scholarly journals Dangerous Stops: Nonsense Mutations Can Dramatically Increase Frequency of Prion Conversion

2021 ◽  
Vol 22 (4) ◽  
pp. 1542
Author(s):  
Alexander A. Dergalev ◽  
Valery N. Urakov ◽  
Michael O. Agaphonov ◽  
Alexander I. Alexandrov ◽  
Vitaly V. Kushnirov
Keyword(s):  
De Novo ◽  
Yeast Cells ◽  
Functional Domain ◽  
Amyloid Formation ◽  
Nonsense Mutations ◽  
C Terminus ◽  
Sporadic Cases ◽  
Amyloidogenic Region ◽  
Prion Conversion

Amyloid formation is associated with many incurable diseases. For some of these, sporadic cases are much more common than familial ones. Some reports point to the role of somatic cell mosaicism in these cases via origination of amyloids in a limited number of cells, which can then spread through tissues. However, specific types of sporadic mutations responsible for such effects are unknown. In order to identify mutations capable of increasing the de novo appearance of amyloids, we searched for such mutants in the yeast prionogenic protein Sup35. We introduced to yeast cells an additional copy of the SUP35 gene with mutated amyloidogenic domain and observed that some nonsense mutations increased the incidence of prions by several orders of magnitude. This effect was related to exposure at the C-terminus of an internal amyloidogenic region of Sup35. We also discovered that SUP35 mRNA could undergo splicing, although inefficiently, causing appearance of a shortened Sup35 isoform lacking its functional domain, which was also highly prionogenic. Our data suggest that truncated forms of amyloidogenic proteins, resulting from nonsense mutations or alternative splicing in rare somatic cells, might initiate spontaneous localized formation of amyloids, which can then spread, resulting in sporadic amyloid disease.

scholarly journals Structure of APP-C991-99 and Implications for Role of Extra-Membrane Domains in Function and Oligomerization

2018 ◽  
Author(s):  
George A. Pantelopulos ◽  
John E. Straub ◽  
D. Thirumalai ◽  
Yuji Sugita
Keyword(s):  
Transmembrane Domain ◽  
Chemical Shifts ◽  
Membrane Surface ◽  
Critical Role ◽  
Amyloid Formation ◽  
Cytoplasmic Proteins ◽  
C Terminus ◽  
N Terminus ◽  
Thin Membranes

AbstractThe 99 amino acid C-terminal fragment of Amyloid Precursor Protein APP-C99 (C99) is cleaved by γ-secretase to form Aβ peptide, which plays a critical role in the etiology of Alzheimer’s Disease (AD). The structure of C99 consists of a single transmembrane domain flanked by intra and intercellular domains. While the structure of the transmembrane domain has been well characterized, little is known about the structure of the flanking domains and their role in C99 processing by γ-secretase. To gain insight into the structure of full-length C99, REMD simulations were performed for monomeric C99 in model membranes of varying thickness. We find equilibrium ensembles of C99 from simulation agree with experimentally-inferred residue insertion depths and protein backbone chemical shifts. In thin membranes, the transmembrane domain structure is correlated with extra-membrane structural states. Mean and variance of the transmembrane and G37G38 hinge angles are found to increase with thinning membrane. The N-terminus of C99 forms β-strands that may seed aggregation of Aβ on the membrane surface, promoting amyloid formation. The N-terminus, which forms α-helices that interact with the nicastrin domain of γ-secretase. The C-terminus of C99 becomes more α-helical as the membrane thickens, forming structures that may be suitable for binding by cytoplasmic proteins, while C-terminal residues essential to cytotoxic function become α-helical as the membrane thins. The heterogeneous but discrete extra-membrane domain states analyzed here open the path to new investigations of the role of C99 structure and membrane in amyloidogenesis.

scholarly journals Mutations Outside the Ure2 Amyloid-Forming Region Disrupt [URE3] Prion Propagation and Alter Interactions with Protein Quality Control Factors

2020 ◽  
Vol 40 (21) ◽  
Author(s):  
Shailesh Kumar ◽  
Elliot A. Dine ◽  
Ethan Paddock ◽  
Danielle N. Steinberg ◽  
Lois E. Greene ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
De Novo ◽  
Protein Quality Control ◽  
Functional Domain ◽  
Amyloid Formation ◽  
Yeast Prions ◽  
Control Factors ◽  
Prion Propagation

