200 research outputs found
Unlocked Concanavalin A Forms Amyloid-like Fibrils from Coagulation of Long-lived "Crinkled'' Intermediates
No full textUnderstanding the early events during amyloid aggregation processes is crucial to single out the involved molecular mechanisms and for designing ad hoc strategies to prevent and reverse amyloidogenic disorders. Here, we show that, in conditions in which the protein is positively charged and its conformational flexibility is enhanced, Concanavalin A leads to fibril formation via a non-conventional aggregation pathway. Using a combination of light scattering, circular dichroism, small angle X-ray scattering, intrinsic (Tryptophan) and extrinsic (ANS) fluorescence and confocal and 2-photon fluorescence microscopy we characterize the aggregation process as a function of the temperature. We highlight a multi-step pathway with the formation of an on-pathway long-lived intermediate and a subsequent coagulation of such "crinkled'' precursors into amyloid-like fibrils. The process results in a temperature-dependent aggregation-coagulation pathway, with the late phase of coagulation determined by the interplay between hydrophobic and electrostatic forces. Our data provide evidence for the complex aggregation pathway for a protein with a highly flexible native conformation. We demonstrate the possibility to generate a long-lived intermediate whose proportion and occurrence are easily tunable by experimental parameters (i.e. temperature). As a consequence, in the case of aggregation processes developing through well-defined energy barriers, our results can open the way to new strategies to induce more stable in vitro on-pathway intermediate species through a minute change in the initial conformational flexibility of the protein. This will allow isolating and experimentally studying such transient species, often indicated as relevant in neurodegenerative diseases, both in terms of structural and cytotoxic properties
Proinflammatory and Amyloidogenic S100A9 Induced by Traumatic Brain Injury in Mouse Model
No full textTraumatic brain injury (TBI) represents a significant risk factor for development of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. The S100A9-driven amyloid-neuroinflammatory cascade occurring during primary and secondary TBI events can serve as a mechanistic link between TBI and Alzheimer’s as demonstrated recently in the human brain tissues. Here by using immunohistochemistry in the controlled cortical impact TBI mouse model we have found pro-inflammatory S100A9 in the brain tissues of all mice on the first and third post-TBI days, while 70% of mice did not show any S100A9 presence on seventh post-TBI day similar to controls. This indicates that defensive mechanisms effectively cleared S100A9 in these mouse brain tissues during post-TBI recovery. By using sequential immunohistochemistry we have shown that S100A9 was produced by both neuronal and microglial cells. However, Aβ peptide deposits characteristic for Alzheimer’s disease were not detected in any post-TBI animals. On the first and third post-TBI days S100A9 was found to aggregate intracellularly into amyloid oligomers, similar to what was previously observed in human TBI tissues. Complementary, by using Rayleigh scatting, intrinsic fluorescence and atomic force microscopy we demonstrated that in vitro S100A9 self-assembles into amyloid oligomers within minutes. Its amyloid aggregation is highly dependent on changes of environmental conditions such as variation of calcium levels, pH, temperature and reduction/oxidation, which might be relevant to perturbation of cellular and tissues homeostasis under TBI. Present results demonstrate that S100A9 induction mechanisms in TBI are similar in mice and humans, emphasizing that S100A9 is an important marker of brain injury and therefore can be a potential therapeutic target
Amyloid formation by the pro-inflammatory S100A8/A9 proteins in the ageing prostate.
