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51.
Ghazaleh Eskandari-Sedighi Nathalie Daude Hristina Gapeshina David W. Sanders Razieh Kamali-Jamil Jing Yang Beipei Shi Holger Wille Bernardino Ghetti Marc I. Diamond Christopher Janus David Westaway 《Molecular neurodegeneration》2017,12(1):72
Background
MAPT mutations cause neurodegenerative diseases such as frontotemporal dementia but, strikingly, patients with the same mutation may have different clinical phenotypes.Methods
Given heterogeneities observed in a transgenic (Tg) mouse line expressing low levels of human (2 N, 4R) P301L Tau, we backcrossed founder stocks of mice to C57BL/6Tac, 129/SvEvTac and FVB/NJ inbred backgrounds to discern the role of genetic versus environmental effects on disease-related phenotypes.Results
Three inbred derivatives of a TgTauP301L founder line had similar quality and steady-state quantity of Tau production, accumulation of abnormally phosphorylated 64–68 kDa Tau species from 90 days of age onwards and neuronal loss in aged Tg mice. Variegation was not seen in the pattern of transgene expression and seeding properties in a fluorescence-based cellular assay indicated a single “strain” of misfolded Tau. However, in other regards, the aged Tg mice were heterogeneous; there was incomplete penetrance for Tau deposition despite maintained transgene expression in aged animals and, for animals with Tau deposits, distinctions were noted even within each subline. Three classes of rostral deposition in the cortex, hippocampus and striatum accounted for 75% of pathology-positive mice yet the mean ages of mice scored as class I, II or III were not significantly different and, hence, did not fit with a predictable progression from one class to another defined by chronological age. Two other patterns of Tau deposition designated as classes IV and V, occurred in caudal structures. Other pathology-positive Tg mice of similar age not falling within classes I-V presented with focal accumulations in additional caudal neuroanatomical areas including the locus coeruleus. Electron microscopy revealed that brains of Classes I, II and IV animals all exhibit straight filaments, but with coiled filaments and occasional twisted filaments apparent in Class I. Most strikingly, Class I, II and IV animals presented with distinct western blot signatures after trypsin digestion of sarkosyl-insoluble Tau.Conclusions
Qualitative variations in the neuroanatomy of Tau deposition in genetically constrained slow models of primary Tauopathy establish that non-synchronous, focal events contribute to the pathogenic process. Phenotypic diversity in these models suggests a potential parallel to the phenotypic variation seen in P301L patients.52.
Feng BY Toyama BH Wille H Colby DW Collins SR May BC Prusiner SB Weissman J Shoichet BK 《Nature chemical biology》2008,4(3):197-199
Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited beta-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus. 相似文献
53.
54.
Insulin, a small hormone protein comprising 51 residues in two disulfide-linked polypeptide chains, adopts a predominantly α-helical conformation in its native state. It readily undergoes protein misfolding and aggregates into amyloid fibrils under a variety of conditions. Insulin is a unique model system in which to study protein fibrillization, since its three disulfide bridges are retained in the fibrillar state and thus limit the conformational space available to the polypeptide chains during misfolding and fibrillization. Taking into account this unique conformational restriction, we modeled possible monomeric subunits of the insulin amyloid fibrils using β-solenoid folds, namely, the β-helix and β-roll. Both models agreed with currently available biophysical data. We performed molecular dynamics simulations, which allowed some limited insights into the relative structural stability, suggesting that the β-roll subunit model may be more stable than the β-helix subunit model. We also constructed β-solenoid-based insulin fibril models and conducted fiber diffraction simulation to identify plausible fibril architectures of insulin amyloid. A comparison of simulated fiber diffraction patterns of the fibril models to the experimental insulin x-ray fiber diffraction data suggests that the model fibers composed of six twisted β-roll protofilaments provide the most reasonable fit to available experimental diffraction patterns and previous biophysical studies. 相似文献
55.
