RESUMEN
Aggregates of medin amyloid (a fragment of the protein MFG-E8, also known as lactadherin) are found in the vasculature of almost all humans over 50 years of age1,2, making it the most common amyloid currently known. We recently reported that medin also aggregates in blood vessels of ageing wild-type mice, causing cerebrovascular dysfunction3. Here we demonstrate in amyloid-ß precursor protein (APP) transgenic mice and in patients with Alzheimer's disease that medin co-localizes with vascular amyloid-ß deposits, and that in mice, medin deficiency reduces vascular amyloid-ß deposition by half. Moreover, in both the mouse and human brain, MFG-E8 is highly enriched in the vasculature and both MFG-E8 and medin levels increase with the severity of vascular amyloid-ß burden. Additionally, analysing data from 566 individuals in the ROSMAP cohort, we find that patients with Alzheimer's disease have higher MFGE8 expression levels, which are attributable to vascular cells and are associated with increased measures of cognitive decline, independent of plaque and tau pathology. Mechanistically, we demonstrate that medin interacts directly with amyloid-ß to promote its aggregation, as medin forms heterologous fibrils with amyloid-ß, affects amyloid-ß fibril structure, and cross-seeds amyloid-ß aggregation both in vitro and in vivo. Thus, medin could be a therapeutic target for prevention of vascular damage and cognitive decline resulting from amyloid-ß deposition in the blood vessels of the brain.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide , Animales , Humanos , Ratones , Persona de Mediana Edad , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Disfunción Cognitiva , Ratones Transgénicos , Placa Amiloide/metabolismo , Proteínas tau/metabolismoRESUMEN
Repeat proteins are common in all domains of life and exhibit a wide range of functions. One class of repeat protein contains solenoid folds where the repeating unit consists of ß-strands separated by tight turns. ß-solenoids have distinguishing structural features such as handedness, twist, oligomerisation state, coil shape and size which give rise to their diversity. Characterised ß-solenoid repeat proteins are known to form regions in bacterial and viral virulence factors, antifreeze proteins and functional amyloids. For many of these proteins, the experimental structure has not been solved, as they are difficult to crystallise or model. Here we use various deep learning-based structure-modelling methods to discover novel predicted ß-solenoids, perform structural database searches to mine further structural neighbours and relate their predicted structure to possible functions. We find both eukaryotic and prokaryotic adhesins, confirming a known functional linkage between adhesin function and the ß-solenoid fold. We further identify exceptionally long, flat ß-solenoid folds as possible structures of mucin tandem repeat regions and unprecedentedly small ß-solenoid structures. Additionally, we characterise a novel ß-solenoid coil shape, the FapC Greek key ß-solenoid as well as plausible complexes between it and other proteins involved in Pseudomonas functional amyloid fibres.
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Aprendizaje Profundo , Amiloide , Adhesinas BacterianasRESUMEN
BACKGROUND: Patients with thoracic aortopathy are at increased risk of catastrophic aortic dissection, carrying with it substantial mortality and morbidity. Although granular medial calcinosis (medial microcalcification) has been associated with thoracic aortopathy, its relationship to disease severity has yet to be established. METHODS: One hundred one thoracic aortic specimens were collected from 57 patients with thoracic aortopathy and 18 control subjects. Standardized histopathologic scores, immunohistochemistry, and nanoindentation (tissue elastic modulus) were compared with the extent of microcalcification on von Kossa histology and 18F-sodium fluoride autoradiography. RESULTS: Microcalcification content was higher in thoracic aortopathy samples with mild (n=28; 6.17 [2.71-10.39]; P≤0.00010) or moderate histopathologic degeneration (n=30; 3.74 [0.87-11.80]; P<0.042) compared with control samples (n=18; 0.79 [0.36-1.90]). Alkaline phosphatase (n=26; P=0.0019) and OPN (osteopontin; n=26; P=0.0045) staining were increased in tissue with early aortopathy. Increasingly severe histopathologic degeneration was related to reduced microcalcification (n=82; Spearman ρ, -0.51; P<0.0001)-a process closely linked with elastin loss (n=82; Spearman ρ, -0.43; P<0.0001) and lower tissue elastic modulus (n=28; Spearman ρ, 0.43; P=0.026).18F-sodium fluoride autoradiography demonstrated good correlation with histologically quantified microcalcification (n=66; r=0.76; P<0.001) and identified areas of focal weakness in vivo. CONCLUSIONS: Medial microcalcification is a marker of aortopathy, although progression to severe aortopathy is associated with loss of both elastin fibers and microcalcification.18F-sodium fluoride positron emission tomography quantifies medial microcalcification and is a feasible noninvasive imaging modality for identifying aortic wall disruption with major translational promise.
