Construction of a mouse Aos1-Uba2 chimeric SUMO-E1 enzyme, mAU, and its expression in baculovirus-insect cells

Abstract

Small ubiquitin-related modifier (SUMO) is a highly conserved protein that is covalently attached to target proteins. This posttranslational modification, designated SUMOylation, is a major protein-conjugation-driven strategy designed to regulate structure and function of cellular proteins. SUMOylation consists of an enzymatic cascade involving the E1-activating enzyme and the E2-conjugating enzyme. The SUMO-E1 enzyme consists of two subunits, a heterodimer of activation of Smt3p 1 (Aos1) and ubiquitin activating enzyme 2 (Uba2), which resembles the N- and C-terminal halves of ubiquitin E1 (Uba1). Herein, we describe the rational design of a single polypeptide version of SUMO-E1, a chimera of mouse Aos1 and Uba2 subunits, termed mAU, in which the functional domains appear to be arranged in a fashion similar to Uba1. We also describe the construction of a mAU plasmid for expression in a baculovirus-insect cell system and present an in situ SUMOylation assay using the recombinant mAU. Our results showed that mAU has SUMO-E1 activity, thereby indicating that mAU can be expressed in baculovirus-insect cells and represents a suitable source of SUMO-E1.

Identifying UBA2 as a proliferation and cell cycle regulator in lung cancer A549 cells

“…Although the mechanisms of their attachment with target proteins are similar, the final effects may be different . SUMOylation refers to the process of posttranslational modification of proteins mediated by SUMO molecules through covalently attached to the lysine residues of the target proteins . The molecular modification of proteins by SUMO plays an important roles in regulating many cell functions, such as cell cycle and apoptosis regulation, and repair of DNA injury .…”

“…2 SUMOylation refers to the process of posttranslational modification of proteins mediated by SUMO molecules through covalently attached to the lysine residues of the target proteins. 1,3 The molecular modification of proteins by SUMO plays an important roles in regulating many cell functions, such as cell cycle and apoptosis regulation, and repair of DNA injury. 4 SUMO has been discovered to regulate the functions of some important molecules including P53 and inhibitor of κBα.…”

“…Small ubiquitin-related modifier (SUMO) is a kind of molecules similar to ubiquitin structurally and involved in posttranslational modification of protein. 1 Unlike ubiquitin, SUMO does not mediate the degradation of proteins. 2 Four major SUMO molecules were found at present, including SUMO-1, -2, -3 and -4.…”

“…2 Two major enzymes were involved in this molecular signaling, including E1-activating enzyme and the E2-conjugating enzyme. 1 The E2-conjugating enzyme consists of two subunits, namely activation of Smt3p 1 (Aos1) and ubiquitin activating enzyme 2 (Uba2). 1,5 Current research focuses on the mechanism of interaction of UBA2 as an important component of E1 with SUMO, but how it functions in human tissues including malignant tumors was rarely studied.…”

“…1 The E2-conjugating enzyme consists of two subunits, namely activation of Smt3p 1 (Aos1) and ubiquitin activating enzyme 2 (Uba2). 1,5 Current research focuses on the mechanism of interaction of UBA2 as an important component of E1 with SUMO, but how it functions in human tissues including malignant tumors was rarely studied. In this study, we investigate the expression and function of UBA2 in non-small-cell lung cancer (NSCLC).…”