SENP1 protects cisplatin-induced AKI by attenuating apoptosis through regulation of HIF-1α.

BACKGROUND: Acute kidney injury is a clinical syndrome with both high morbidity and mortality. However, the underlying molecular mechanism of AKI is still largely unknown. The role of SENP1 in AKI is unclear, while one of its substrates, HIF-1α possesses nephroprotective effect in AKI. Herein, this study aimed to reveal the role of SENP1/HIF-1α axis in AKI by using both cell and animal models. METHODS: We investigated the effects of AKI on SENP1 expression using clinical samples, and cisplatin-induced AKI model based on mice or HK-2 cells. The influence of SENP1 knockdown or over-expression on cisplatin-induced AKI was studied in vitro and in vivo. Following the exploration of the change in HIF-1α expression brought by AKI, the synergistic effects of SENP1 knockdown and HIF-1α over-expression on AKI were examined. RESULTS: The results showed the up-regulation of SENP1 in clinical specimens, as well as cell and animal models. The knockdown or over-expression of SENP1 in HK-2 cells could promote or inhibit AKI through regulating cell apoptosis, respectively. Moreover, SENP1+/- mice suffered from much more serious AKI compared with mice in wild type group. Furthermore, we found that HIF-1α over-expression could attenuate the promoted cell apoptosis as well as AKI induced by SENP1 knockdown. CONCLUSIONS: we showed that SENP1 provided protection for kidney in AKI via regulating cell apoptosis and through the regulation of HIF-1α. This study could benefit for the understanding of the pathogenesis of AKI and provide potential therapeutic target for AKI treatment.

Screening ssDNA Aptamers Against Human Vascular Endothelial Factor 165 via Semirational Design.

As a valid tumor marker, vascular endothelial growth factor 165 (VEGF165) is an effective therapeutic target for anticancer treatments. Aptamers hold great promise for the development of anti-VEGF strategies. In this study, anti-VEGF165 ssDNA aptamers were screened using a semirational design and a multilevel screening strategy. Recombinant human VEGF165 protein was used as a target for the construction of an ssDNA virtual aptamer library with ssDNA that had one sole secondary structure. After silicon-assisted prescreening, circular dichroism and isothermal titration calorimetry were used to further screen for candidates. Three aptamers (nos. 524, 529, and 64) with one sole secondary and tertiary structure, showing a high affinity for VEGF165, were identified. The KD values obtained using surface plasmon resonance analysis were 36.3, 288, and 79.3 nM for aptamers 524, 529, and 64, respectively. Cytological tests revealed that the three aptamers inhibit rhVEGF165-induced proliferation of HUVECs. Specifically, aptamer 529 had the strongest inhibitory effect (nearly 100% inhibition). The screening strategy used in our study showed improved screening efficiency relative to other methods and resulted in aptamers with one sole conformation. The aptamers had an advantage in ensuring the uniqueness of aptamer targeting. This semirational design and multilevel screening strategy provide a reference for the screening of other aptamers.

Emodin ameliorates tubulointerstitial fibrosis in obstructed kidneys by inhibiting EZH2.

Emodin, a major component of Chinese herbal rhubarb, delays the progression of chronic renal failure. However, the effect and working mechanisms of Emodin on renal tubulointerstitial fibrosis remains elusive. We hypothesized that emodin inhibits renal tubulointerstitial fibrosis through EZH2, a histone methyltransferase. Our in vivo and in vitro studies demonstrate that emodin reduced extracellular collagen deposition and inhibited Smad3 and CTGF pro-fibrotic signaling pathways, which were correlated with the down-regulation of EZH2 and reduced trimethylation of histone H3 on lysine 27 (H3k27me3) in NRK-49F fibrotic cells and UUO kidneys. Inhibition of EZH2 by 3-DZNeP blocked or attenuated the anti-fibrotic effect of emodin in UUO kidneys and NRK-49F cells. These data indicate that emodin inhibits renal tubulointerstitial fibrosis in obstructed kidneys and this effect is mediated through EZH2.

The endophytic bacterium Bacillus koreensis 181-22 promotes rice growth and alleviates cadmium stress under cadmium exposure.

