title: Isolation, characterization, and structure analysis of a vacuolar processing enzyme gene (MhVPEγ) from Malus hupehensis (Pamp) Rehd.
abstract: Vacuolar processing enzymes (VPEs) have received considerable attention recently, as they exhibit caspase-1-like cleavage activity and regulate the process of PCD. However, knowledge about their detailed characteristics and structures is relatively limited. In this study, a gamma vacuolar processing enzyme gene, MhVPEγ, has been isolated from the leaves of Malus hupehensis (Ramp) Rehd. var pinyiensis Jiang. MhVPEγ coded-translated protein sequence comprised of 494 amino acids with a signal peptide and a transmembrane helix structure at N-terminal, peptidase_C13 domain, and vacuolar sorting signal at C-terminal. Consequently, genomic walking approach was performed for the isolation of its upstream sequence. Computational analysis demonstrated several motifs of the promoter exhibiting hypothetic MeJA, ABA, and light-induced characteristics, as well as some typical domains universally discovered in promoter, such as TATA-box and CAAT-box. MhVPEγ transcript level was enhanced during wounding treatment, and WUN-motif, as one of the cis-acting regulatory elements existing in the upstream sequence perhaps regulates its expression. In silico-constructed 3D models revealed that MhCPYL successively interacts with MhVPEγ like that of "Induced Fit-Lock and Key" model, providing molecular conformation evidence that CPY is a direct substrate of VPEγ. This study is the first stride to understand the molecular mechanism of VPEγ and CPYL interactions.
authors.affiliation: College of Horticulture Science and Engineering, Shandong Agricultural University, Daizong Street 61, Tai'an, 271000, Shandong, People's Republic of China.
authors.forename: Kun
authors.initials: K
authors.lastname: Ran
authors.forename: Hongqiang
authors.initials: H
authors.lastname: Yang
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Sun
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Jiang
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Shen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002268:Carboxypeptidases / Q000378:metabolism; D020170:Caspase 1 / Q000737:chemistry / Q000378:metabolism; D043402:Cathepsin A / Q000378:metabolism; D003198:Computer Simulation; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D027845:Malus / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D002268:Carboxypeptidases; D043402:Cathepsin A; C055587:carboxypeptidase L; D003546:Cysteine Endopeptidases; C089278:vacuolar processing enzyme; D020170:Caspase 1