ABSTRACT The yeast prion [URE3] propagates as a misfolded amyloid form of the Ure2 protein. Propagation of amyloid-based yeast prions requires protein quality control (PQC) factors, and altering PQC abundance or activity can cure cells of prions. Yeast antiprion systems composed of PQC factors act at normal abundance to restrict establishment of the majority of prion variants that arise de novo. While these systems are well described, how they or other PQC factors interact with prion proteins remains unclear. To gain insight into such interactions, we identified mutations outside the Ure2 prion-determining region that destabilize [URE3]. Despite residing in the functional domain, 16 of 17 mutants retained Ure2 activity. Four characterized mutations caused rapid loss of [URE3] yet allowed [URE3] to propagate under prion-selecting conditions. Two sensitized [URE3] to Btn2, Cur1, and Hsp42, but in different ways. Two others reduced amyloid formation in vitro. Of these, one impaired prion replication and the other apparently impaired transmission. Thus, widely dispersed sites outside a prion’s amyloid-forming region can contribute to prion character, and altering such sites can disrupt prion propagation by altering interactions with PQC factors.

scholarly journals Mechanism of De Novo Initiation by the Hepatitis C Virus RNA-Dependent RNA Polymerase: Role of Divalent Metals

2002 ◽  
Vol 76 (24) ◽  
pp. 12513-12525 ◽  
Author(s):  
C. T. Ranjith-Kumar ◽  
Young-Chan Kim ◽  
Les Gutshall ◽  
Carol Silverman ◽  
Sanjay Khandekar ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
De Novo ◽  
Primer Extension ◽  
Diarrhea Virus ◽  
C Terminus ◽  
Virus Rna ◽  
Tryptophan Residues

ABSTRACT We functionally analyzed the role of metal ions in RNA-dependent RNA synthesis by three recombinant RNA-dependent RNA polymerases (RdRps) from GB virus-B (GBV), bovine viral diarrhea virus (BVDV), and hepatitis C virus (HCV), with emphasis on the HCV RdRp. Using templates capable of both de novo initiation and primer extension and RdRps purified in the absence of metal, we found that only reactions with exogenously provided Mg2+ and Mn2+ gave rise to significant amounts of synthesis. Mg2+ and Mn2+ affected the mode of RNA synthesis by the three RdRps. Both metals supported primer-dependent and de novo-initiated RNA by the GBV RdRp, while Mn2+ significantly increased the amount of de novo-initiated products by the HCV and BVDV RdRps. For the HCV RdRp, Mn2+ reduced the Km for the initiation nucleotide, a GTP, from 103 to 3 μM. However, it increased de novo initiation even at GTP concentrations that are comparable to physiological levels. We hypothesize that a change in RdRp structure occurs upon GTP binding to prevent primer extension. Analysis of deleted proteins revealed that the C terminus of the HCV RdRp plays a role in Mn2+-induced de novo initiation and can contribute to the suppression of primer extension. Spectroscopy examining the intrinsic fluorescence of tyrosine and tryptophan residues in the HCV RdRp produced results consistent with the protein undergoing a conformational change in the presence of metal. These results document the fact that metal can affect de novo initiation or primer extension by flaviviral RdRps.

scholarly journals The Hsc70 disaggregation machinery removes monomer units directly from α-synuclein fibril ends

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Matthias M. Schneider ◽  
Saurabh Gautam ◽  
Therese W. Herling ◽  
Ewa Andrzejewska ◽  
Georg Krainer ◽  
...  
Keyword(s):  
Molecular Chaperones ◽  
De Novo ◽  
Cellular Protein ◽  
Amyloid Formation ◽  
First Order ◽  
Hsp70 Family ◽  
First Order Kinetics ◽  
Reaction Proceeds ◽  
Kinetics Of

AbstractMolecular chaperones contribute to the maintenance of cellular protein homoeostasis through assisting de novo protein folding and preventing amyloid formation. Chaperones of the Hsp70 family can further disaggregate otherwise irreversible aggregate species such as α-synuclein fibrils, which accumulate in Parkinson’s disease. However, the mechanisms and kinetics of this key functionality are only partially understood. Here, we combine microfluidic measurements with chemical kinetics to study α-synuclein disaggregation. We show that Hsc70 together with its co-chaperones DnaJB1 and Apg2 can completely reverse α-synuclein aggregation back to its soluble monomeric state. This reaction proceeds through first-order kinetics where monomer units are removed directly from the fibril ends with little contribution from intermediate fibril fragmentation steps. These findings extend our mechanistic understanding of the role of chaperones in the suppression of amyloid proliferation and in aggregate clearance, and inform on possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies.