Recently, cadmium (Cd) contamination in paddy soils has become a highly concerning pollution problem. Endophytic microbes in rice not only affect the plant growth but also contribute to ion absorption by the roots. Therefore, they are a promising, ecologically sound means of reducing the Cd transport from soils to shoots and grains of the plant. In this study, a Cd-resistant endophytic bacterium, named 181-22, with high Cd absorption capacity (90.8%) was isolated from the roots of rice planting in heavily Cd-contaminated paddy soils and was identified as Bacillus koreensis CGMCC 19,468. The strain significantly increased fresh weight of roots and shoots (44.4% and 42.7%) and dry weight of roots and shoots (71.3% and 39.9%) and decreased Cd content in the rice roots (12.8%), shoots (34.3%), and grains (39.1%) under Cd stress compared to uninoculated plant by colonizing rice roots via seed inoculation. Moreover, colonization of 181-22 reprogrammed rice physiology to alleviate Cd stress by increasing pigment and total protein content, regulating Cd-induced oxidative stress enzymes such as superoxide dismutase and catalase and reducing malondialdehyde. Thus, B. koreensis 181-22 has the potential to protect rice against Cd stress and can be used as a biofertilizer to bioremediate paddy soils contaminated with Cd. KEY POINTS: • Bacillus koreensis 181-22 colonized the inside of rice roots at high numbers via seed inoculation. • B. koreensis 181-22 promoted rice growth and decreased Cd accumulation in grains. • B. koreensis 181-22 regulated the physiological response to alleviated Cd stress in rice.

A Sequence-Indexed Mutator Insertional Library for Maize Functional Genomics Study.

Sequence-indexed insertional libraries are important resources for functional gene study in model plants. However, the maize (Zea mays) UniformMu library covers only 36% of the annotated maize genes. Here, we generated a new sequence-indexed maize Mutator insertional library named ChinaMu through high-throughput sequencing of enriched Mu-tagged sequences. A total of 2,581 Mu F2 lines were analyzed, and 311,924 nonredundant Mu insertion sites were obtained. Based on experimental validation, ChinaMu contains about 97,000 germinal Mu insertions, about twice as many as UniformMu. About two-thirds (66,565) of the insertions are high-quality germinal insertions (positive rate > 90%), 89.6% of which are located in genic regions. Furthermore, 45.7% (20,244) of the 44,300 annotated maize genes are effectively tagged and about two-thirds (13,425) of these genes harbor multiple insertions. We tested the utility of ChinaMu using pentatricopeptide repeat (PPR) genes. For published PPR genes with defective kernel phenotypes, 17 out of 20 were tagged, 11 of which had the previously reported mutant phenotype. For 16 unstudied PPR genes with both Mu insertions and defective kernel phenotypes, 6 contained insertions that cosegregated with the mutant phenotype. Our sequence-indexed Mu insertional library provides an important resource for functional genomics study in maize.

18ß-Glycyrrhetinic acid acts through hepatocyte nuclear factor 4 alpha to modulate lipid and carbohydrate metabolism.

Hepatocyte nuclear factor 4 alpha (HNF4α) regulates the expression of essential genes involved in very-low-density lipoprotein (VLDL) homeostasis and gluconeogenesis.　18ß-glycyrrhetinic acid (GA) is an active ingredient of Glycyrrhiza uralensis an herbal medicine used for treating liver aliments. In this study, we established that GA functions as a partial antagonist of HNF4α through HNF4α-driven reporter luciferase assay and co-immunoprecipitation experiments with co-activator PGC1α. By virtual docking and site-directed mutagenesis analysis, we confirmed that serine 190 and arginine 235 of HNF4α are both essential for GA to exert its antagonistic action on HNF4α. Importantly, GA suppressed the expression of HNF4α target genes such as apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTP) and phospholipase A2 G12B (PLA2G12B) modulating hepatic VLDL secretion in mice fed on a high fat diet. In addition, GA also suppressed gluconeogenesis and ameliorated glucose intolerance via down-regulating the expression of HNF4α target genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (Pepck). Furthermore, GA significantly lowered blood glucose and improved insulin resistance in db/db mice. In all, we established that GA acts as a partial HNF4α antagonist modulating lipid and carbohydrate metabolism.

Maize opaque10 Encodes a Cereal-Specific Protein That Is Essential for the Proper Distribution of Zeins in Endosperm Protein Bodies.

