Proteome-Wide Analysis of Lysine 2-Hydroxyisobutyrylated Proteins in Fusarium oxysporum.

Protein lysine 2-hydroxyisobutyrylation (K hib ), a new type of post-translational modification, occurs in histones and non-histone proteins and plays an important role in almost all aspects of both eukaryotic and prokaryotic living cells. Fusarium oxysporum, a soil-borne fungal pathogen, can cause disease in more than 150 plants. However, little is currently known about the functions of K hib in this plant pathogenic fungus. Here, we report a systematic analysis of 2-hydroxyisobutyrylated proteins in F. oxysporum. In this study, 3782 K hib sites in 1299 proteins were identified in F. oxysporum. The bioinformatics analysis showed that 2-hydroxyisobutyrylated proteins are involved in different biological processes and functions and are located in diverse subcellular localizations. The enrichment analysis revealed that K hib participates in a variety of pathways, including the ribosome, oxidative phosphorylation, and proteasome pathways. The protein interaction network analysis showed that 2-hydroxyisobutyrylated protein complexes are involved in diverse interactions. Notably, several 2-hydroxyisobutyrylated proteins, including three kinds of protein kinases, were involved in the virulence or conidiation of F. oxysporum, suggesting that K hib plays regulatory roles in pathogenesis. Moreover, our study shows that there are different K hib levels of F. oxysporum in conidial and mycelial stages. These findings provide evidence of K hib in F. oxysporum, an important filamentous plant pathogenic fungus, and serve as a resource for further exploration of the potential functions of K hib in Fusarium species and other filamentous pathogenic fungi.

Systematic identification of mitochondrial lysine succinylome in silkworm (Bombyx mori) midgut during the larval gluttonous stage.

Lysine succinylation is a newly identified protein post-translational modification (PTM) of lysine residues. Increasing evidences demonstrate that this modification is prevalent in mitochondria and regulates many vital cellular processes, especially metabolism. Here, we determined the succinylome of the silkworm (Bombyx mori) midgut mitochondria during the larval gluttonous stage (the fifth instar) using succinylated peptides enrichment coupled with nano HPLC/MS/MS. A total of 1884 lysine succinylation sites on 373 mitochondrial proteins were identified. The bioinformatic analysis reveal that succinylated proteins are significantly enriched in central metabolic processes and mitochondrial protein synthesis. Several apoptosis and detoxification related enzymes or proteins are succinylated. The findings suggest the crucial role of lysine succinylation in silkworm midgut metabolism and resistance. Our data provide a rich resource for further analysis of lysine succinylation in silkworm. SIGNIFICANCE: Insect midgut is the vital tissue for nutrient metabolism and also for xenobiotic metabolism. There is a growing body of knowledge on regulation of midgut function at the gene or protein levels in silkworm, however, the regulation at post-translation modification level remains largely unknown. We provide a first global analysis of the mitochondrial lysine succinylome in silkworm midgut. A total of 1884 lysine succinylation sites on 373 mitochondrial proteins were identified. Bioinformatics results suggest an important role of this modification in regulating metabolism and mitochondrial protein synthesis. Our data greatly expand the catalog of lysine succinylation substrates and sites in insects, and represents an important resource for understanding the physiological function of lysine succinylation in insect midgut.

[Cloning, expression and activity analysis of full-length gene encoding thioredoxin peroxidase from Oncomelania hupensis].

OBJECTIVE: To clone and express full-length thioredoxin peroxidase (TPx) gene of Oncomelania hupensis and study on the peroxidase activity of the recombinant protein. METHODS: Total RNA was obtained from the cultivated O. hupensis and a cDNA sequence of the TPx gene was cloned by RT-PCR. The TPx cDNA ends were amplified by the SMARTer RACE cDNA Amplification Kit. After sequencing, blasting and matching, the full-length cDNA of the TPx gene was obtained. The TPx cDNA was ligated with the pGEM-Teasy and transformed into E. coli DH5alpha. After sequencing and blasting, the characteristics of biological information of the TPx gene was analyzed. The positive recombinants with pGEM-Teasy/TPx and expression vector pET-28a were digested by the double restriction enzymes, ligated each other, transformed into E. coli BL21 (DE3), and induced by IPTG for expression. The recombinant TPx was expressed as a histidine fusion protein and was purified with Ni chromatography and NTA cation exchange chromatography. The expressed and purified TPx was analyzed by SDS-PAGE. The different concentrations of TPx recombinant protein (10, 20, 30, 40, and 50 microg/ml) were added into hydrogen peroxide (H2O2) reduction test in vitro to calculate the clearance rate of H2O2, each concentration with parallel control of dithiothreitol sugar alcohol (DTT). In the protection test of super-coiled DNA, the TPx protein was added with a concentration of 2.5, 5.0, and 10 microg/ml, respectively, to observe the protective effect of super-coiled DNA in metal-catalyzed oxidation (MCO). RESULTS: The complete cDNA encoding TPx was 992 bp. ORF was 747 bp with GenBank accession number of JN831437. The ORF encoded 249 amino acids, and the relative molecular weight (M(r)) of predicted protein was 27 000. The recombinant plasmid pET28a/TPx was built, and the soluble recombinant protein was obtained by induction and purification. The results of SDS-PAGE showed that the M(r) was 27 000. H2O2 reduction test in vitro showed that the H2O2 clearance rate of reaction system containing DTT was significantly higher than the clearance rate without DTT (P < 0.05), the difference among various concentrations was not statistically significant (P > 0.05). The protection of super-coiled DNA showed that the protective effect of 5.0 microg/ml group was better than 2.5 microg/ml group, but there was no difference between the protective effect of 5.0 microg/ml group and 10 microg/ml group. CONCLUSION: The full-length cDNA of the TPx gene of O. hupensis is obtained, and the recombinant TPx protein shows a certain antioxidant activity.

[Progress of research on peroxiredoxin in invertebrates].

Peroxiredoxin (Prx) belongs to a peroxidase family of antioxidant enzymes distributed ubiquitously in aerobic organisms such as yeast, fungi, parasites, mammals and humans. It plays an important role in the defense of reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by aerobic organisms. This article introduces the classification, mechanism of Prx and the progress of research on Prx in invertebrates.