Steffen Kutter Manfred S. Weiss Georg Wille Ralph Golbik Michael Spinka Stephan K?nig 《The Journal of biological chemistry》2009,284(18):12136-12144
The mechanism by which the enzyme pyruvate decarboxylase from two yeast
species is activated allosterically has been elucidated. A total of seven
three-dimensional structures of the enzyme, of enzyme variants, or of enzyme
complexes from two yeast species, three of them reported here for the first
time, provide detailed atomic resolution snapshots along the activation
coordinate. The prime event is the covalent binding of the substrate pyruvate
to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction
causes the shift of a neighboring amino acid, which eventually leads to the
rigidification of two otherwise flexible loops, one of which provides two
histidine residues necessary to complete the enzymatically competent active
site architecture. The structural data are complemented and supported by
kinetic investigations and binding studies, providing a consistent picture of
the structural changes occurring upon enzyme activation.Pyruvate decarboxylases (EC 4.1.1.1) catalyze the non-oxidative
decarboxylation of pyruvate, yielding acetaldehyde and carbon dioxide.
Together with the enzyme alcohol dehydrogenase (EC 1.1.1.1), which reduces the
acetaldehyde to ethanol with the help of the co-substrate NADH, it represents
the metabolic pathway of alcoholic fermentation.
PDC3 is localized in
the cytosol of cells from yeasts, plant seeds, and a few bacteria. The
catalytic activity of PDC depends on the presence of the cofactor thiamine
diphosphate (ThDP), which is bound mainly via a divalent metal ion (magnesium
in most cases) to the protein moiety. Many detailed kinetic studies have been
published on yeast PDC wild types
(1–9).
A number of ScPDC variants were analyzed, too
(1–9).
Some active site variants (E51A, D28A, E477Q) proved to be almost
catalytically inactive. PDCs are multisubunit enzymes. The typical molecular
mass of one subunit is 59–61 kDa. The tetramer is the catalytically
active state of most PDCs. Higher oligomers (octamers) have been described for
PDCs from plant seeds (10,
11) or some fungi
(12). However, studies on
structure function relationships of yeast PDCs showed that the dimer is the
minimum functional unit of the enzyme displaying considerable catalytic
activity (13,
14). The two closely related
pyruvate decarboxylases from Saccharomyces cerevisiae
(ScPDC) and Kluyveromyces lactis (KlPDC) are well
characterized ThDP-dependent enzymes, which share 86.3% identical amino acid
residues. They have been studied in great detail by means of kinetic
investigations and spectroscopic studies. Both enzymes are allosterically
regulated as reflected by sigmoid steady state kinetics and lag phases in
their progress curves. The substrate PYR activates the initially inactive
yeast PDCs in a time-dependent manner. Kinetic studies reveal a slow
isomerization as triggered by substrate binding to a separate regulatory site
(15). A number of substrate
surrogates have been identified, which are able to activate PDC as well. The
effects of pyruvamide (PA; for the chemical structure, see
Scheme 1) on the activation
kinetics have been studied in detail for ScPDC
(15) and for KlPDC
(16). Phosphonate analogues
(among them methyl acetylphosphonate, MAP,
Scheme 1) of pyruvate have been
applied to elucidate the catalytic cycle
(17–21)
or to trap reaction intermediates in crystal structures
(22–24).
Chemical modification of PDCs with group-specific reagents pointed to an
important role of cysteine residues
(25). Site-directed
mutagenesis of cysteine residues to alanine or serine demonstrated that
residue Cys-221 might be the decisive one for enzyme activation
(1,
4,
26,
27). Consequently, it was
postulated that the region around Cys-221 is the regulatory site of PDC, and
formation of a thiohemiketal at this side chain was proposed. However, a
number of questions remained elusive. (i) How is the activator fixed at the
regulatory site? (ii) What are the prime structural properties of the active
state as compared with the inactive state? (iii) How is the signal transmitted
from the regulatory to the active site? (iv) Which are the decisive features
of the active site in the activated state that render efficient catalysis
possible? To answer these questions, we present here the crystal structures of
KlPDC with the bound substrate surrogate MAP and of the
ScPDC variants D28E and E477Q with bound substrate PYR along with
kinetic studies on the activating effect of both activators and binding
studies using the small angle x-ray solution scattering (SAXS) method.Open in a separate windowSCHEME 1.Chemical structures of the substrate pyruvate, the activators pyruvamide
and methyl acetylphosphonate, and the thiohemiketal from pyruvate and
cysteine, respectively. 相似文献
56.