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Calcinosis , Elastina , Aorta , Calcinosis/diagnóstico por imagen , Humanos , Índice de Severidad de la Enfermedad , Fluoruro de SodioRESUMEN
Medin is the most common amyloid known in humans, as it can be found in blood vessels of the upper body in virtually everybody over 50 years of age. However, it remains unknown whether deposition of Medin plays a causal role in age-related vascular dysfunction. We now report that aggregates of Medin also develop in the aorta and brain vasculature of wild-type mice in an age-dependent manner. Strikingly, genetic deficiency of the Medin precursor protein, MFG-E8, eliminates not only vascular aggregates but also prevents age-associated decline of cerebrovascular function in mice. Given the prevalence of Medin aggregates in the general population and its role in vascular dysfunction with aging, targeting Medin may become a novel approach to sustain healthy aging.
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Envejecimiento/metabolismo , Amiloide/metabolismo , Antígenos de Superficie/metabolismo , Proteínas de la Leche/metabolismo , Enfermedades Vasculares/metabolismo , Anciano de 80 o más Años , Amiloide/genética , Animales , Antígenos de Superficie/genética , Aorta/metabolismo , Aorta/patología , Química Encefálica/fisiología , Circulación Cerebrovascular/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de la Leche/genética , Enfermedades Vasculares/patologíaRESUMEN
Altered proteoglycan (PG) and glycosaminoglycan (GAG) distribution within the aortic wall has been implicated in thoracic aortic aneurysm and dissection (TAAD). This review was conducted to identify literature reporting the presence, distribution and role of PGs and GAGs in the normal aorta and differences associated with sporadic TAAD to address the question; is there enough evidence to establish the role of GAGs/PGs in TAAD? 75 studies were included, divided into normal aorta (n = 51) and TAAD (n = 24). There is contradictory data regarding changes in GAGs upon ageing; most studies reported an increase in GAG sub-types, often followed by a decrease upon further ageing. Fourteen studies reported changes in PG/GAG or associated degradation enzyme levels in TAAD, with most increased in disease tissue or serum. We conclude that despite being present at relatively low abundance in the aortic wall, PGs and GAGs play an important role in extracellular matrix maintenance, with differences observed upon ageing and in association with TAAD. However, there is currently insufficient information to establish a cause-effect relationship with an underlying mechanistic understanding of these changes requiring further investigation. Increased PG presence in serum associated with aortic disease highlights the future potential of these biomolecules as diagnostic or prognostic biomarkers.
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Aneurisma de la Aorta Torácica , Disección Aórtica , Disección Aórtica/metabolismo , Animales , Aneurisma de la Aorta Torácica/metabolismo , Modelos Animales de Enfermedad , Glicosaminoglicanos , Humanos , Proteoglicanos/metabolismoRESUMEN
Under physiological conditions, the cornea is exposed to various enzymes, some of them have digestive actions, such as amylase and collagenase that may change the ultrastructure (collagen morphology) and sequentially change the mechanical response of the cornea and distort vision, such as in keratoconus. This study investigates the ultrastructure and nanomechanical properties of porcine cornea following incubation with α-amylase and collagenase. Atomic force microscopy (AFM) was used to capture nanoscale topographical details of stromal collagen fibrils (diameter and D-periodicity) and calculate their elastic modulus. Samples were incubated with varying concentrations of α-amylase and collagenase (crude and purified). Dimethylmethylene blue (DMMB) assay was utilised to detect depleted glycosaminoglycans (GAGs) following incubation with amylase. Collagen fibril diameters were decreased following incubation with amylase, but not D-periodicity. Elastic modulus was gradually decreased with enzyme concentration in amylase-treated samples. Elastic modulus, diameter, and D-periodicity were greatly reduced in collagenase-treated samples. The effect of crude collagenase on corneal samples was more pronounced than purified collagenase. Amylase was found to deplete GAGs from the samples. This enzymatic treatment may help in answering some questions related to keratoconus, and possibly be used to build an empirical animal model of keratoconic corneas with different progression levels.
