scholarly journals 2.7 Å cryo-EM structure of ex vivo RML prion fibrils

2021 ◽  
Author(s):  
Szymon W. Manka ◽  
Wenjuan Zhang ◽  
Adam Wenborn ◽  
Jemma Betts ◽  
Susan Joiner ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Structural Features ◽  
Prion Strain ◽  
Conformational Variations ◽  
Molecular Determinants ◽  
Prion Propagation ◽  
Distinguishing Features ◽  
The Brain

Mammalian prions are lethal infectious agents that propagate as distinct strains and are composed of multichain assemblies of misfolded host-encoded prion protein (PrP), often referred to as prion rods. The structural features that define infectious prion rods and the molecular determinants of prion strain diversity are poorly understood. Here, we present a near-atomic resolution cryo-EM structure of PrP fibrils present in highly infectious prion rod preparations isolated from the brains of RML prion-infected mice. We found that prion rods comprise single-protofilament helical amyloid fibrils that coexist with twisted pairs of the same protofilaments. Each rung of the protofilament is formed by a single PrP monomer with the ordered core comprising PrP residues 94-225, which folds to create two asymmetric lobes with the N-linked glycans and the glycosylphosphatidylinositol anchor projecting from the C-terminal lobe. The overall architecture is comparable to that of recently reported PrP fibrils isolated from the brain of hamsters infected with the 263K prion strain. However, there are marked conformational variations that could result from differences in PrP primary sequence and/or represent distinguishing features of the distinct prion strains. These conformational changes impact the overall geometry of the fibrils and may also impact fibril pairing, one or both of which may critically influence PrP glycoform selection that occurs during strain-specific prion propagation.

scholarly journals Molecular determinants of the physicochemical properties of a critical prion protein region comprising residues 106–126

1999 ◽  
Vol 342 (1) ◽  
pp. 207-214 ◽  
Author(s):  
Mario SALMONA ◽  
Paolo MALESANI ◽  
Luca DE GIOIA ◽  
Stefano GORLA ◽  
Maurizio BRUSCHI ◽  
...  
Keyword(s):  
Prion Protein ◽  
Conformational Changes ◽  
Amyloid Fibrils ◽  
Physicochemical Characteristics ◽  
Cellular Prion Protein ◽  
Random Coil ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
C Terminus ◽  
Molecular Determinants

Prion diseases are marked by the cerebral accumulation of conformationally modified forms of the cellular prion protein (PrPC), known as PrPres. The region comprising the residues 106-126 of human PrP seems to have a key role in this conformational conversion, because a synthetic peptide homologous with this sequence (PrP106-126) adopts different secondary structures in different environments. To investigate the molecular determinants of the physicochemical characteristics of PrP106-126, we synthesized a series of analogues including PrP106-126 HD, PrP106-126 A and PrP106-126 K, with L-His → D-His, His → Ala and His → Lys substitutions respectively at position 111, PrP106-126 NH2 with amidation of the C-terminus, PrP106-126 V with an Ala → Val substition at position 117, and PrP106-126 VNH2 with an Ala → Val substitution at position 117 and amidation of the C-terminus. The analysis of the secondary structure and aggregation properties of PrP106-126 and its analogues showed the following. (1) His111 is central to the conformational changes of PrP peptides. (2) Amidation of the C-terminal Gly126 yields a predominantly random coil structure, abolishes the molecular polymorphism and decreases the propensity of PrP106-126 to generate amyloid fibrils. (3) PrP106-126 V, carrying an Ala → Val substitution at position 117, does not demonstrate a fibrillogenic ability superior to that of PrP106-126. However, the presence of Val at position 117 increases the aggregation properties of the amidated peptide. (4) Amyloid fibrils are not required for neurotoxicity because the effects of PrP106-126 NH2 on primary neuronal cultures were similar to those of the wild-type sequence. Conversely, astroglial proliferation is related to the presence of amyloid fibrils, suggesting that astrogliosis in prion encephalopathies without amyloid deposits is a mediated effect rather than a direct effect of disease-specific PrP isoforms.