scholarly journals Lipids and Trehalose Actively Cooperate in Heat Stress Management of Schizosaccharomyces pombe

2021 ◽  
Vol 22 (24) ◽  
pp. 13272
Author(s):  
Mária Péter ◽  
Péter Gudmann ◽  
Zoltán Kóta ◽  
Zsolt Török ◽  
László Vígh ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Heat Stress ◽  
Schizosaccharomyces Pombe ◽  
De Novo ◽  
Acyl Chain ◽  
Membrane Lipid ◽  
Yeast Cells ◽  
Storage Lipids ◽  
Acyl Chain Length

Homeostatic maintenance of the physicochemical properties of cellular membranes is essential for life. In yeast, trehalose accumulation and lipid remodeling enable rapid adaptation to perturbations, but their crosstalk was not investigated. Here we report about the first in-depth, mass spectrometry-based lipidomic analysis on heat-stressed Schizosaccharomyces pombe mutants which are unable to synthesize (tps1Δ) or degrade (ntp1Δ) trehalose. Our experiments provide data about the role of trehalose as a membrane protectant in heat stress. We show that under conditions of trehalose deficiency, heat stress induced a comprehensive, distinctively high-degree lipidome reshaping in which structural, signaling and storage lipids acted in concert. In the absence of trehalose, membrane lipid remodeling was more pronounced and increased with increasing stress dose. It could be characterized by decreasing unsaturation and increasing acyl chain length, and required de novo synthesis of stearic acid (18:0) and very long-chain fatty acids to serve membrane rigidification. In addition, we detected enhanced and sustained signaling lipid generation to ensure transient cell cycle arrest as well as more intense triglyceride synthesis to accommodate membrane lipid-derived oleic acid (18:1) and newly synthesized but unused fatty acids. We also demonstrate that these changes were able to partially substitute for the missing role of trehalose and conferred measurable stress tolerance to fission yeast cells.

A KCNC1 mutation in Epilepsy of Infancy with Focal Migrating Seizures produces functional channels that fail to be regulated by phosphorylation

2021 ◽  
Author(s):  
Yalan Zhang ◽  
Syed R Ali ◽  
Rima Nabbout ◽  
Giulia Barcia ◽  
Leonard K. Kaczmarek
Keyword(s):  
De Novo ◽  
Channel Modulation ◽  
Progressive Myoclonus Epilepsy ◽  
C Terminus ◽  
Myoclonus Epilepsy ◽  
Voltage Dependent ◽  
Genes Encoding ◽  
Clear Change

Channelopathies caused by mutations in genes encoding ion channels generally produce a clear change in channel function. Accordingly, mutations in KCNC1, which encodes the voltage-dependent Kv3.1 potassium channel, result in Progressive Myoclonus Epilepsy as well as other Developmental and Epileptic Encephalopathies, and these have been shown to reduce or fully abolish current amplitude. One exception to this is the mutation A513V Kv3.1b, located in the cytoplasmic C-terminal domain of the channel protein. This de novo variant was detected in a patient with Epilepsy of Infancy with Focal Migrating Seizures (EIFMS) but no difference could be detected between A513V Kv3.1 current and that of wild type Kv3.1. Using both biochemical and electrophysiological approaches, we have now confirmed that this variant produces functional channels but find that the A513V mutation renders the channel completely insensitive to regulation by phosphorylation at S503, a nearby regulatory site in the C-terminus. In this respect, the mutation resembles those in another channel, KCNT1, which are the major cause of EIFMS. Because the amplitude of Kv3.1 current is constantly adjusted by phosphorylation in vivo, our findings suggest that loss of such regulation contributes to EIFMS phenotype and emphasize the role of channel modulation for normal neuronal function.

scholarly journals Role of Force-Sensitive Amyloid-Like Interactions in Fungal Catch Bonding and Biofilms

2014 ◽  
Vol 13 (9) ◽  
pp. 1136-1142 ◽  
Author(s):  
Cho X. J. Chan ◽  
Peter N. Lipke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Laminar Flow ◽  
Biofilm Formation ◽  
Amyloid Peptide ◽  
Yeast Cells ◽  
Amyloid Formation ◽  
Cell Binding ◽  
Flow Shear ◽  
Content Type

