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  • 1
    Keywords: CELLS ; IN-VITRO ; INHIBITOR ; CELL ; Germany ; human ; PATHWAY ; VITRO ; DISEASE ; DISEASES ; PROTEIN ; PROTEINS ; ACCUMULATION ; COMPLEX ; COMPLEXES ; culture ; resistance ; DEGRADATION ; SEGMENTS ; OXIDATIVE STRESS ; ANTAGONIST ; INHIBITORS ; INCREASE ; methods ; USA ; function ; DEGENERATION ; retinal ; DYSFUNCTION ; AGE PIGMENT ; FUNDUS AUTOFLUORESCENCE ; GEOGRAPHIC ATROPHY ; LIPOFUSCIN ; MACULAR DEGENERATION ; OUTER SEGMENTS ; RAT RETINA ; RETINAL-PIGMENT EPITHELIUM
    Abstract: PURPOSE. Lipofuscin accumulation in the RPE is a common downstream pathogenic pathway in various monogenic and complex retinal diseases including age-related macular degeneration (AMD). Lipid peroxidation-induced modification of proteins is thought to play a role in lipofuscinogenesis and may contribute to RPE dysfunction. A prior study demonstrated that a variety of lipofuscin-associated proteins are damaged by aberrant covalent modifications of malondialdehyde (MDA) and 4-hydroxynonenal (HNE). The present study was conducted to test the hypothesis that these damaged proteins are more resistant to proteolytic attack and act as protease inhibitors. METHODS. Isolated photoreceptor outer segments (POS) were radioactively labeled and in vitro modified with MDA and HNE. Pure lysosomal fractions isolated from human RPE were tested for their proteolytic activities toward modified and unmodified POS proteins. In parallel, modified and radiolabeled POS were fed to RPE cell cultures for phagocytosis and their lysosomal degradation as well as intracellular accumulation was compared with unmodified POS. RESULTS. Both experimental approaches revealed that MDA or FINE modifications strikingly increase the resistance of POS proteins to the attack by lysosomal proteases. When cultured RPE cells were fed with modified or unmodified POS the amount of degraded POS proteins was reduced by approximately 60% to 70% for the modified POS compared with those in normal control subjects. Some of the modified proteins remained undegraded in the lysosomal compartment of cultured RPE cells and were still detectable 3 weeks after feeding, whereas unmodified POS were completely degraded within.1 week after feeding. Moreover, modified proteins had the potential to impair degradation of unmodified proteins, indicating their efficacy as proteolytic antagonists. CONCLUSIONS. The results indicate that lipid peroxidation-derived protein modifications are involved in lipofuscinogenesis and may contribute to cell damaging effects of lipofuscin in retinal diseases such as AMD
    Type of Publication: Journal article published
    PubMed ID: 17325182
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  • 2
    Abstract: In age-related macular degeneration (AMD), reduced lysosomal capacity may contribute to lipofuscinogenesis and progressive dysfunction of the retinal pigment epithelium (RPE). We previously demonstrated that lipid peroxidation-related protein modifications inhibit lysosomal degradation of photoreceptor outer segment (POS) proteins in RPE cells. Herein, we investigate the effects of lipid peroxidation products on activities of key RPE lysosomal proteases. In lysosomes isolated from primary human RPE cells, lipid peroxidation products 4-hydroxynonenal (HNE) and malondialdehyde (MDA) exerted a dose-dependent inhibitory effect on cysteine proteases cathepsin B and L, with biologically relevant concentrations of 1 muM resulting in a reduction of enzyme activities by 88-94%. This effect was confirmed in cultured RPE cells. Using mass spectrometry, covalent HNE and MDA adducts were detected in the active center region of inactivated cathepsins. POS previously modified with HNE and MDA likewise caused a dose-dependent reduction of cathepsin B and L activities in isolated lysosomes and, in addition, inhibited the aspartic protease cathepsin D. Our results indicate that lipid peroxidation products in vitro interfere with RPE lysosomal protease activities by two different mechanisms of action: (i) HNE and MDA directly inactivate lysosomal cysteine proteases by covalent binding to the active center; (ii) HNE- and MDA-mediated protein modifications convert proteolytic substrates into competitive inhibitors of lysosomal proteases. Via these mechanisms, lipid peroxidation products may induce lysosomal dysfunction and lipofuscinogenesis in the aging RPE and thus contribute to the pathogenesis of AMD.