de Giuseppe PO Ullah A Silva DT Gremski LH Wille AC Chaves Moreira D Ribeiro AS Chaim OM Murakami MT Veiga SS Arni RK 《Biochemical and biophysical research communications》2011,409(4):6064-627
Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7 Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference. 相似文献
57.
The simultaneous use of the repellent DEET, pyridostigmine, and organophosphorus pesticides has been assumed as a potential cause for the Gulf War Illness and combinations have been tested in different animal models. However, human in vitro data on interactions of DEET with other compounds are scarce and provoked the present in vitro study scrutinizing the interactions of DEET, pyridostigmine and pesticides with human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE). DEET showed to be a weak and reversible inhibitor of hAChE and hBChE. The IC(50) of DEET was calculated to be 21.7mM DEET for hAChE and 3.2mM DEET for hBChE. The determination of the inhibition kinetics of pyridostigmine, malaoxon and chlorpyrifos oxon with hAChE in the presence of 5mM DEET resulted in a moderate reduction of the inhibition rate constant k(i). The decarbamoylation velocity of pyridostigmine-inhibited hAChE was not affected by DEET. In conclusion, the in vitro investigation of interactions between human cholinesterases, DEET, pyridostigmine, malaoxon and chlorpyrifos oxon showed a weak inhibition of hAChE and hBChE by DEET. The inhibitory potency of the tested cholinesterase inhibitors was not enhanced by DEET and it did not affect the regeneration velocity of pyridostigmine-inhibited AChE. Hence, this in vitro study does not give any evidence of a synergistic effect of the tested compounds on human cholinesterases. 相似文献
58.
59.
Lubov Petkova Nedialkova Rémy Denzler Martin B. Koeppel Manuel Diehl Diana Ring Thorsten Wille Roman G. Gerlach B?rbel Stecher 《PLoS pathogens》2014,10(1)
The host''s immune system plays a key role in modulating growth of pathogens and the intestinal microbiota in the gut. In particular, inflammatory bowel disorders and pathogen infections induce shifts of the resident commensal microbiota which can result in overgrowth of Enterobacteriaceae (“inflammation-inflicted blooms”). Here, we investigated competition of the human pathogenic Salmonella enterica serovar Typhimurium strain SL1344 (S. Tm) and commensal E. coli in inflammation-inflicted blooms. S. Tm produces colicin Ib (ColIb), which is a narrow-spectrum protein toxin active against related Enterobacteriaceae. Production of ColIb conferred a competitive advantage to S. Tm over sensitive E. coli strains in the inflamed gut. In contrast, an avirulent S. Tm mutant strain defective in triggering gut inflammation did not benefit from ColIb. Expression of ColIb (cib) is regulated by iron limitation and the SOS response. CirA, the cognate outer membrane receptor of ColIb on colicin-sensitive E. coli, is induced upon iron limitation. We demonstrate that growth in inflammation-induced blooms favours expression of both S. Tm ColIb and the receptor CirA, thereby fuelling ColIb dependent competition of S. Tm and commensal E. coli in the gut. In conclusion, this study uncovers a so-far unappreciated role of inflammation-inflicted blooms as an environment favouring ColIb-dependent competition of pathogenic and commensal representatives of the Enterobacteriaceae family. 相似文献
60.
N. Wille 《Plant Systematics and Evolution》1903,53(3):89-95
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