ABSTRACTTheCandida albicansAls adhesin Als5p has an amyloid-forming sequence that is required for aggregation and formation of model biofilms on polystyrene. Because amyloid formation can be triggered by force, we investigated whether laminar flow could activate amyloid formation and increase binding to surfaces. ShearingSaccharomyces cerevisiaecells expressing Als5p orC. albicansat 0.8 dyne/cm2increased the quantity and strength of cell-to-surface and cell-to-cell binding compared to that at 0.02 dyne/cm2. Thioflavin T fluorescence showed that the laminar flow also induced adhesin aggregation into surface amyloid nanodomains in Als5p-expressing cells. Inhibitory concentrations of the amyloid dyes thioflavin S and Congo red or a sequence-specific anti-amyloid peptide decreased binding and biofilm formation under flow. Shear-induced binding also led to formation of robust biofilms. There was less shear-activated increase in adhesion, thioflavin fluorescence, and biofilm formation in cells expressing the amyloid-impaired V326N-substituted Als5p. Similarly,S. cerevisiaecells expressing Flo1p or Flo11p flocculins also showed shear-dependent binding, amyloid formation, biofilm formation, and inhibition by anti-amyloid compounds. Together, these results show that laminar flow activated amyloid formation and led to enhanced adhesion of yeast cells to surfaces and to biofilm formation.

Posttransplant CMV infection and the role of immunosuppression (Sponsor: Novartis Pharma GmbH, Nürnberg)

2016 ◽  
Vol 04 (01) ◽  
pp. 4-10
Keyword(s):  
Lower Incidence ◽  
Paradigm Shift ◽  
Mtor Inhibitor ◽  
De Novo ◽  
Expert Panel ◽  
Risk Of Infection ◽  
Cmv Infection ◽  
Expert Meeting ◽  
Selection Of

AbstractImmunosuppression permits graft survival after transplantation and consequently a longer and better life. On the other hand, it increases the risk of infection, for instance with cytomegalovirus (CMV). However, the various available immunosuppressive therapies differ in this regard. One of the first clinical trials using de novo everolimus after kidney transplantation [1] already revealed a considerably lower incidence of CMV infection in the everolimus arms than in the mycophenolate mofetil (MMF) arm. This result was repeatedly confirmed in later studies [2–4]. Everolimus is now considered a substance with antiviral properties. This article is based on the expert meeting “Posttransplant CMV infection and the role of immunosuppression”. The expert panel called for a paradigm shift: In a CMV prevention strategy the targeted selection of the immunosuppressive therapy is also a key element. For patients with elevated risk of CMV, mTOR inhibitor-based immunosuppression is advantageous as it is associated with a significantly lower incidence of CMV events.

Factor Xa Enhances the Binding of Tissue Factor Pathway Inhibitor to Acidic Phospholipids

1996 ◽  
Vol 75 (05) ◽  
pp. 796-800 ◽  
Author(s):  
Sanne Valentin ◽  
Inger Schousboe
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Microtitre Plate ◽  
C Terminus ◽  
Microtitre Plate Assay ◽  
Kunitz Domain ◽  
Tissue Factor Pathway ◽  
Acidic Phospholipids

SummaryIn the present study, the interaction between tissue factor pathway inhibitor (TFPI) and phospholipids has been characterized using a microtitre plate assay. TFPI was shown to bind calcium-independently to an acidic phospholipid surface composed of phosphatidylserine, but not a surface composed of the neutral phosphatidylcholine. The interaction was demonstrated to be dependent on the presence of the TFPI C-terminus. The presence of heparin (1 U/ml, unfractionated) was able to significantly reduce the binding of TFPI to phospholipid. The interaction of TFPI with phosphatidylserine was significantly decreased in the presence of calcium, but this was counteracted, and even enhanced, following complex formation of TFPI with factor Xa prior to incubation with the phospholipid surface. Moreover, a TFPI variant, not containing the third Kunitz domain and the C-terminus, was unable to bind to phospholipid. However, following the formation of a TFPI/factor Xa-complex this TFPI variant was capable of interacting with the phospholipid surface. This indicates that the role of factor Xa as a TFPI cofactor, at least in part, is to mediate the binding of TFPI to the phospholipid surface.

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