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91.
Nora Wender Jan Hegermann Bettina Brunner Brigitte Nuscher Tim Bartels Armin Giese Klaus Beyer Stefan Eimer Konstanze F Winklhofer Christian Haass 《The EMBO journal》2010,29(20):3571-3589
Aggregation of α‐synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age‐dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA‐mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ‐1 but not the PD‐associated mutations PINK1 G309D and parkin Δ1–79 or by DJ‐1 C106A. 相似文献
92.
Bartels T Ahlstrom LS Leftin A Kamp F Haass C Brown MF Beyer K 《Biophysical journal》2010,99(7):2116-2124
Alpha-synuclein (αS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although αS is abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length αS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the αS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu. 相似文献
93.
Biologically active ingredients and excipients are the essentials of a drug formulation, such as a tablet, dragee, solution, etc. Quality control of such substances thus plays a pivotal role in the production process of pharmaceutical drugs. Since these agents often exhibit complex structures, consist of multiple components, or lack of a chromophore, traditional means of characterization are often not feasible. Furthermore, substances of small molecular weight or strong polar character generally exhibit poor chromatographic properties, thus, conventional procedures such as high-performance liquid chromatography are often not applicable. Instead, quantitative nuclear magnetic resonance (qNMR) spectroscopy has emerged as an alternative or orthogonal method in drug analysis. In this review, we elaborate on the application of qNMR to three important classes of biological substances, namely polysaccharides, amino acids, and lipids, and demonstrate the benefits of this modern tool in contrast to traditional techniques. 相似文献
94.
Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids.
However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage
dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile
compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2),
a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was
introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations.
Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable
to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were
determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed
with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that
apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta. 相似文献
95.
96.
Qian YW Schmidt RJ Zhang Y Chu S Lin A Wang H Wang X Beyer TP Bensch WR Li W Ehsani ME Lu D Konrad RJ Eacho PI Moller DE Karathanasis SK Cao G 《Journal of lipid research》2007,48(7):1488-1498
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR. 相似文献
97.
98.
Dirk Pohlers Andreas Beyer Dirk Koczan Thomas Wilhelm Hans-Jürgen Thiesen Raimund W Kinne 《Arthritis research & therapy》2007,9(3):R59
Genome-wide gene expression was comparatively investigated in early-passage rheumatoid arthritis (RA) and osteoarthritis (OA)
synovial fibroblasts (SFBs; n = 6 each) using oligonucleotide microarrays; mRNA/protein data were validated by quantitative PCR (qPCR) and western blotting
and immunohistochemistry, respectively. Gene set enrichment analysis (GSEA) of the microarray data suggested constitutive
upregulation of components of the transforming growth factor (TGF)-β pathway in RA SFBs, with 2 hits in the top 30 regulated
pathways. The growth factor TGF-β1, its receptor TGFBR1, the TGF-β binding proteins LTBP1/2, the TGF-β-releasing thrombospondin
1 (THBS1), the negative effector SkiL, and the smad-associated molecule SARA were upregulated in RA SFBs compared to OA SFBs,
whereas TGF-β2 was downregulated. Upregulation of TGF-β1 and THBS1 mRNA (both positively correlated with clinical markers
of disease activity/severity) and downregulation of TGF-β2 mRNA in RA SFBs were confirmed by qPCR. TGFBR1 mRNA (only numerically
upregulated in RA SFBs) and SkiL mRNA were not differentially expressed. At the protein level, TGF-β1 showed a slightly higher
expression, and the signal-transducing TGFBR1 and the TGF-β-activating THBS1 a significantly higher expression in RA SFBs
than in OA SFBs. Consistent with the upregulated TGF-β pathway in RA SFBs, stimulation with TGF-β1 resulted in a significantly
enhanced expression of matrix-metalloproteinase (MMP)-11 mRNA and protein in RA SFBs, but not in OA SFBs. In conclusion, RA
SFBs show broad, constitutive alterations of the TGF-β pathway. The abundance of TGF-β, in conjunction with an augmented mRNA
and/or protein expression of TGF-β-releasing THBS1 and TGFBR1, suggests a pathogenetic role of TGF-β-induced effects on SFBs
in RA, for example, the augmentation of MMP-mediated matrix degradation/remodeling. 相似文献
99.
100.