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Córnea , Glicosaminoglicanos , Queratocono , Animales , Colágeno , Microscopía de Fuerza Atómica , PorcinosRESUMEN
Medin, a 50-amino-acid cleavage product of the milk fat globule-EGF factor 8 protein, is one of the most common forms of localized amyloid found in the vasculature of individuals older than 50 years. Medin induces endothelial dysfunction and vascular inflammation, yet despite its prevalence in the human aorta and multiple arterial beds, little is known about the nature of its pathology. Medin oligomers have been implicated in the pathology of aortic aneurysm, aortic dissection, and more recently, vascular dementia. Recent in vitro biomechanical measurements found increased oligomer levels in aneurysm patients with altered aortic wall integrity. Our results suggest an oligomer-mediated toxicity mechanism for medin pathology. Using lipid bilayer electrophysiology, we show that medin oligomers induce ionic membrane permeability by pore formation. Pore activity was primarily observed for preaggregated medin species from the growth-phase and rarely for lag-phase species. Atomic force microscopy (AFM) imaging of medin aggregates at different stages of aggregation revealed the gradual formation of flat domains resembling the morphology of supported lipid bilayers. Transmission electron microscopy images showed the coexistence of compact oligomers, largely consistent with the AFM data, and larger protofibrillar structures. Circular dichroism spectroscopy revealed the presence of largely disordered species and suggested the presence of ß-sheets. This observation and the significantly lower thioflavin T fluorescence emitted by medin aggregates compared to amyloid-ß fibrils, along with the absence of amyloid fibers in the AFM and transmission electron microscopy images, suggest that medin aggregation into pores follows a nonamyloidogenic pathway. In silico modeling by molecular dynamics simulations provides atomic-level structural detail of medin pores with the CNpNC barrel topology and diameters comparable to values estimated from experimental pore conductances.
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Amiloide , Aorta , Péptidos beta-Amiloides , Humanos , Membrana Dobles de Lípidos , Microscopía de Fuerza AtómicaRESUMEN
Clinical and preclinical studies have suggested a link between cardiovascular disease and dementia disorders, but the role of the collateral brain circulation in cognitive dysfunction remains unknown. We aimed to test the hypothesis that leptomeningeal arteriole (LMA) function and response to metabolic stressors differ among subjects with dementia, mild cognitive impairment (MCI), and normal cognition (CN). After rapid autopsy, LMAs were isolated from subjects with CN ( n = 10), MCI ( n = 12), or dementia [ n = 42, Alzheimer's disease (AD), vascular dementia (VaD), or other dementia], and endothelial and smooth muscle-dependent function were measured at baseline and after exposure to ß-amyloid (2 µM), palmitic acid (150 µM), or medin (5 µM) and compared. There were no differences among the groups in baseline endothelial function (maximum dilation to acetylcholine, CN: 74.1 ± 9.7%, MCI: 67.1 ± 4.8%, AD: 74.7 ± 2.8%, VaD: 72.0 ± 5.3%, and other dementia: 68.0 ± 8.0%) and smooth muscle-dependent function (CN: 93.4 ± 3.0%, MCI: 83.3 ± 4.1%, AD: 91.8 ± 1.7%, VaD: 91.7 ± 2.4%, and other dementia: 87.9 ± 4.9%). There was no correlation between last cognitive function score and baseline endothelial or smooth muscle-dependent function. LMA endothelial function and, to a lesser extent, smooth muscle-dependent function were impaired posttreatment with ß-amyloid, palmitic acid, and medin. Posttreatment LMA responses were not different between subjects with CN/MCI vs. dementia. Baseline responses and impaired vasoreactivity after treatment with metabolic stressors did not differ among subjects with CN, MCI, and dementia. The results suggest that the cognitive dysfunction in dementia disorders is not attributable to differences in baseline brain collateral circulation function but may be influenced by exposure of the vasculature to metabolic stressors.
