1,721,061 research outputs found
Equine lysozyme: The molecular basis of folding, self-assembly and innate amyloid toxicity
No full textAbstractCalcium-binding equine lysozyme (EL) combines the structural and folding properties of c-type lysozymes and α-lactalbumins, connecting these two most studied subfamilies. The structural insight into its native and partially folded states is particularly illuminating in revealing the general principles of protein folding, amyloid formation and its inhibition. Among lysozymes EL forms one of the most stable molten globules and shows the most uncooperative refolding kinetics. Its partially-folded states serve as precursors for calcium-dependent self-assembly into ring-shaped and linear amyloids. The innate amyloid cytotoxicity of the ubiquitous lysozyme highlights the universality of this phenomenon and necessitates stringent measures for its prevention
Proinflammatory S100A8/A9 in amyloid formation in the aging prostate : risk factor for cancer
No full text
Pro-inflammatory S100a9 protein involved in the amyloid-neuroinflammatory cascade in Alzheimer's disease serves as a robust biomarker differentiating early stages of dementia
No full textWe have demonstrated that pro-inflammatory protein S100A9 plays a critical role in the amyloid-neuroinflammatory cascade leading to amyloid depositions and amyloid neurotoxicity in Alzheimer’s disease (AD). S100A9 proved to be as a robust biomarker differentiating early stages of cognitive impairment in AD in conjunction with others such as Ab(142) and tau-proteins. CSF samples from 104 stringently diagnosed individuals divided into five subgroups were analyzed, including nondemented controls, stable mild cognitive impairment (SMCI), mild cognitive impairment due to AD (MCI-AD), AD, and vascular dementia (VaD) patients. ELISA, dot-blotting, and electrochemical impedance spectroscopy were used as research methods. The S100A9 and Ab(142) levels correlated with each other: their CSF content decreased already at the SMCI stage and declined further under MCIAD, AD, and VaD conditions. Immunohistochemical analysis also revealed involvement of both Ab142 and S100A9 in the amyloid-neuroinflammatory cascade already during SMCI. Tau proteins were not yet altered in SMCI; however their contents increased during MCI-AD and AD, diagnosing later dementia stages. Thus, S100A9 and three other biomarkers taken together and reflecting different underlying pathological causes can accurately differentiate dementia progression and also distinguish AD from VaD.</p
Equine lysozyme: the molecular basis of folding, self-assembly and innate amyloid toxicity
No full textA false paradise - mixed blessings in the protein universe: the amyloid as a new challenge in drug development
No full textThe Stabilization of S100A9 Structure by Calcium Inhibits the Formation of Amyloid Fibrils
No full textThe calcium-binding protein S100A9 is recognized as an important component of the brain neuroinflammatory response to the onset and development of neurodegenerative disease. S100A9 is intrinsically amyloidogenic and in vivo co-aggregates with amyloid-β peptide and α-synuclein in Alzheimer’s and Parkinson’s diseases, respectively. It is widely accepted that calcium dyshomeostasis plays an important role in the onset and development of these diseases, and studies have shown that elevated levels of calcium limit the potential for S100A9 to adopt a fibrillar structure. The exact mechanism by which calcium exerts its influence on the aggregation process remains unclear. Here we demonstrate that despite S100A9 exhibiting α-helical secondary structure in the absence of calcium, the protein exhibits significant plasticity with interconversion between different conformational states occurring on the micro- to milli-second timescale. This plasticity allows the population of conformational states that favour the onset of fibril formation. Magic-angle spinning solid-state NMR studies of the resulting S100A9 fibrils reveal that the S100A9 adopts a single structurally well-defined rigid fibrillar core surrounded by a shell of approximately 15–20 mobile residues, a structure that persists even when fibrils are produced in the presence of calcium ions. These studies highlight how the dysregulation of metal ion concentrations can influence the conformational equilibria of this important neuroinflammatory protein to influence the rate and nature of the amyloid deposits formed
Structural studies of heterogeneous amyloid species of lysozymes and de novo protein albebetin and their cytotoxicity [Elektronisk resurs]
No full textA number of diseases are linked to protein folding problems which lead to the deposition of insoluble protein plaques in the brain or other organs. These diseases include prion diseases such as Creutzfeld-Jakob disease, Alzheimer's disease, Parkinson's disease and type II (non-insulin dependent) diabetes. The protein plaques are found to consist of amyloid fibrils - cross-beta-sheet polymers with the beta-strands arranged perpendicular to the long axis of the fibre. Studies of ex vivo fibrils and fibrils produced in vitro showed that amyloid structures possess similar tinctorial and morphological properties. These suggest that the ability to form amyloid fibrils is an inherent property of polypeptide chains. The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the involved mechanisms. The model proteins used in the studies were the equine and hen lysozymes and de novo designed protein albebetin. Lysozymes are naturally ubiquitous proteins. Equine lysozyme belongs to an extended family of structurally related lysozymes and α-lactalbumins and can be considered as an evolutional bridge between them. Hen lysozyme is one of the most characterized protein and its amyloidogenic properties were described earlier. De novo protein albebetin and its constructs are designed to perform the function of grafted polypeptide sequence. Fibrils of equine lysozyme are formed at acidic pH and elevated temperatures where a partially folded molten globule state is populated. We have shown that lysozyme assembles into annular and linear protofilaments in a calcium-dependent manner. We showed that albebetin and its constructs are inherently highly amyloidogenic under physiological conditions. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils, among which two distinct types of oligomeric intermediates were characterized. Pivotal oligomers comprise of 10-12 monomers and on-pathway amyloid-prone oligomers constitute of 26-30 molecules. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in albebetin fibrillation. Cytotoxic studies of albebetin amyloid species have revealed that initial, pivotal oligomers do not effect on cell viability while amyloid-prone ones induce cell death. We suggest that oligomeric size is important for the stabilizing cross-beta-sheet core which is crucial for cell toxicity. Cytotoxic studies of both oligomers and fibrils of hen lysozyme have revealed that both species induce cell death. The amyloid sample containing cross-β-sheet oligomers induces an apoptosis-like cell death. The oligomers without cross-β-sheet appeared to be non-toxic, indicating that the stabilization of this structural pattern is critical for the induced toxicity. In contrast, the fibrils induce more rapid, necrosis-like death. These studies gained insights into a structure–function relationship of different forms of amyloid and general pathways of cell death. This is an important step in understanding the mechanisms of amyloid-associated degeneration and defining specific therapeutic targets.</p
Islet Amyloid Polypeptide: Interaction with Amyloid Beta, Alpha-Synuclein and BRICHOS [Elektronisk resurs]
No full text Amyloid, the congophilic deposits of misfolded protein, are pathological hallmarks of many common diseases, among others, Alzheimer’s disease (AD), Parkinson’s disease (PD), and type 2 diabetes (T2D). Amyloid beta (Aβ) forms senile plaques in AD, alpha-synuclein (aSyn) forms Lewy bodies and Lewy neurites in PD, islet amyloid polypeptide (IAPP) forms islet amyloid in T2D. Amyloid fibril formation is a nucleation-dependent process, which can be accelerated via the addition of preformed seeds. This mechanism is referred to as seeding or cross-seeding, depending on whether homologous or heterologous seeds are added. The present thesis has investigated the interaction between IAPP and Aβ, IAPP and aSyn, and IAPP and anti-amyloid chaperone Bri2 BRICHOS. The possibility of using luminescent conjugated oligothiophenes (LCOs) to detect islet amyloid formation has also been examined.IAPP and Aβ interacted at molecular level in living cells and preferred parallel binding over anti-parallel binding. Interaction of IAPP and Aβ led to the formation of intracellular amyloid, increased lysosomal area, increased superoxide production, and increased susceptibility to cell death. In addition, co-expression of IAPP and Aβ in brain of Drosophila melanogaster resulted in co-deposition of proteins and reduced fly lifespan. These results provide a molecular link between AD and T2D.ASyn expressed in pancreatic β cells, co-localized with IAPP but was absent in extracellular islet amyloid. Preformed aSyn seeds triggered IAPP fibril formation, but not vice versa. Neither knockdown nor overexpression of aSyn affected β cell viability. These results suggest that the proximity of aSyn and IAPP does not guarantee functional interference.Bri2 BRICHOS expressed in pancreatic β cells and co-localized with both intracellular IAPP and extracellular islet amyloid. Bri2 BRICHOS inhibited IAPP fibril formation and reduced IAPP-induced cytotoxicity. Knockdown endogenous Bri2 increased the susceptibility of β cells death under stressed conditions, but overexpression of BRICHOS rescued β cells. These results suggest BRICHOS be a potential endogenous chaperone in preventing islet amyloid formation in β cells.The LCO pFTAA-CN detected islet amyloid in fixed islets, unfixed frozen islets, and living islets and could be used to monitor islet amyloid formation in real-time without interfering with the formation of islet amyloid.In conclusion, the interaction between IAPP and Aβ results in increased toxicity on cells, while the impact of the interaction between IAPP and aSyn is still an open question, Bri2 BRICHOS is an endogenous inhibitor of IAPP fibrillation and toxicity. PFTAA-CN is suitable for detecting islet amyloid under various conditions. </p
The amyloid [Elektronisk resurs] : structure, properties and application
No full textProtein aggregation, leading to the formation and depositions of amyloids, is a cause for a number of diseases such as Alzheimer’s and Creutzfeld-Jacob’s disease, systemic amyloidoses, type II diabetes and others . More than 20 proteins are associated with protein misfolding diseases and even a larger number of proteins can self-assemble into amyloid in vitro. Relating structural and functional properties of amyloid is of particular interest, as this will lead to the identification of the main factors and mechanisms involved in the process of protein misfolding and aggregation; consequently, this will provide a basis for developing new strategies to treat protein misfolding diseases. The aim of the thesis is to investigate structural aspects of amyloid formation and relate that to the functional properties of amyloid. The first paper describes the amyloid formation of equine lysozyme (EL). We have demonstrated that EL enters an amyloid forming pathways under conditions where the molten globule state is populated. We have found that the morphology of the amyloids depend on the calcium-binding to lysozyme, specifically the holo-protein assembles into short, linear protofilaments, while the apo-EL forms ring-shaped structures. The morphology of EL amyloid significantly differs from the amyloid fibrils of human and hen lysozymes. We have suggested that the stable alpha-helical core of EL, which remains structured in the molten globule intermediate, may obstruct the formation of fibrilar interface and therefore leads to assembly of short, curly fibrils and rings.In the second paper, we describe the cytotoxicity of EL amyloids. We have analysed the amyloid intermediates on the pathway towards amyloid fibrils. The sizes of amyloid oligomers were determined by atomic force microscopy (AFM) and the formation of cross-beta sheet was shown by thioflavin T (ThT) binding. The toxicity studies show that the oligomers formed during amyloid growth phase are toxic to a range of cell lines and cultures and the toxicity is size-dependant.The last manuscript describes a novel method for manufacturing of silver nanowires by the biotemplating using amyloid fibrils. The amyloid assembled from an abundant and cheap hen egg white lysozyme was used as a scaffold for casting ultrathin silver nanowires. We have manufactured nanowires with a diameter of 1.0-2.5 nm and up to 2 micrometers in length. Up to date, it is the thinnest silver nanowires produced by using biotemplating and at least one order of magnitude thinner than nanowires manufactured by chemical synthesis.</p
- …
