de Péptidos/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Animales , Fármacos Anti-VIH/uso terapéutico , Química Encefálica , Espacio Extracelular , Infecciones por VIH/tratamiento farmacológico , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/química , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , Ratones , Modelos Moleculares , Neprilisina/química , Neprilisina/metabolismo , Ovillos Neurofibrilares/metabolismo , Neuroglía/metabolismo , Neuroglía/virología , Fragmentos de Péptidos/química , Fagocitosis , Fosforilación , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Relación Estructura-Actividad , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/toxicidad , Proteínas tau/químicaRESUMEN
Nodding syndrome is an epileptic disorder of unknown etiology that occurs in children in East Africa. There is an epidemiological association with Onchocerca volvulus, the parasitic worm that causes onchocerciasis (river blindness), but there is limited evidence that the parasite itself is neuroinvasive. We hypothesized that nodding syndrome may be an autoimmune-mediated disease. Using protein chip methodology, we detected autoantibodies to leiomodin-1 more abundantly in patients with nodding syndrome compared to unaffected controls from the same village. Leiomodin-1 autoantibodies were found in both the sera and cerebrospinal fluid of patients with nodding syndrome. Leiomodin-1 was found to be expressed in mature and developing human neurons in vitro and was localized in mouse brain to the CA3 region of the hippocampus, Purkinje cells in the cerebellum, and cortical neurons, structures that also appear to be affected in patients with nodding syndrome. Antibodies targeting leiomodin-1 were neurotoxic in vitro, and leiomodin-1 antibodies purified from patients with nodding syndrome were cross-reactive with O. volvulus antigens. This study provides initial evidence supporting the hypothesis that nodding syndrome is an autoimmune epileptic disorder caused by molecular mimicry with O. volvulus antigens and suggests that patients may benefit from immunomodulatory therapies.
Asunto(s)
Enfermedades Autoinmunes/parasitología , Síndrome del Cabeceo/inmunología , Síndrome del Cabeceo/parasitología , Onchocerca volvulus/fisiología , Secuencia de Aminoácidos , Animales , Autoanticuerpos/sangre , Autoanticuerpos/líquido cefalorraquídeo , Autoantígenos/química , Autoantígenos/inmunología , Enfermedades Autoinmunes/sangre , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Niño , Preescolar , Reacciones Cruzadas/inmunología , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/inmunología , Femenino , Proteínas del Helminto/metabolismo , Humanos , Masculino , Síndrome del Cabeceo/sangre , Síndrome del Cabeceo/líquido cefalorraquídeoRESUMEN
Deposition of amyloid-ß plaques is increased in the brains of HIV-infected individuals, and the HIV transactivator of transcription (Tat) protein affects amyloidogenesis through several indirect mechanisms. Here, we investigated direct interactions between Tat and amyloid-ß peptide. Our in vitro studies showed that in the presence of Tat, uniform amyloid fibrils become double twisted fibrils and further form populations of thick unstructured filaments and aggregates. Specifically, Tat binding to the exterior surfaces of the Aß fibrils increases ß-sheet formation and lateral aggregation into thick multifibrillar structures, thus producing fibers with increased rigidity and mechanical resistance. Furthermore, Tat and Aß aggregates in complex synergistically induced neurotoxicity both in vitro and in animal models. Increased rigidity and mechanical resistance of the amyloid-ß-Tat complexes coupled with stronger adhesion due to the presence of Tat in the fibrils may account for increased damage, potentially through pore formation in membranes.
Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Amiloide/toxicidad , Neurotoxinas/toxicidad , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/toxicidad , Amiloide/química , Péptidos beta-Amiloides/metabolismo , Animales , Células Cultivadas , Dicroismo Circular , Técnica del Anticuerpo Fluorescente , Humanos , Ratones Transgénicos , Microscopía de Fuerza Atómica , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotoxinas/química , Agregado de Proteínas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Ratas Sprague-Dawley , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Individual fluorescently labeled fibrin(ogen) molecules and their assembly to make a clot were observed by total internal reflection fluorescence microscopy (TIRFM). We used the bleaching of the fluorescent labels to determine the number of active fluorophores attached nonspecifically to each molecule. From the total intensity of bleaching steps, as single-molecule signature events, and the distribution of active labeling, we developed a new single-molecule intensity calibration, which accounts for all molecules, including those "not seen." Live observation of fibrin polymerization in TIRFM by diffusive mixing of thrombin and plasma revealed the real-time growth kinetics of individual fibrin fibers quantitatively at the molecular level. Some fibers thickened in time to thousands of molecules across, equivalent to hundreds of nanometers in diameter, whereas others reached an early stationary state at smaller diameters. This new approach to determine the molecular dynamics of fiber growth provides information important for understanding clotting mechanisms and the associated clinical implications.