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Encéfalo/irrigación sanguínea , Disfunción Cognitiva/fisiopatología , Demencia/fisiopatología , Anciano de 80 o más Años , Arteriolas/fisiopatología , Encéfalo/fisiopatología , Endotelio Vascular/fisiopatología , Femenino , Hemodinámica , Humanos , Masculino , Miocitos del Músculo Liso/fisiologíaRESUMEN
Incubation conditions are an important factor to consider when studying protein aggregation in vitro. Here, we employed biophysical methods and atomic force microscopy to show that agitation dramatically alters the morphology of medin, an amyloid protein deposited in the aorta. Agitation reduces the lag time for fibrillation by ~18-fold, suggesting that the rate of fibril formation plays a key role in directing the protein packing arrangement within fibrils. Utilising preformed sonicated fibrils as seeds, we probed the role of seeding on medin fibrillation and revealed three distinct fibril morphologies, with biophysical modelling explaining the salient features of experimental observations. We showed that nucleation pathways to distinct fibril morphologies may be switched on and off depending on the properties of the seeding fibrils and growth conditions. These findings may impact on the development of amyloid-based biomaterials and enhance understanding of seeding as a pathological mechanism.
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Microscopía de Fuerza Atómica/métodos , Modelos Teóricos , Amiloide/química , Cinética , Semillas/químicaRESUMEN
Peptides derived from apolipoprotein A-I (apoA-I), the main component of high-density lipoprotein (HDL), constitute the main component of amyloid deposits that colocalize with atherosclerotic plaques. Here we investigate the molecular details of full-length, lipid-deprived apoA-I after assembly into insoluble aggregates under physiologically relevant conditions known to induce aggregation in vitro. Unmodified apoA-I is shown to remain soluble at pH 7 for at least 3 days, retaining its native α-helical-rich structure. Upon acidification to pH 4, apoA-I rapidly assembles into insoluble nonfibrillar aggregates lacking the characteristic cross-ß features of amyloid. In the presence of heparin, the rate and thioflavin T responsiveness of the aggregates formed at pH 4 increase and short amyloid-like fibrils are observed, which give rise to amyloid-characteristic X-ray reflections at 4.7 and 10 Å. Solid-state nuclear magnetic resonance (SSNMR) and synchrotron radiation circular dichroism spectroscopy of fibrils formed in the presence of heparin show they retain some α-helical characteristics together with new ß-sheet structures. Interestingly, SSNMR indicates a similar molecular structure of aggregates formed in the absence of heparin at pH 6 after oxidation of the three methionine residues, although their morphology is rather different from that of the heparin-derived fibrils. We propose a model for apoA-I aggregation in which perturbations of a four-helix bundle-like structure, induced by interactions of heparin or methionine oxidation, cause the partially helical N-terminal residues to disengage from the remaining, intact helices, thereby allowing self-assembly via ß-strand associations.
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Proteínas Amiloidogénicas/química , Apolipoproteína A-I/química , Heparina/química , Metionina/química , Agregado de Proteínas , Proteínas Amiloidogénicas/metabolismo , Proteínas Amiloidogénicas/ultraestructura , Apolipoproteína A-I/metabolismo , Apolipoproteína A-I/ultraestructura , Benzotiazoles , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Heparina/metabolismo , Humanos , Peróxido de Hidrógeno/química , Concentración de Iones de Hidrógeno , Metionina/metabolismo , Oxidación-Reducción , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Espectrometría de Fluorescencia , Tiazoles/químicaRESUMEN
Aortic medial amyloid (AMA) is the most common localized human amyloid, occurring in virtually all of the Caucasian population over the age of 50. The main protein component of AMA, medin, readily assembles into amyloid-like fibrils in vitro. Despite the prevalence of AMA, little is known about the self-assembly mechanism of medin or the molecular architecture of the fibrils. The amino acid sequence of medin is strikingly similar to the sequence of the Alzheimer disease (AD) amyloid-ß (Aß) polypeptides around the structural turn region of Aß, where mutations associated with familial, early onset AD, have been identified. Asp(25) and Lys(30) of medin align with residues Asp(23) and Lys(28) of Aß, which are known to form a stabilizing salt bridge in some fibril morphologies. Here we show that substituting Asp(25) of medin with asparagine (D25N) impedes assembly into fibrils and stabilizes non-cytotoxic oligomers. Wild-type medin, by contrast, aggregates into ß-sheet-rich amyloid-like fibrils within 50 h. A structural analysis of wild-type fibrils by solid-state NMR suggests a molecular repeat unit comprising at least two extended ß-strands, separated by a turn stabilized by a Asp(25)-Lys(30) salt bridge. We propose that Asp(25) drives the assembly of medin by stabilizing the fibrillar conformation of the peptide and is thus reminiscent of the influence of Asp(23) on the aggregation of Aß. Pharmacological comparisons of wild-type medin and D25N will help to ascertain the pathological significance of this poorly understood protein.