No full textBACKGROUND: The conversion of soluble peptides and proteins into polymeric amyloid structures is a hallmark of many age-related degenerative disorders, including Alzheimer's disease, type II diabetes and a variety of systemic amyloidoses. We report here that amyloid formation is linked to another major age-related phenomenon--prostate tissue remodelling in middle-aged and elderly men. METHODOLOGY/PRINCIPAL FINDINGS: By using multidisciplinary analysis of corpora amylacea inclusions in prostate glands of patients diagnosed with prostate cancer we have revealed that their major components are the amyloid forms of S100A8 and S100A9 proteins associated with numerous inflammatory conditions and types of cancer. In prostate protease rich environment the amyloids are stabilized by dystrophic calcification and lateral thickening. We have demonstrated that material closely resembling CA can be produced from S100A8/A9 in vitro under native and acidic conditions and shows the characters of amyloids. This process is facilitated by calcium or zinc, both of which are abundant in ex vivo inclusions. These observations were supported by computational analysis of the S100A8/A9 calcium-dependent aggregation propensity profiles. We found DNA and proteins from Escherichia coli in CA bodies, suggesting that their formation is likely to be associated with bacterial infection. CA inclusions were also accompanied by the activation of macrophages and by an increase in the concentration of S100A8/A9 in the surrounding tissues, indicating inflammatory reactions. CONCLUSIONS/SIGNIFICANCE: These findings, taken together, suggest a link between bacterial infection, inflammation and amyloid deposition of pro-inflammatory proteins S100A8/A9 in the prostate gland, such that a self-perpetuating cycle can be triggered and may increase the risk of malignancy in the ageing prostate. The results provide strong support for the prediction that the generic ability of polypeptide chains to convert into amyloids could lead to their involvement in an increasing number of otherwise apparently unrelated diseases, particularly those associated with ageing.Original Publication:Kiran Yanamandra, Oleg Alexeyev, Vladimir Zamotin, Vaibhav Srivastava, Andrei Shchukarev, Ann-Christin Brorsson, Gian Gaetano Tartaglia, Thomas Vogl, Rakez Kayed, Gunnar Wingsle, Jan Olsson, Christopher M Dobson, Anders Bergh, Fredrik Elgh and Ludmilla A Morozova-Roche, Amyloid formation by the pro-inflammatory S100A8/A9 proteins in the ageing prostate., 2009, PloS one, (4), 5, e5562.http://dx.doi.org/10.1371/journal.pone.000556
The 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
Observation of the Early Structural Changes Leading to the Formation of Protein Superstructures.
No full textFormation of superstructures in protein aggregation processes has been indicated as a general pathway for several proteins, possibly playing a role in human pathologies. There is a severe lack of knowledge on the origin of such species in terms of both mechanisms of formation and structural features. We use equine lysozyme as a model protein, and by combining spectroscopic techniques and microscopy with X-ray fiber diffraction and ab initio modeling of Small Angle X-ray Scattering data, we isolate the partially unfolded state from which one of these superstructures (i.e., particulate) originates. We reveal the low-resolution structure of the unfolded state and its mechanism of formation, highlighting the physicochemical features and the possible pathway of formation of the particulate structure. Our findings provide a novel detailed knowledge of such a general and alternative aggregation pathway for proteins, this being crucial for a basic and broader understanding of the aggregation phenomena
Protein misfolding and self-assembly: acquired functions and their therapeutic and pathological significance
No full textEquine 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
AMYLOID-NEUROINFLAMMATORY CASCADE IN NEURODEGENERATIVE DISEASES – ROLE OF PRO-INFLAMMATORY S100 PROTEINS
No full textPro-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
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WORKSHOP 1 Green Building Certification Systems / Energy Standards in US
No full textLudmilla Pavlova is Senior Facilities Planner in Campus Planning at UMass Amherst and is responsible for master plan programming and planning for research, academic and administrative facilities and developing design guidelines.
Ludmilla has managed the planning of major capital projects including Harold Alfond Hall/ ISOM Addition, the Integrated Science Building, the Berkshire Dining Commons Renovation, the Southwest Residential Area Concourse, the Comprehensive Academic and Classroom Facilities Plan and the Library Master Plan.
Passionate about campus sustainability, Ludmilla often lectures on the topic and helped author the first UMass Amherst Sustainability Plan. Prior to coming to UMass she worked for design firms in New York City most notably for John M.Y. Lee/ Michael Timchula Associates, managing the design and construction of the Curtis/Balkan Factory in Sofia, Bulgaria, and as a member of the team that designed the Shenzhen City Center Urban Design and City Hall, Shenzhen, China.
Ludmilla has a Bachelor of Arts in Architecture and European Cultural Studies from Princeton University and a Masters of Architecture from Columbia University. She is Chair of the USGBC MA Chapter Western MA Committee and Secretary of the Western MA AIA.Learning objective: to learn about energy standards and how to design advanced energy efficient building envelopes. Passive house principals and fundamentals on thermal, moisture issues, and air tightness will be covere
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