scholarly journals Human transthyretin in complex with iododiflunisal: structural features associated with a potent amyloid inhibitor

2005 ◽  
Vol 388 (2) ◽  
pp. 615-621 ◽  
Author(s):  
Luís GALES ◽  
Sandra MACEDO-RIBEIRO ◽  
Gemma ARSEQUELL ◽  
Gregorio VALENCIA ◽  
Maria João SARAIVA ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Structural Features ◽  
Familial Amyloidotic Polyneuropathy ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
The Stability ◽  
New Interactions

Ex vivo and in vitro studies have revealed the remarkable amyloid inhibitory potency and specificity of iododiflunisal in relation to transthyretin [Almeida, Macedo, Cardoso, Alves, Valencia, Arsequell, Planas and Saraiva (2004) Biochem. J. 381, 351–356], a protein implicated in familial amyloidotic polyneuropathy. In the present paper, the crystal structure of transthyretin complexed with this diflunisal derivative is reported, which enables a detailed analysis of the protein–ligand interactions. Iododiflunisal binds very deep in the hormone-binding channel. The iodine substituent is tightly anchored into a pocket of the binding site and the fluorine atoms provide extra hydrophobic contacts with the protein. The carboxylate substituent is involved in an electrostatic interaction with the Nζ of a lysine residue. Moreover, ligand-induced conformational alterations in the side chain of some residues result in the formation of new intersubunit hydrogen bonds. All these new interactions, induced by iododiflunisal, increase the stability of the tetramer impairing the formation of amyloid fibrils. The crystal structure of this complex opens perspectives for the design of more specific and effective drugs for familial amyloidotic polyneuropathy patients.

pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase

2019 ◽  
Vol 26 (10) ◽  
pp. 743-750 ◽  
Author(s):  
Remya Radha ◽  
Sathyanarayana N. Gummadi
Keyword(s):  
Vibrio Cholerae ◽  
Conformational Changes ◽  
Kinetic Studies ◽  
Structural Features ◽  
Structure Stability ◽  
Kcal Mole ◽  
Scanning Calorimetry ◽  
Wide Range ◽  
Ph Dependent ◽  
Ph Range

Background:pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.Objective:The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.Methods:Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.Results:The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.Conclusion:The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.

scholarly journals Synthesis and pharmacokinetic characterisation of a fluorine-18 labelled brain shuttle peptide fusion dimeric affibody

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takahiro Morito ◽  
Ryuichi Harada ◽  
Ren Iwata ◽  
Yiqing Du ◽  
Nobuyuki Okamura ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Post Injection ◽  
Affibody Molecule ◽  
Positron Emission ◽  
A Cell ◽  
Protein Binders ◽  
Rapid Clearance ◽  
Labelling Method ◽  
The Brain ◽  
Ex Vivo Study

AbstractBrain positron emission tomography (PET) imaging with radiolabelled proteins is an emerging concept that potentially enables visualization of unique molecular targets in the brain. However, the pharmacokinetics and protein radiolabelling methods remain challenging. Here, we report the performance of an engineered, blood–brain barrier (BBB)-permeable affibody molecule that exhibits rapid clearance from the brain, which was radiolabelled using a unique fluorine-18 labelling method, a cell-free protein radiosynthesis (CFPRS) system. AS69, a small (14 kDa) dimeric affibody molecule that binds to the monomeric and oligomeric states of α-synuclein, was newly designed for brain delivery with an apolipoprotein E (ApoE)-derived brain shuttle peptide as AS69-ApoE (22 kDa). The radiolabelled products 18F-AS69 and 18F-AS69-ApoE were successfully synthesised using the CFPRS system. Notably, 18F-AS69-ApoE showed higher BBB permeability than 18F-AS69 in an ex vivo study at 10 and 30 min post injection and was partially cleared from the brain at 120 min post injection. These results suggest that small, a brain shuttle peptide-fused fluorine-18 labelled protein binders can potentially be utilised for brain molecular imaging.