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Antígenos de Superficie/metabolismo , Aorta/metabolismo , Ácido Aspártico/metabolismo , Proteínas de la Leche/metabolismo , Secuencia de Aminoácidos , Antígenos de Superficie/química , Ácido Aspártico/química , Dicroismo Circular , Humanos , Proteínas de la Leche/química , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The aggregation and fibril deposition of amyloid proteins have been implicated in a range of neurodegenerative and vascular diseases, and yet the underlying molecular mechanisms are poorly understood. Here, we use a combination of cell-based assays, biophysical analysis, and atomic force microscopy to investigate the potential involvement of oxidative stress in aortic medial amyloid (AMA) pathogenesis and deposition. We show that medin, the main constituent of AMA, can induce an environment rich in oxidative species, increasing superoxide and reducing bioavailable nitric oxide in human cells. We investigate the role that this oxidative environment may play in altering the aggregation process of medin and identify potential posttranslational modification sites where site-specific modification and interaction can be unambiguously demonstrated. In an oxidizing environment, medin is nitrated at tyrosine and tryptophan residues, with resultant effects on morphology that lead to longer fibrils with increased toxicity. This provides further motivation to investigate the role of oxidative stress in AMA pathogenicity.
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Antígenos de Superficie/toxicidad , Aorta/metabolismo , Proteínas de la Leche/toxicidad , Estrés Oxidativo/efectos de los fármacos , Antígenos de Superficie/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas de la Leche/metabolismo , Nitratos/metabolismoRESUMEN
Recent studies have indicated that the human amyloidogenic protein medin is associated with a range of vascular diseases, including aortic aneurysms, vascular dementia, and Alzheimer's disease. Medin accumulates in the vasculature with age, leading to endothelial dysfunction through oxidative and nitrative stress and inducing pro-inflammatory activation. Medin is a cleavage product from the C2 domain of MfgE8. The exact mechanism of medin production from MfgE8 is unknown, with crystal structures of homologous C2 domains suggesting that the cleavage sites are buried, requiring a conformational transition for medin production. Molecular dynamics simulations can explore a wide range of conformations, from small-scale bond rotations to large-scale changes like protein folding or ligand binding. This study employed a combination of full-atom and coarse-grained molecular dynamics simulations, along with CONCOORD- and AlphaFold2-generated models, to investigate MfgE8 conformations and their implications for medin cleavage site accessibility. The simulations revealed that MfgE8 tends to adopt a compact conformation with the RGD motif, important for cell attachment within the N-terminal domain, and the medin region in the C-terminal domain close in proximity. Formation of this compact structure is facilitated by interdomain electrostatic interactions that promote stability and in turn decrease the solvent-accessible surface area of the medin region and particularly the C-terminal medin cleavage site. This data enhances current knowledge on medin generation to propose that alterations in local environmental conditions, possibly through changes in glycosylation or other post-translational modifications are required to induce MfgE8 to unfold partially or fully: this would result in enhanced accessibility of the cleavage sites and therefore enable medin generation.
RESUMEN
Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.
RESUMEN
The dietary consumption of extra virgin olive oil (EVOO) is believed to slow the progression of Alzheimer's disease (AD) symptoms. Its protective mechanisms are unclear, but specific EVOO phenolic compounds can individually impede the aggregation of amyloid-ß (Aß) peptides and the microtubule-associated protein tau, two important pathological manifestations of AD. It is unknown, however, whether the numerous and variable phenolic compounds that are consumed in dietary EVOO can collectively alter tau and Aß aggregation as effectively as the individual compounds. The activity of these complex mixtures against Aß and tau may be moderated by competition between active and nonactive phenolic components and by extensive derivatizations and isomerization. Here, phenolic mixtures extracted from two different EVOO sources are characterized and tested for how they modulate the aggregation of Aß40 peptide and tau peptides in vitro. The chromatographic and NMR analysis of Greek and Saudi Arabian EVOO phenolic extracts reveals that they have different concentration profiles, and over 30 compounds are identified. Thioflavin T fluorescence and circular dichroism measurements show that relatively low concentrations (<20 µg/mL) of the Greek and Saudi extracts reduce the rate of Aß40 aggregation and fibril mass, despite the extracts having different phenolic profiles. By contrast, the Greek extract reduces the rate of tau aggregation only at very high phenolic concentrations (>100 µg/mL). Most compounds in the extracts bind to preformed Aß40 fibrils and release soluble Aß oligomers that are mildly toxic to SH-SY5Y cells. Much higher (500 µg/mL) extract concentrations are required to remodel tau filaments into oligomers, and a minimal binding of phenolic compounds to the preformed filaments is observed. It is concluded that EVOO extracts having different phenol profiles are similarly capable of modulating Aß40 aggregation and fibril morphology in vitro at relatively low concentrations but are less efficient at modulating tau aggregation. Over 2 M tonnes of EVOO are consumed globally each year as part of the Mediterranean diet, and the results here provide motivation for further clinical interrogation of the antiaggregation properties of EVOO as a potential protective mechanism against AD.