Asunto(s)
Coagulación Sanguínea/fisiología , Fibrina/metabolismo , Fibrinógeno/metabolismo , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Calibración , Colorantes Fluorescentes , Humanos , Cinética , Plasma/metabolismo , Polimerizacion , Trombina/metabolismoRESUMEN
Myosin-II's rod-like tail drives filament assembly with a head arrangement that is often considered to be a symmetric bipole that generates equal and opposite contractile forces on actin. Self-assembled myosin filaments are shown here to be asymmetric in physiological buffer based on cross-correlated images from both atomic force microscopy and total internal reflection fluorescence. Quantitative cross-correlation of these orthogonal methods produces structural information unavailable to either method alone in showing that fluorescence intensity along the filament length is proportional to height. This implies that myosin heads form a shell around the filament axis, consistent with F-actin binding. A motor density of approximately 50-100 heads/micrometer is further estimated but with an average of 32% more motors on one half of any given filament compared to the other, regardless of length. A purely entropic pyramidal lattice model is developed and mapped onto the Dyck paths problem that qualitatively captures this lack of length dependence and the distribution of filament asymmetries. Such strongly asymmetric bipoles are likely to produce an unbalanced contractile force in cells and in actin-myosin gels and thereby contribute to motility as well as cytoskeletal tension.
Asunto(s)
Miosinas/química , Animales , Fluorescencia , Microscopía de Fuerza Atómica , Modelos Químicos , Músculo Esquelético/química , Miosinas/aislamiento & purificación , Conformación Proteica , ConejosRESUMEN
Strong adhesion of highly active cells often nucleates focal adhesions, synapses, and related structures. Red cells lack such complex adhesion systems and are also nonmotile, but they are shown here to dynamically evolve complex spatial patterns beyond an electrostatic threshold for strong adhesion. Spreading of the cell onto a dense, homogeneous poly-L-lysine surface appears complete in <1 s with occasional blisters that form and dissipate on a similar timescale; distinct rippled or stippled patterns in fluorescently labeled membrane components emerge later, however, on timescales more typical of long-range lipid diffusion (approximately minutes). Within the contact zone, the anionic fluorescent lipid fluorescein phosphoethanolamine is seen to rearrange, forming worm-like rippled or stippled domains of <500 nm that prove independent of whether the cell is intact and sustaining a tension or ruptured. Lipid patterns are accompanied by visible perturbations in Band 3 distribution and weaker perturbations in membrane skeleton actin. Pressing down on the membrane quenches the lipid patterns, revealing a clear topographical basis for pattern formation. Counterion screening and membrane fluctuations likely contribute, but the results primarily highlight the fact that even in adhesion of a passive red cell, regions of strong contact slowly evolve to become interspersed with regions where the membrane is more distant from the surface.
Asunto(s)
Adhesión Celular/fisiología , Agregación Celular/fisiología , Movimiento Celular/fisiología , Membrana Eritrocítica/fisiología , Membrana Eritrocítica/ultraestructura , Fusión de Membrana/fisiología , Polilisina/química , Células Cultivadas , Eritrocitos/fisiología , Eritrocitos/ultraestructura , Humanos , Fluidez de la Membrana/fisiologíaRESUMEN
Substrate stiffness is emerging as an important physical factor in the response of many cell types. In agreement with findings on other anchorage-dependent cell lineages, aortic smooth muscle cells are found to spread and organize their cytoskeleton and focal adhesions much more so on "rigid" glass or "stiff" gels than on "soft" gels. Whereas these cells generally show maximal spreading on intermediate collagen densities, the limited spreading on soft gels is surprisingly insensitive to adhesive ligand density. Bell-shaped cell spreading curves encompassing all substrates are modeled by simple functions that couple ligand density to substrate stiffness. Although smooth muscle cells spread minimally on soft gels regardless of collagen, GFP-actin gives a slight overexpression of total actin that can override the soft gel response and drive spreading; GFP and GFP-paxillin do not have the same effect. The GFP-actin cells invariably show an organized filamentous cytoskeleton and clearly indicate that the cytoskeleton is at least one structural node in a signaling network that can override spreading limits typically dictated by soft gels. Based on such results, we hypothesize a central structural role for the cytoskeleton in driving the membrane outward during spreading whereas adhesion reinforces the spreading.
Asunto(s)
Movimiento Celular/fisiología , Colágeno/metabolismo , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Geles , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Animales , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula/métodos , División Celular/fisiología , Línea Celular , Tamaño de la Célula/fisiología , Elasticidad , RatasRESUMEN
Membrane tension underlies a range of cell physiological processes. Strong adhesion of the simple red cell is used as a simple model of a spread cell with a finite membrane tension-a state which proves useful for studies of both membrane rupture kinetics and atomic force microscopy (AFM) probing of native structure. In agreement with theories of strong adhesion, the cell takes the form of a spherical cap on a substrate densely coated with poly-L-lysine. The spreading-induced tension, sigma, in the membrane is approximately 1 mN/m, which leads to rupture over many minutes; and sigma is estimated from comparable rupture times in separate micropipette aspiration experiments. Under the sharpened tip of an AFM probe, nano-Newton impingement forces (10-30 nN) are needed to penetrate the tensed erythrocyte membrane, and these forces increase exponentially with tip velocity ( approximately nm/ms). We use the results to clarify how tapping-mode AFM imaging works at high enough tip velocities to avoid rupturing the membrane while progressively compressing it to a approximately 20-nm steric core of lipid and protein. We also demonstrate novel, reproducible AFM imaging of tension-supported membranes in physiological buffer, and we describe a stable, distended network consistent with the spectrin cytoskeleton. Additionally, slow retraction of the AFM tip from the tensed membrane yields tether-extended, multipeak sawtooth patterns of average force approximately 200 pN. In sum we show how adhesive tensioning of the red cell can be used to gain novel insights into native membrane dynamics and structure.