scholarly journals Cryo-EM structure of amyloid fibrils formed by the entire low complexity domain of TDP-43

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qiuye Li ◽  
W. Michael Babinchak ◽  
Witold K. Surewicz
Keyword(s):  
Amyloid Fibrils ◽  
Low Complexity ◽  
Structural Features ◽  
Protein Fragments ◽  
Hydrophobic Residues ◽  
Backbone Conformation ◽  
Hydrophobic Amino Acids ◽  
Insight Into ◽  
Lateral Sclerosis

AbstractAmyotrophic lateral sclerosis and several other neurodegenerative diseases are associated with brain deposits of amyloid-like aggregates formed by the C-terminal fragments of TDP-43 that contain the low complexity domain of the protein. Here, we report the cryo-EM structure of amyloid formed from the entire TDP-43 low complexity domain in vitro at pH 4. This structure reveals single protofilament fibrils containing a large (139-residue), tightly packed core. While the C-terminal part of this core region is largely planar and characterized by a small proportion of hydrophobic amino acids, the N-terminal region contains numerous hydrophobic residues and has a non-planar backbone conformation, resulting in rugged surfaces of fibril ends. The structural features found in these fibrils differ from those previously found for fibrils generated from short protein fragments. The present atomic model for TDP-43 LCD fibrils provides insight into potential structural perturbations caused by phosphorylation and disease-related mutations.

scholarly journals Co-factor-free aggregation of tau into seeding-competent RNA-sequestering amyloid fibrils

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pijush Chakraborty ◽  
Gwladys Rivière ◽  
Shu Liu ◽  
Alain Ibáñez de Opakua ◽  
Rıza Dervişoğlu ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Corticobasal Degeneration ◽  
Binding Studies ◽  
Rigid Core ◽  
Tau Aggregation ◽  
The Brain ◽  
Tau Aggregate ◽  
Disease Specific ◽  
Tau Fibrils

AbstractPathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.

scholarly journals Shear-Induced Amyloid Aggregation in the Brain: V. Are Alzheimer’s and Other Amyloid Diseases Initiated in the Lower Brain and Brainstem by Cerebrospinal Fluid Flow Stresses?

2020 ◽  
pp. 1-24
Author(s):  
Conrad N. Trumbore
Keyword(s):  
Cerebrospinal Fluid ◽  
Conformational Changes ◽  
Extensional Flow ◽  
Amyloid Β ◽  
High Energy ◽  
Mechanical Agitation ◽  
Cerebrospinal Fluid Flow ◽  
Pulse Waves ◽  
Amyloid Diseases ◽  
The Brain

Amyloid-β (Aβ) and tau oligomers have been identified as neurotoxic agents responsible for causing Alzheimer’s disease (AD). Clinical trials using Aβ and tau as targets have failed, giving rise to calls for new research approaches to combat AD. This paper provides such an approach. Most basic AD research has involved quiescent Aβ and tau solutions. However, studies involving laminar and extensional flow of proteins have demonstrated that mechanical agitation of proteins induces or accelerates protein aggregation. Recent MRI brain studies have revealed high energy, chaotic motion of cerebrospinal fluid (CSF) in lower brain and brainstem regions. These and studies showing CSF flow within the brain have shown that there are two energetic hot spots. These are within the third and fourth brain ventricles and in the neighborhood of the circle of Willis blood vessel region. These two regions are also the same locations as those of the earliest Aβ and tau AD pathology. In this paper, it is proposed that cardiac systolic pulse waves that emanate from the major brain arteries in the lower brain and brainstem regions and whose pulse waves drive CSF flows within the brain are responsible for initiating AD and possibly other amyloid diseases. It is further proposed that the triggering of these diseases comes about because of the strengthening of systolic pulses due to major artery hardening that generates intense CSF extensional flow stress. Such stress provides the activation energy needed to induce conformational changes of both Aβ and tau within the lower brain and brainstem region, producing unique neurotoxic oligomer molecule conformations that induce AD.