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Amyloid deposits in vivo are complex mixtures composed of protein fibrils and nonfibrillar components, including polysaccharides of the glycosaminoglycan (GAG) class. It has been widely documented that GAGs influence the initiation and progress of self-assembly by several disease-associated amyloidogenic proteins and peptides in vitro. Here we investigated whether the GAG heparin can serve as a cofactor to induce amyloid-like fibril formation in a peptide predicted to have a weak propensity to aggregate and not associated with amyloid disorders. We selected the 23-residue peptide PLB(1-23), which corresponds to the acetylated cytoplasmic domain of the phospholamban transmembrane protein. PLB(1-23) remains unfolded in aqueous solution for >24 h and does not bind thioflavin T over this time period, in agreement with computer predictions that the peptide has a very low intrinsic amyloidogenicity. In the presence of low-molecular mass (5 kDa) heparin, which binds PLB(1-23) with micromolar affinity, the peptide undergoes spontaneous and rapid assembly into amyloid-like fibrils, the effect being more pronounced at pH 5.5 than at pH 7.4. At the lower pH, peptide aggregation is accompanied by a transition to a ß-sheet rich structure. These results are consistent with the polyanionic heparin serving as a scaffold to enhance aggregation by aligning the peptide molecules in the correct orientation and with the appropriate periodicity. PLB(1-23) is toxic to cells when added in isolation, and promotion of fibril formation by heparin can reduce the toxicity of this peptide, consistent with the notion that amyloid-like fibrils represent a benign end stage of fibrillization. This work provides insight into the role that heparin and other glycosaminoglycans may play in amyloid formation and provides therapeutic avenues targeting the reduction of cytotoxicity of species along the amyloid formation pathway.
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Amiloide/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Péptidos/metabolismo , Amiloide/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glicosaminoglicanos/química , Heparina/química , Humanos , Concentración de Iones de Hidrógeno , Tamaño de la Partícula , Péptidos/química , Péptidos/farmacología , Relación Estructura-Actividad , Propiedades de Superficie , VolumetríaRESUMEN
Queuing up: Molecular orientation within macroscopically aligned nanotubes of the peptide AAAAAAK can be studied by solid-state NMR and IR spectroscopy. Line shape analysis of the NMR spectra indicates that the peptide N-H bonds are tilted 65-70° relative to the nanotube long axis. Re-evaluation of earlier X-ray fiber diffraction data suggests that the peptide molecules are hydrogen-bonded in a helical arrangement along the nanotube axis.
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Nanotubos de Péptidos/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular/instrumentación , Resonancia Magnética Nuclear Biomolecular/métodos , Oligopéptidos/química , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Vascular amyloidosis, caused when peptide monomers aggregate into insoluble amyloid, is a prevalent age-associated pathology. Aortic medial amyloid (AMA) is the most common human amyloid and is composed of medin, a 50-amino acid peptide. Emerging evidence has implicated extracellular vesicles (EVs) as mediators of pathological amyloid accumulation in the extracellular matrix (ECM). To determine the mechanisms of AMA formation with age, we explored the impact of vascular smooth muscle cell (VSMC) senescence, EV secretion, and ECM remodeling on medin accumulation. Medin was detected in EVs secreted from primary VSMCs. Small, round medin aggregates colocalized with EV markers in decellularized ECM in vitro and medin was shown on the surface of EVs deposited in the ECM. Decreasing EV secretion with an inhibitor attenuated aggregation and deposition of medin in the ECM. Medin accumulation in the aortic wall of human subjects was strongly correlated with age and VSMC senescence increased EV secretion, increased EV medin loading and triggered deposition of fibril-like medin. Proteomic analysis showed VSMC senescence induced changes in EV cargo and ECM composition, which led to enhanced EV-ECM binding and accelerated medin aggregation. Abundance of the proteoglycan, HSPG2, was increased in the senescent ECM and colocalized with EVs and medin. Isolated EVs selectively bound to HSPG2 in the ECM and its knock-down decreased formation of fibril-like medin structures. These data identify VSMC-derived EVs and HSPG2 in the ECM as key mediators of medin accumulation, contributing to age-associated AMA development.
