Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome.

The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants.

A chloroplast membrane protein LTO1/AtVKOR involving in redox regulation and ROS homeostasis.

KEY MESSAGE: The role of LTO1/ At VKOR-DsbA in ROS homeostasis and in redox regulation of cysteine-containing proteins in chloroplast was studied in lto1 - 2 mutant, and a potential target of LTO1 was captured. A chloroplast membrane protein LTO1/AtVKOR-DsbA encoded by the gene At4g35760 was recently found to be an oxidoreductase and involved in assembly of PSII. Here, the growth of a mutant lto1-2 line of Arabidopsis was found to be severely stunted and transgenic complementation ultimately demonstrated the phenotype changes were due to this gene. A proteomic experiment identified 23 proteins presenting a differential abundance in lto1-2 compared with wild-type plants, including components in PSII and proteins scavenging active oxygen. Three scavengers of active oxygen, L-ascorbate peroxidase 1, peroxisomal catalase 2, dehydroascorbate reductase 1, are reduced in lto1-2 plants, corresponding to high levels of accumulation of reactive oxygen species (ROS). The photosynthetic activities of PSII and the quantity of core protein D1 decreased significantly in lto1-2. Further investigation showed the synthesis of D1 was not affected in mutants both at transcription and translation levels. The soluble DsbA-like domain of LTO1 was found to have reduction, oxidation and isomerization activities, and could promote the formation of disulfide bonds in a lumenal protein, FKBP13. A potential target of LTO1 was captured which was involving in chlorophyll degradation and photooxidative stress response. Experimental results imply that LTO1 plays important roles in redox regulation, ROS homeostasis and maintenance of PSII.

Development and validation of a preoperative nomogram for predicting positive surgical margins after laparoscopic radical prostatectomy.

BACKGROUND: Positive surgical margins are independent risk factor for biochemical recurrence, local recurrence, and distant metastasis after radical prostatectomy. However, limited predictive tools are available. This study aimed to develop and validate a preoperative nomogram for predicting positive surgical margins after laparoscopic radical prostatectomy (LRP). METHODS: From January 2010 to March 2016, a total of 418 patients who underwent LRP without receiving neoadjuvant therapy at Peking University Third Hospital were retrospectively involved in this study. Clinical and pathological results of each patient were collected for further analysis. Univariable and multivariable logistic regression (backward stepwise method) were used for the nomogram development. The concordance index (CI), calibration curve analysis and decision curve analysis were used to evaluate the performance of our model. RESULTS: Of 418 patients involved in this study, 142 patients (34.0%) had a positive surgical margin on final pathology. Based on the backward selection, four variables were included in the final multivariable regression model, including the percentage of positive cores in preoperative biopsy, clinical stage, free prostate specific antigen (fPSA)/total PSA (tPSA), and age. A nomogram was developed using these four variables. The concordance index (C-index) of the nomogram was 0.722 in the development cohort and 0.700 in the bootstrap validations. The bias-corrected calibration plot showed a limited departure from the ideal line with a mean absolute error of 2.0%. In decision curve analyses, the nomogram showed net benefits in the range from 0.2 to 0.7. CONCLUSION: A nomogram to predict positive surgical margins after LRP was developed and validated, which could help urologists plan surgical procedures.

The conservative cysteines in transmembrane domain of AtVKOR/LTO1 are critical for photosynthetic growth and photosystem II activity in Arabidopsis.

