Regulation of intracellular protein degradation by post-translational modification and low-molecular-mass ligands

许多细胞功能都是通过蛋白质水解来调节的，细胞内和细胞外蛋白质的选择性蛋白质水解机制已被深入研究。虽然翻译后修饰决定蛋白质半衰期的观点早已提出，但没有令人信服的实验验证。该项目已经确定，与谷胱甘肽（生理上最重要的巯基化合物）形成混合二硫化物，大大增加了细胞脂肪酸结合蛋白的蛋白水解敏感性。许多细胞蛋白含有未知功能的游离半胱氨酸残基，这些半胱氨酸残基上的巯基-二硫键交换可能是细胞蛋白降解的普遍机制。我们还发现大鼠皮肤脂肪酸结合蛋白和鱼肝脂肪酸结合蛋白含有二硫键，尽管它们的胞质起源。这一发现表明，分子内巯基-二硫键交换机制调节蛋白质水解和功能的蛋白质。大鼠肝脏脂肪酸结合蛋白在Asn-Gly（残基105和106）处发生转肽反应。修饰的蛋白质也容易被蛋白酶消化。由于异天冬氨酸形成的速率取决于标记序列，这种修饰也可能决定单个蛋白质的半衰期。正常可溶性蛋白质的有限蛋白水解有时导致淀粉样原纤维形成。影响这种蛋白水解的因素也进行了研究，淀粉样蛋白轻链的结构分析。本项目还发现了一种新的蛋白酶抑制剂的活性-非活性构象转变的蛋白水解调节机制。

A number of cellular functions are regulated through proteolysis, and mechanism of selective proteolysis of intra-and extra-cellular proteins has been intensively studied. Although the idea that post-translational modifications determine half-lives of proteins has long been proposed, there is no convincing experimental verification. This project has established that mixed disulfide formation with glutathione, the physiologically most important thiol compound, greatly increases proteolytic susceptibility of cellular fatty acid-binding proteins. Many cellular proteins contain free cysteine residues of unknown function, and thiol-disulfide interchange at these cysteine residues may be a ubiquitous mechanism for cellular protein degradation. We also found rat cutaneous fatty acid-binding protein and a fish liver fatty acid-binding protein contain disulfide bonds despite their cytosolic origin. This finding suggests an intramolecular thiol-disulfide interchange mechanism for regulation of the proteolysis and functions of such proteins. Rat liver fatty acid-binding protein undergoes transpeptidation reaction at Asn-Gly (residue105 and 106). The modified protein also readily digested by proteinases. Because rate of the isoaspartate formation depends onthe franking sequence, this modification may also determine the half-life of individual protein. Limited proteolysis of a normally soluble protein sometimes leads to amyloid fibril formation. Factors affecting such proteolysis was also investigated by structural analysis of an amyloidgenic light chain. A novel regulatory mechanism of proteolysis by the active-inactive conformational transition of a proteinase inhibitor was also found by the present project.