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Vesículas Extracelulares , Músculo Liso Vascular , Humanos , Músculo Liso Vascular/metabolismo , Proteómica , Vesículas Extracelulares/metabolismo , Péptidos/metabolismo , Matriz Extracelular/metabolismo , Amiloide , Senescencia Celular , Miocitos del Músculo Liso/metabolismoRESUMEN
BACKGROUND: An association with aortic aneurysm has been reported among patients with atrial fibrillation (AF). The aims of this study were to investigate the prevalence of thoracic aorta aneurysm (TAA) among patients with AF and to assess whether the co-presence of TAA is associated with a higher risk of adverse clinical outcomes. METHODS AND RESULTS: Using TriNetX, a global federated health research network of anonymised electronic medical records, all adult patients with AF, were categorised into two groups based on the presence of AF and TAA or AF alone. Between 1 January 2017 and 1 January 2019, 874,212 people aged ≥ 18 years with AF were identified. Of these 17,806 (2.04%) had a TAA. After propensity score matching (PSM), 17,805 patients were included in each of the two cohorts. During the 3 years of follow-up, 3079 (17.3%) AF patients with TAA and 2772 (15.6%) patients with AF alone, developed an ischemic stroke or transient ischemic attack (TIA). The risk of ischemic stroke/TIA was significantly higher in patients with AF and TAA (HR 1.09, 95% CI 1.04-1.15; log-rank p value < 0.001) The risk of major bleeding was higher in patients with AF and TAA (OR 1.07, 95% CI 1.01-1.14), but not significant in time-dependent analysis (HR 1.04, 95% CI 0.98-1.10; log-rank p value = 0.187), CONCLUSION: This retrospective analysis reports a clinical concomitance of the two medical conditions, and shows in a PSM analysis an increased risk of ischemic events in patients affected by TAA and AF compared to AF alone.
Asunto(s)
Aneurisma de la Aorta Torácica , Fibrilación Atrial , Ataque Isquémico Transitorio , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Adulto , Humanos , Fibrilación Atrial/complicaciones , Fibrilación Atrial/epidemiología , Ataque Isquémico Transitorio/epidemiología , Estudios Retrospectivos , Factores de Riesgo , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/complicaciones , Aneurisma de la Aorta Torácica/complicaciones , Accidente Cerebrovascular Isquémico/complicacionesRESUMEN
Medin is a common vascular amyloidogenic peptide recently implicated in Alzheimer's disease (AD) and vascular dementia and its pathology remains unknown. We aim to identify changes in transcriptomic profiles and pathways in human brain microvascular endothelial cells (HBMVECs) exposed to medin, compare that to exposure to ß-amyloid (Aß) and evaluate protection by monosialoganglioside-containing nanoliposomes (NL). HBMVECs were exposed for 20 h to medin (5 µM) without or with Aß(1-42) (2 µM) or NL (300 µg/mL), and RNA-seq with signaling pathway analyses were performed. Separately, reverse transcription polymerase chain reaction of select identified genes was done in HBMVECs treated with medin (5 µM) without or with NFκB inhibitor RO106-9920 (10 µM) or NL (300 µg/mL). Medin caused upregulation of pro-inflammatory genes that was not aggravated by Aß42 co-treatment but reversed by NL. Pathway analysis on differentially expressed genes revealed multiple pro-inflammatory signaling pathways, such as the tumor necrosis factor (TNF) and the nuclear factor-κB (NFkB) signaling pathways, were affected specifically by medin treatment. RO106-9920 and NL reduced medin-induced pro-inflammatory activation. Medin induced endothelial cell pro-inflammatory signaling in part via NFκB that was reversed by NL. This could have potential implications in the pathogenesis and treatment of vascular aging, AD and vascular dementia.