Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110 067, India.
Gene. 2011 Mar 15;474(1-2):29-38. doi: 10.1016/j.gene.2010.12.004. Epub 2010 Dec 22.
Heat shock protein 90 (Hsp90) is an abundant and highly conserved molecular chaperone that is essential for viability in eukaryotes. They have a crucial role in the folding of a set of proteins involved in the regulation of many essential cellular pathways and also re-folding of stress-denatured polypeptides. However, their exact function is still not clearly elucidated. In this study the full-length cDNA encoding for Hsp90 polypeptide and its corresponding gene was isolated from Pennisetum glaucum (designated PgHsp90). PgHsp90 cDNA encoded for a polypeptide of 698 amino acids with a predicted molecular mass of 80.3kDa and shared a high sequence homology (97-81%) to other plant cytosolic Hsp90s and shared less sequence homology (40-45%) to organelle and endoplasmic reticulum specific Hsp90 isoforms. A deduced amino acid sequence possessed three structural domains: N-terminus (1-211) ATP binding domain, middle (281-540) client protein interacting domain and C-terminus (541-698) dimerization domain; the N-terminus and middle domain is linked by a charged linker domain (212-280). It possesses the five-conserved amino acid signature sequence motifs characteristic of the Hsp90 family and a C-terminus MEEVD penta-peptide characteristic of the cytosolic Hsp90 isoform. The predicted quaternary architecture generated for PgHsp90 through molecular modeling was globally akin to that of yeast Hsp90. The PgHsp90 gene consists of 3 exons and 2 introns. The position and phasing of these introns were conserved in other plant cytosolic Hsp90 genes. Recombinant PgHsp90 protein was expressed in E. coli and purified to homogeneity, which possessed in vitro chaperone activity. E. coli expressing PgHsp90 protein showed enhanced tolerance to heat, salt and dehydration stresses. The quantitative up-regulation of PgHsp90 gene expression positively correlates in response to different stresses to meet the additional demand for protein folding support. Cumulatively, the in vivo and in vitro experiments indicated that PgHsp90 plays an adaptive or protective role to counter the stress induced protein damage.
热休克蛋白 90（Hsp90）是一种丰富且高度保守的分子伴侣，对于真核生物的存活至关重要。它们在一组参与调节许多基本细胞途径的蛋白质的折叠以及应激变性多肽的重折叠中发挥着关键作用。然而，其确切功能仍未得到明确阐明。在这项研究中，从 Pennisetum glaucum（命名为 PgHsp90）中分离出 Hsp90 多肽的全长 cDNA 及其相应基因。PgHsp90 cDNA 编码一个 698 个氨基酸的多肽，预测分子量为 80.3kDa，与其他植物细胞质 Hsp90 具有高度序列同源性（97-81%），与细胞器和内质网特异性 Hsp90 同工型的序列同源性较低（40-45%）。推导的氨基酸序列具有三个结构域：N 端（1-211）ATP 结合域、中间（281-540）与客户蛋白相互作用域和 C 端（541-698）二聚化域；N 端和中间域由带电连接域（212-280）连接。它具有 HSP90 家族的五个保守氨基酸特征序列基序和细胞质 Hsp90 同工型的 C 端 MEEVD 五肽特征序列基序。通过分子建模为 PgHsp90 生成的预测四级结构在全局上类似于酵母 Hsp90。PgHsp90 基因由 3 个外显子和 2 个内含子组成。这些内含子的位置和相位在其他植物细胞质 Hsp90 基因中是保守的。重组 PgHsp90 蛋白在大肠杆菌中表达并纯化至均一性，具有体外伴侣活性。表达 PgHsp90 蛋白的大肠杆菌对热、盐和脱水胁迫表现出增强的耐受性。PgHsp90 基因表达的定量上调与不同胁迫下的反应呈正相关，以满足对蛋白质折叠支持的额外需求。总之，体内和体外实验表明，PgHsp90 在应对应激诱导的蛋白质损伤时发挥适应性或保护作用。
