Chattopadhyay Madhab K, Kern Renée, Mistou Michel-Yves, Dandekar Abhaya M, Uratsu Sandra L, Richarme Gilbert
Molécules de Stress, Institut Jacques Monod, Université Paris 7, 2 place Jussieu, 75251 Paris Cedex 05, France.
J Bacteriol. 2004 Dec;186(23):8149-52. doi: 10.1128/JB.186.23.8149-8152.2004.
Since, like other osmolytes, proline can act as a protein stabilizer, we investigated the thermoprotectant properties of proline in vitro and in vivo. In vivo, elevated proline pools in Escherichia coli (obtained by altering the feedback inhibition by proline of gamma-glutamylkinase, the first enzyme of the proline biosynthesis pathway) restore the viability of a dnaK-deficient mutant at 42 degrees C, suggesting that proline can act as a thermoprotectant for E. coli cells. Furthermore, analysis of aggregated proteins in the dnaK-deficient strain at 42 degrees C by two-dimensional gel electrophoresis shows that high proline pools reduce the protein aggregation defect of the dnaK-deficient strain. In vitro, like other "chemical chaperones," and like the DnaK chaperone, proline protects citrate synthase against thermodenaturation and stimulates citrate synthase renaturation after urea denaturation. These results show that a protein aggregation defect can be compensated for by a single mutation in an amino acid biosynthetic pathway and that an ubiquitously producible chemical chaperone can compensate for a defect in one of the major chaperones involved in protein folding and aggregation.
由于脯氨酸与其他渗透溶质一样可作为蛋白质稳定剂，我们对脯氨酸在体外和体内的热保护特性进行了研究。在体内，大肠杆菌中脯氨酸库增加（通过改变脯氨酸对脯氨酸生物合成途径的首个酶γ-谷氨酰激酶的反馈抑制来实现）可恢复dnaK缺陷型突变体在42℃时的活力，这表明脯氨酸可作为大肠杆菌细胞的热保护剂。此外，通过二维凝胶电泳分析42℃时dnaK缺陷型菌株中的聚集蛋白发现，高脯氨酸库可减少dnaK缺陷型菌株的蛋白质聚集缺陷。在体外，与其他“化学伴侣”以及DnaK伴侣一样，脯氨酸可保护柠檬酸合酶免受热变性影响，并在尿素变性后刺激柠檬酸合酶复性。这些结果表明，氨基酸生物合成途径中的单个突变可弥补蛋白质聚集缺陷，并且一种普遍可产生的化学伴侣可弥补参与蛋白质折叠和聚集的主要伴侣之一的缺陷。
