Oxidative stress: enzymatic detoxification of reactive carbonyl compounds in simple model organisms and man

细胞呼吸持续产生活性氧，通过脂质过氧化作用，活性氧可产生具有攻击性的羰基化合物，导致人体衰老和疾病。短链脱氢酶/还原酶（SDR）超家族的还原性代谢可有效保护人体免受氧化应激的影响。此外，SDR在其他内源性和外源性化合物的生物转化过程中也发挥着重要作用，羰基还原在不同疾病发病机制中的重要性可以通过模式生物得到显著证明。研究D.黑腹果蝇的研究表明，羰基还原酶嗅探器可以防止年龄依赖性神经变性，并延长这些动物的寿命。我们最终可以证明嗅探器可以解毒一种神经毒性脂质过氧化产物，这种产物通常在退行性疾病期间积累在组织中。本项目的目的是在模式生物秀丽隐杆线虫、蚤状/大型水蚤和水螅中调查嗅探器和人类羰基还原酶1的可能的功能同源物。生物信息学预分析表明，嗅探器和羰基还原酶确实存在于这些生物体的许多变体中，最有可能是基因复制的结果。特别是，蚤状蚤（Daphnia pulex）是一种经典的测试生物，具有许多环境响应基因，这使其成为生态毒理学中有趣的模式生物，但其蛋白质的特征仍然很差。另一种方法集中于人羰基还原酶CBR 1和CBR 3。通过将来自模式生物的酶与人野生型及其相关的CBR 3> CBR 1转换突变体的结构-活性比较，将鉴定酶活性位点的重要结构决定因素。基于我们的生物信息学分析，将克隆酶，在原核和真核系统中重组产生酶，并进行生物化学表征。特别感兴趣的是它们可能对氧化应激（脂质过氧化产物或AGEs前体的代谢）的保护作用。通过结晶和X射线分析确定酶的催化热点。这些信息可能会让我们深入了解导致嗅探酶和羰基还原酶出现的进化路线，并可能有助于破译它们的生理作用。借助模式生物获得的结果可能有助于阐明有效保护人体免受氧化应激的基本细胞生物学/生化机制，从而有助于预防与年龄和疾病相关的神经退行性变。

Cellular respiration continuously produces reactive oxygen species, which can, by lipid peroxidation, generate aggressive carbonyl compounds, contributing to ageing processes and diseases in humans.The reductive metabolism by enzymes from the short-chain dehydrogenase/reductase (SDR) superfamily effectively protects against these effects of oxidative stress. Moreover, SDR play a central role in the biotransformation of other endo- and xenobiotics.The importance of carbonyl reduction in the pathogenesis of different diseases could be strikingly demonstrated by employing model organisms. Studies on D. melanogaster show that the carbonyl reductase sniffer protects against age-dependent neurodegeneration and prolongs the lifespan of those animals. We could finally demonstrate that sniffer detoxifies a neurotoxic lipid peroxidation product, which often accumulates in tissues during degenerative diseases. This project aims at the investigation of possible functional homologs of sniffer and human carbonyl reductase 1 in the model organisms Caenorhabditis elegans, Daphnia pulex/magna and Hydra vulgaris. Bioinformatic pre-analyses show that sniffer and carbonyl reductases do occur in numerous variants in these organisms, most likely as a result of gene duplications. Particularly, Daphnia pulex, a classical test-organism, harbours numerous environment-responsive genes which predisposes it as interesting model organism in ecotoxicology but its proteins are still poorly characterised. A further approach focuses on human carbonyl reductases CBR1 and CBR3. By structure-activity comparison of the enzymes from the model organisms with both human wildtype forms and relevant CBR3>CBR1 transition mutants thereof, important structural determinants of the enzymes active sites will be identified.Based on our bioinformatics analyses, the enzymes will be cloned, recombinantly produced in both prokaryotic and eukaryotic systems and biochemically characterised. Of special interest will be their possible protective role against oxidative stress (metabolism of lipid peroxidation products or AGEs precursors). The enzymes catalytic hotspots will be determined by crystallisation and X-ray analysis. This information might give insight into the evolutionary route that led to the emergence of sniffer and carbonyl reductases and will possibly contribute to decipher their physiologic role. The results obtained with the aid of model organisms might help to elucidate basic cell-biological/biochemical mechanisms that effectively protect the human body against oxidative stress and thereby help to prevent age- and disease-associated neurodegeneration.