Enzymatic deglycation of nonenzymatic glycation products

描述（由申请人提供）：糖尿病并发症仍然是糖尿病的一个严重问题。DCCT和UKPDS研究清楚地表明，血糖控制是决定并发症发生率的关键变量。然而，将高血糖与长期并发症联系起来的机制仍然知之甚少。关于糖尿病并发症病因学的一个很有前景的假说是非酶糖基化假说，该假说认为糖尿病相关的病理改变至少部分是葡萄糖及其反应副产物对蛋白质和其他一些大分子进行非酶修饰的结果。在这个过程中主要的初始中间体是葡萄糖赖氨酸（GL）和果糖赖氨酸（FL）（又称早期糖基化产物- EGP's），它们经过一系列的重排和反应形成不可逆的晚期糖基化终产物（AGE's）。在我们过去的研究中，我们已经获得证据表明，这种非酶糖基化过程在哺乳动物细胞中通过活性糖基化机制被抵消。基于这一证据，我们建议将糖尿病并发症的非酶糖基化假说修改为非酶糖基化/酶去糖基化假说。我们的第一个假设是，在恒温生物中，蛋白质和一些其他大分子的非酶糖基化是不可避免的和有害的。因此，我们的第二个假设是，这些生物体通过活性糖基化系统控制非酶糖基化不可避免的破坏性影响。根据我们的假设，在糖尿病中，特别是在周期性的极端高血糖发作期间，如在餐后观察到的，非酶糖基化过程不受控制，随着时间的推移，对必需蛋白质造成累积和不可逆转的损害，最终导致细胞功能和活力的丧失。我们目前的研究主要集中在两个方面：1)在合适的模型系统中通过FN3K的过表达、mRNA的敲除和基因敲除来检测FN3K的功能；2)研究与FN3K无关的去糖基化机制。为了FN3K的研究，我们已经向NIDDK起草了R01授权提案（DK62315-01）。为了进行高风险的去糖基化研究，我们提交了R21拨款提案，其中我们建议研究转糖基化反应，其中葡萄糖胺的葡萄糖部分（如葡萄糖赖氨酸）被更亲核的化合物（如还原型谷胱甘肽）清除。我们已经有证据表明，这种反应在体外很容易发生，我们也有数据表明，这一过程的主要稳定副产物，葡萄糖半胱氨酸，存在于尿液中，在糖尿病患者中升高。这笔拨款将使我们能够进一步研究转糖基化反应途径，并确定其在正常血糖和糖尿病个体中的重要性。

DESCRIPTION (provided by applicant): Diabetic complications remain a serious issue in diabetes mellitus. The DCCT and UKPDS studies demonstrate clearly that glycemic control is a key variable in determining the rate of development of complications. However, mechanisms linking hyperglycemia with long-term complications remain poorly understood. One promising hypothesis on the etiology of diabetic complications is the nonenzymatic glycation hypothesis which postulates that diabetes associated pathological changes are a result, at least in part, of the nonenzymatic modification of proteins and some other macromolecules by glucose and its reactive byproducts. The predominant initial intermediates in this process are glucoselysine (GL) and fructoselysine (FL) (a.k.a Early Glycation Products - EGP's) which undergo a series of further rearrangements and reactions to form the irreversible Advanced Glycation Endproducts (AGE's). In our past studies we have obtained evidence that this nonenzymatic glycation process is counteracted in mammalian cells by active deglycation mechanisms. Based on this evidence, we are proposing to modify the nonenzymatic glycation hypothesis of diabetic complications to a Nonenzymatic Glycation/Enzymatic Deglycation hypothesis. Our first postulate is that, in homeothermic organisms nonenzymatic glycation of proteins and some other macromolecules is unavoidable and deleterious. Consequently, our second postulate is that these organisms control the unavodable and damaging effects of non-enzymatic glycation by active deglycation systems. According to our hypothesis, in diabetes, especially during periodic episodes of extreme hyperglycemia, such as observed in the postprandial period, the process of nonenzymatic glycation proceeds unchecked and results, over time, in cumulative and irreversible damage to essential proteins leading ultimately to loss of cell function and viability. Our present research is focused in two areas: 1) Testing the function of FN3K by its overexpression, knockdown of its mRNA and knockout of its gene in appropriate model systems and 2) Study of the FN3K-independent deglycation mechanisms. For the study of FN3K we have drafted a R01 grant proposal to NIDDK (DK62315-01). In order to pursue the higher-risk studies on deglycation we are submitting this R21 grant proposal in which we propose to investigate transglycosylation reactions in which the glucose moiety of glucosamine such as glucoselysine is scavenged away by a more nucleophilic compounds such a reduced glutathione. We already have evidence that such reactions occur readily in vitro and we also have data that the principal stable byproduct of this process, glucosecysteine, is present in urine and is elevated in diabetic individuals. This grant will enable us to further study the transglycation reaction pathway and to determine its importance in normoglycemic and diabetic individuals.