Thylakoid protein vitamin K epoxide reductase (AtVKOR/LTO1) is involved in oxidoreduction. The deficiency of this compound causes pleiotropic defects in Arabidopsis thaliana, such as severely stunted growth, smaller sized leaves, and delay of flowering. Transgenic complementation of wild-type AtVKOR (VKORWT) to vkor mutant lines ultimately demonstrates that the phenotype changes are due to this gene. However, whether AtVKOR functions in Arabidopsis through its protein oxidoreduction is unknown. To further study the redox-active sites of AtVKOR in vivo, a series of plasmids containing cysteine-mutant VKORs were constructed and transformed into vkor deficient lines. Compared with transgenic AtVKORWT plants, the size of the transgenic plants with a single conservative cysteine mutation (VKORC109A, VKORC116A, VKORC195A, and VKORC198A) were smaller, and two double-cysteine mutations (VKORC109AC116A and VKORC195AC198A) showed significantly stunted growth, similar with the vkor mutant line. However, mutations of two non-conservative cysteines (VKORC46A and VKORC230A) displayed little obvious changes in the phenotypes of Arabidopsis. Consistently, the maximum and actual efficiency of photosystem II (PSII) in double-cysteine mutation plants decreased significantly to the level similar to that of the vkor mutant line both under normal growth light and high light. A significantly decreased amount of D1 protein and increased accumulation of reactive oxygen species were observed in two double-cysteine mutations under high light. All of the results above indicated that the conservative cysteines in transmembrane domains were the functional sites of AtVKOR in Arabidopsis and that the oxidoreductase activities of AtVKOR were directly related to the autotrophic photosynthetic growth and PSII activity of Arabidopsis thaliana.

Key amino acids of arabidopsis VKOR in the activity of phylloquinone reduction and disulfide bond formation.

Many proteins in chloroplast are regulated through the disulfide bond/thiol transformation to realize their activities. A homologue of VKOR (Vitamin K epoxide reductase) in Arabidopsis chloroplast is found to catalyze the disulfide bond formation in vivo and to mediate the specific phylloquinone reduction in vitro. It is also called LTO1 (Lumen Thiol Oxidoreductase 1). Investigations about functions and essential amino acid residues of AtVKOR have important theoretical significance to clarify the chloroplast redox regulation mechanism. In this study, several amino acids in the VKOR domain of AtVKOR were identified to be involved in binding of phylloquinone. Site-directed mutagenesis was used to study the function of these positions. The results suggested that residues Ser77, Leu87, Phe137 and Leu141 were quite important in the binding and catalyzing the reduction of phylloquinone. These residues were also involved in the electron transferring and disulfide bond formation of substrate proteins by motility assays in vivo, suggesting that the binding of phylloquinone not only affected the delivery of electrons to phylloquinone but also affected the whole electron transfer process. The conserved cysteines in the AtVKOR domain also played critical roles in phylloquinone reduction. When each of the four conserved cysteines was mutated to alanine, the mutants lost reduction activity entirely, suggesting that the four conserved cysteines played crucial roles in the electron transfer process.

Identification and functional analysis of a novel parvulin-type peptidyl-prolyl isomerase from Gossypium hirsutum.

Plants have developed a variety of adaptive mechanisms to cope with stresses. A novel salt-induced gene was isolated during the screening of a NaCl-induced cDNA library of cotton seedlings. The gene was registered as accession number AY972810 in GenBank. Phylogenetic analysis suggested that the protein encoded by the gene belongs to the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases, EC 5.2.1.8). Northern blot analysis indicated that the mRNA accumulation of GhPPI was induced by salt stress. Subcellular localization revealed that GhPPI (Gossypium hirsutum peptidyl-prolyl isomerase) was localized in the nucleus. The purified recombinant GhPPI could accelerate the initial velocity of the cis-trans conversion of peptidyl-prolyl bonds of a tetrapeptide in a GhPPI concentration-dependent manner. Recombinant GhPPI also suppressed protein aggregation under denaturing conditions using Gdn-HCl (guanidine hydrochloride), suggesting an additional chaperone activity. Several amino acid residues in GhPPI were speculated to be involved in substrate binding or catalysis based on molecular modeling and docking results. The activity of the peptidyl-prolyl isomerase was affected when the relevant amino acids were mutated. Among the 11 mutants, five amino acids mutations led to the enzyme activities decreased to 30% as that of wild type, and two reduced to approximately 60%. To the best of our knowledge, this is the first report of a plant parvulin PPIase involved in the salt stress response.