    Type of Publication: Journal article published
    PubMed ID: 19895809
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  • 3
    Keywords: p53 ; CANCER-CELLS ; COLON-CANCER ; MASS-SPECTROMETRY ; GEL-ELECTROPHORESIS ; HUMAN SMALL-INTESTINE ; LIPID-METABOLISM ; LIPOTOXICITY ; ACYL-COA SYNTHETASE-5 ; FATTY-ACID REGULATION
    Abstract: Acyl-CoA synthetase 5 (ACSL5), a mitochondrially localized enzyme, catalyzes the synthesis of long-chain fatty acid thioesters and is physiologically involved in pro-apoptotic sensing of enterocytes. The aim of the present study is to identify an ACSL5-dependent regulation of mitochondrially expressed proteins and the characterization of related pathways in normal and diseased human intestinal mucosa. Proteomics of isolated mitochondria from ACSL5 transfectants and CaCo2 controls were performed. ACSL5-dependent protein synthesis was verified with quantitative reverse transcription plus the polymerase chain reaction, Western blotting, short-interfering-RNA-mediated gene silencing and additional cell culture experiments. Lipid changes were analyzed with tandem mass spectrometry. ACSL5-related pathways were characterized in normal mucosa and sporadic adenocarcinomas of the human intestine. In CaCo2 cells transfected with ACSL5, mortalin (HSPA9) was about two-fold increased in mitochondria, whereas cytoplasmic mortalin levels were unchanged. Disturbance of acyl-CoA/sphingolipid metabolism, induced by ACSL5 over-expression, was characterized as crucial. ACSL5-related over-expression of mitochondrial mortalin was found in HEK293 and Lovo (wild-type TP53 [tumor protein p53]) and CaCo2 (p53-negative; TP53 mutated) cells but not in Colo320DM cells (mutated TP53). In normal human intestinal mucosa, an increasing gradient of both ACSL5 and mortalin from bottom to top was observed, whereas p53 (wild-type TP53) decreased. In sporadic intestinal adenocarcinomas with strong p53 immunostaining (mutated TP53), ACSL5-related mortalin expression was heterogeneous. ACSL5-induced mitochondrial mortalin expression is assumed to be a stress response to ACSL5-related changes in lipid metabolism and is regulated by the TP53 status. Uncoupling of ACSL5 and mitochondrial mortalin by mutated TP53 could be important in colorectal carcinogenesis.
    Type of Publication: Journal article published
    PubMed ID: 24770931
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  • 4
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    German Medical Science; Düsseldorf, Köln
    In:  Evidenzbasierte Medizin - Anspruch und Wirklichkeit; 102. Jahrestagung der Deutschen Ophthalmologischen Gesellschaft; 20040923-20040926; Berlin; DOC04dogSA.07.02 /20040922/
    Publication Date: 2004-09-21
    Keywords: ddc: 610
    Language: English
    Type: conferenceObject
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  • 5
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    German Medical Science; Düsseldorf, Köln
    In:  Evidenzbasierte Medizin - Anspruch und Wirklichkeit; 102. Jahrestagung der Deutschen Ophthalmologischen Gesellschaft; 20040923-20040926; Berlin; DOC04dogP 140 /20040922/
    Publication Date: 2004-09-21
    Keywords: ddc: 610
    Language: English
    Type: conferenceObject
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Acta neurochirurgica 18 (1968), S. 129-169 
    ISSN: 0942-0940
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 0044-8249
    Keywords: Chemistry ; General Chemistry
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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