scholarly journals Myosinome: A Database of Myosins from Select Eukaryotic Genomes to Facilitate Analysis of Sequence-Structure-Function Relationships

2012 ◽  
Vol 6 ◽  
pp. BBI.S9902 ◽  
Author(s):  
Divya P. Syamaladevi ◽  
Margaret S Sunitha ◽  
S. Kalaimathy ◽  
Chandrashekar C. Reddy ◽  
Mohammed Iftekhar ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Homo Sapiens ◽  
Relevant Literature ◽  
Myosin Ii ◽  
Coiled Coil ◽  
Structural Features ◽  
Model Organisms ◽  
Congenital Diseases ◽  
C Elegans

Myosins are one of the largest protein superfamilies with 24 classes. They have conserved structural features and catalytic domains yet show huge variation at different domains resulting in a variety of functions. Myosins are molecules driving various kinds of cellular processes and motility until the level of organisms. These are ATPases that utilize the chemical energy released by ATP hydrolysis to bring about conformational changes leading to a motor function. Myosins are important as they are involved in almost all cellular activities ranging from cell division to transcriptional regulation. They are crucial due to their involvement in many congenital diseases symptomatized by muscular malfunctions, cardiac diseases, deafness, neural and immunological dysfunction, and so on, many of which lead to death at an early age. We present Myosinome, a database of selected myosin classes (myosin II, V, and VI) from five model organisms. This knowledge base provides the sequences, phylogenetic clustering, domain architectures of myosins and molecular models, structural analyses, and relevant literature of their coiled-coil domains. In the current version of Myosinome, information about 71 myosin sequences belonging to three myosin classes (myosin II, V, and VI) in five model organisms ( Homo Sapiens, Mus musculus, D. melanogaster, C. elegans and S. cereviseae) identified using bioinformatics surveys are presented, and several of them are yet to be functionally characterized. As these proteins are involved in congenital diseases, such a database would be useful in short-listing candidates for gene therapy and drug development. The database can be accessed from http://caps.ncbs.res.in/myosinome .

beta 2-microglobulin can be refolded into a native state from ex vivo amyloid fibrils

1998 ◽  
Vol 258 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Vittorio Bellotti ◽  
Monica Stoppini ◽  
Palma Mangione ◽  
Margaret Sunde ◽  
Carol Robinson ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Native State ◽  
Beta 2 ◽  
Beta 2 Microglobulin

Methods to study the structure of misfolded protein states in systemic amyloidosis

2021 ◽  
Vol 49 (2) ◽  
pp. 977-985
Author(s):  
Marcus Fändrich ◽  
Matthias Schmidt
Keyword(s):  
Protein Misfolding ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Systemic Amyloidosis ◽  
Structural Basis ◽  
Amyloid A ◽  
Serum Amyloid A Protein ◽  
Proteolytic Stability

Systemic amyloidosis is defined as a protein misfolding disease in which the amyloid is not necessarily deposited within the same organ that produces the fibril precursor protein. There are different types of systemic amyloidosis, depending on the protein constructing the fibrils. This review will focus on recent advances made in the understanding of the structural basis of three major forms of systemic amyloidosis: systemic AA, AL and ATTR amyloidosis. The three diseases arise from the misfolding of serum amyloid A protein, immunoglobulin light chains or transthyretin. The presented advances in understanding were enabled by recent progress in the methodology available to study amyloid structures and protein misfolding, in particular concerning cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy. An important observation made with these techniques is that the structures of previously described in vitro formed amyloid fibrils did not correlate with the structures of amyloid fibrils extracted from diseased tissue, and that in vitro fibrils were typically more protease sensitive. It is thus possible that ex vivo fibrils were selected in vivo by their proteolytic stability.

Sign in / Sign up

Export Citation Format

Share Document