RESUMO
The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized alphabeta motif (CS alphabeta) consists of an alpha-helix and an antiparallel triple-stranded beta-sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CS alphabeta motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CS alphabeta motif. The information is valuable to the scaffold with the CS alphabeta motif and protein engineering.
Assuntos
Alanina/metabolismo , Substituição de Aminoácidos/fisiologia , Cisteína/metabolismo , Proteínas/metabolismo , Alanina/química , Alanina/genética , Sequência de Aminoácidos , Dicroísmo Circular , Cisteína/química , Cisteína/genética , Defensinas/química , Defensinas/genética , Defensinas/metabolismo , Fabaceae/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Desnaturação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/genética , Alinhamento de Sequência , Espectrometria de Fluorescência , alfa-Amilases/metabolismoRESUMO
Plant defensins are a family of small, cysteine-rich and basic proteins. Proteins of the family constitute the major innate immune system of plants and insects. The most significant feature of the family is the conserved cysteine scaffold and one alpha-helix and a triple strand anti parallel beta-sheet connected by the scaffold. Proteins of the family display anti-microbial and anti-fungal activities and a broad spectrum of biological functions. The proteins also demonstrate ultra stable proprieties even in extreme environments. In the past twenty years, patents on the proteins and their applications have increased as technologies have been developed. By utilizing protein engineering approach, medical applications of proteins of the family have been developed. By reviewing patents on proteins of the family, the application development trend of the protein family is traced. Currently, the development of proteins of the family is still in the initial and discovery stage. For their excellent physical and chemical proprieties, it is believed that application of the proteins in the biomedical area should have a high potential and the future of proteins of the plant defensin family.
Assuntos
Defensinas/metabolismo , Doenças das Plantas , Defensinas/química , Defensinas/fisiologia , Patentes como Assunto , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologiaRESUMO
The CDA14, a 45kD protein, is currently annotated as PTX1-like protein or ERGIC 32. Over expressing CDA14 can slow PC11 cell proliferation rate. In HepG2 cells, it had been demonstrated that CDA14 is involved in protein transportation. The knowledge about the protein is very limited and not clarified. The CDA14 and its homologous proteins form a family and are restricted to eukaryotes. In the family, there are no homologous sequences with resolved three-dimensional structure and their functions are difficult to predict. Transcriptional expression of CDA14 in three hepatoma cell lines, Hu7, HCC and HepG2, was lower than normal liver tissue and liver carcinoma tissue. In this study, functional proteomic techniques were utilized in searching the interacting counterpart of CDA14. Several proteins involved in protein translation and folding were selectively precipitated with CDA14 and identified mass spectrometry. Interaction of CDA14 and elongation factor 1alpha was confirmed by Western blotting and confocal microscopy. Elongation factor 1alpha is a multiple function protein and involved in several biological mechanisms, including protein synthesis, cell proliferation, apoptosis and tumorigensis. Over-expression of CDA14 down regulated the proliferation of HepG2 cells. These results suggest that CDA14 participated in the elongation factor 1alpha regulated mechanisms.
