HYDROXYNONENAL MODIFICATION OF SUPEROXIDE DISMUTASE

肌萎缩性侧索硬化症（ALS）是一种脊柱、脑干和运动皮层运动神经元的退行性疾病。90%的ALS病例为散发性（SALS）。在其余家族性病例（FALS）中，10-30%与编码Cu，Zn-超氧化物歧化酶（SOD1）的SOD1基因突变有关。SOD的突变使过氧化物酶活性增加；降低金属亲和力；促进聚合;并可能赋予SOD1催化有毒硝化反应的能力。这些发现导致一些人推测ALS患者遭受氧化应激增强，可能是由于携带功能失调的SOD1。其他人推测SALS可能涉及SOD1的翻译后修饰，从而诱导毒性功能；这种修改在此之前尚未查明。我们小组已经确定了这样一种可能的翻译后修饰。我们一直在研究细胞毒性脂质氧化产物4-羟基-2-壬烯醛（HNE），已知其在阿尔茨海默病患者的大脑和ALS患者的中枢神经系统中升高。HNE和相关的α、β不饱和醛与蛋白质形成共价加合物，可能作为异双功能交联剂。我们利用一种针对hne蛋白加合物的抗体，对AD大脑中的hne反应蛋白进行了表征。我们发现，在Western blots中，约95%的HNE免疫反应性定位于一个32 kDa的蛋白，我们已将其确定为SOD1。我们已经开发了一个体外模型系统来研究HNE-SOD1反应，我们已经开始用maldi - tof质谱法表征反应性位点。我们现在假设，fals相关的SOD1与HNE的反应速度更快，因为SOD1同型二聚体界面区域赖氨酸残基对（赖氨酸-9）之间的距离减小，这种反应导致蛋白质聚集和金属从酶中释放出来。我们寻求资金来开始批判性地评估这一假设。我们将采用定点诱变和大肠杆菌表达系统来产生野生型人类SOD1以及与FALS相关的三种突变酶（G93A， G37R和G4V）。这些酶将在体外对其与HNE的反应速率和相关性质进行表征，特别是铜的释放。在平行工作中，将采用分子建模方法来研究突变对二聚体界面和金属配体结合位点的影响。我们希望这些初步研究能够更详细地了解SOD1在体内与HNE交联的机制，并最终可能导致治疗ALS和/或AD的新治疗策略。

Amyotrophic lateral sclerosis (ALS) is a degenerative disorder of motor neurons in the spinal column, brainstem and motor cortex. 90% of ALS cases are sporadic (SALS). Of the remanining familial cases (FALS), 10-30% associate with mutations in the SOD1 gene encoding Cu,Zn- superoxide dismutase (SOD1). The mutations in SOD create a gain-of- peroxidase activity; decrease metal affinity; promote aggregation; and may imbue the SOD1 with the ability to catalyze toxic nitration reactions. These findings have led some to speculate that ALS patients suffer enhanced oxidative stress, perhaps as a result of bearing dysfunctional SOD1. Others have speculated that SALS may involve post-translational modifications to SOD1 that induce toxic functions; such modifications have not been heretofore identified. Our group has identified such a possible post-translational modification. We have been studying the cytotoxic lipid oxidation product 4-hydroxy-2- nonenal (HNE), which is known to be elevated in the AD brain and in the central nervous system of ALS patients. HNE and related alpha, beta-unsaturated aldehydes form covalent adducts with proteins and may act as heterobifunctional crosslinking agents. We have characterized HNE-reactive proteins in the AD brain using an antibody directed against HNE-protein adducts. We find that >95% of the HNE immunoreactivity in Western blots localizes to a single 32 kDa protein that we have identified as SOD1. We have developed an in vitro model system for studying HNE-SOD1 reactions, and we have begun to characterize sites of reactivity by MALDI-TOF-mass spectrometry. We now hypothesize that FALS-associated SOD1 will react more rapidly with HNE because of decreased distance between a key pair of lysine residues (Lys-9) in the interfacial region of the SOD1 homodimer, and that this reaction leads to protein aggregation and metal release from the enzyme. We seek funds to begin critically assessing this hypothesis. We will employ site-directed mutagenesis and an E. coli expression system to generate wild-type human SOD1 along with three mutant enzymes (G93A, G37R and G4V) that are associated with FALS. The enzymes will be characterized in vitro with respect to their rate of reaction with HNE and correlated properties, particularly copper release. In parallel work, a molecular modeling approach will be taken to study the effects of the mutations on the dimer interface and metal ligand binding sites. We hope these pilot studies will lead to more detailed understanding of the mechanism by which SOD1 is crosslinked by HNE in vivo, and perhaps eventually may lead to new therapeutic strategies for the treatment of ALS and/or AD.