Cereal storage proteins are major nitrogen sources for humans and livestock. Prolamins are the most abundant storage protein in most cereals. They are deposited into protein bodies (PBs) in seed endosperm. The inner structure and the storage mechanism for prolamin PBs is poorly understood. Maize opaque10 (o10) is a classic opaque endosperm mutant with misshapen PBs. Through positional cloning, we found that O10 encodes a novel cereal-specific PB protein. Its middle domain contains a seven-repeat sequence that is responsible for its dimerization. Its C terminus contains a transmembrane motif that is required for its ER localization and PB deposition. A cellular fractionation assay indicated that O10 is initially synthesized in the cytoplasm and then anchored to the ER and eventually deposited in the PB. O10 can interact with 19-kD and 22-kD α-zeins and 16-kD and 50-kD γ-zeins through its N-terminal domain. An immunolocalization assay indicated that O10 co-localizes with 16-kD γ-zein and 22-kD α-zein in PBs, forming a ring-shaped structure at the interface between the α-zein-rich core and the γ-zein-rich peripheral region. The loss of O10 function disrupts this ring-shaped distribution of 22-kD and 16-kD zeins, resulting in misshapen PBs. These results showed that O10, as a newly evolved PB protein, is essential for the ring-shaped distribution of 22-kD and 16-kD zeins and controls PB morphology in maize endosperm.

Aging Reduces an ERRalpha-Directed Mitochondrial Glutaminase Expression Suppressing Glutamine Anaplerosis and Osteogenic Differentiation of Mesenchymal Stem Cells.

Aging deteriorates osteogenic capacity of mesenchymal stem/stromal cells (MSCs), contributing to imbalanced bone remodeling and osteoporosis. Glutaminase (Gls) catabolizes glutamine into glutamate at the first step of mitochondrial glutamine (Gln)-dependent anaplerosis which is essential for MSCs upon osteogenic differentiation. Estrogen-related receptor α (ERRα) regulates genes required for mitochondrial function. Here, we found that ERRα and Gls are upregulated by osteogenic induction in human MSCs (hMSCs). In contrast, osteogenic differentiation capacity and glutamine consumption of MSCs, as well as ERRα, Gls and osteogenic marker genes are significantly reduced with age. We demonstrated that ERRα binds to response elements on Gls promoter and affects glutamine anaplerosis through transcriptional induction of Gls. Conversely, mTOR inhibitor rapamycin, ERRα inverse agonist compound 29 or Gls inhibitor BPTES leads to reduced Gln anaplerosis and deteriorated osteogenic differentiation of hMSCs. Importantly, overexpression of ERRα or Gls restored impairment by these inhibitors. Finally, we proved that compensated ERRα or Gls expression indeed potentiated Gln anaplerosis and osteogenic capability of elderly mice MSCs in vitro. Together, we establish that Gls is a novel ERRα target gene and ERRα/Gls signaling pathway plays an important role in osteogenic differentiation of MSCs, providing new sights into novel regenerative therapeutics development. Our findings suggest that restoring age-related mitochondrial Gln-dependent anaplerosis may be beneficial for degenerative bone disorders such as osteoporosis. Stem Cells 2017;35:411-424.

Crystal structures of Aflatoxin-oxidase from Armillariella tabescens reveal a dual activity enzyme.

Aflatoxin-oxidase (AFO), a newly discovered oxidase isolated from Armillariella tabescens, was reported to perform aflatoxin B1 (AFB1) detoxification through breaking the bisfuran ring of AFB1. However, based on sequence alignment, we found that AFO shares high sequence identities with dipeptidyl peptidase III (DPP III) family members. To understand the functions of AFO, we determined its crystal structures in the absence and presence of zinc, copper ion, and employed HPLC to test if AFO could cleave the substrates of DPP III. Our structures reveal that AFO contains the classic DPP III activity center and the HPLC results further confirm that AFO possesses the dipeptidyl peptidase activity. Therefore, AFO should belong to DPP III family. Interestingly, unlike reported classic DPP III structure that has a large domain movement upon substrate binding, the AFO structures all adopt the closed conformation, independent of substrate binding. This conformation characteristic of AFO may be related to its enzyme activities. Taken together, our results demonstrate that AFO is a dual activity enzyme with both aflatoxin-oxidase and dipeptidyl peptidase activities and its unique conformation feature expands our understanding on the mode of reaction for this enzyme family.

Maize reas1 Mutant Stimulates Ribosome Use Efficiency and Triggers Distinct Transcriptional and Translational Responses.

Ribosome biogenesis is a fundamental cellular process in all cells. Impaired ribosome biogenesis causes developmental defects; however, its molecular and cellular bases are not fully understood. We cloned a gene responsible for a maize (Zea mays) small seed mutant, dek* (for defective kernel), and found that it encodes Ribosome export associated1 (ZmReas1). Reas1 is an AAA-ATPase that controls 60S ribosome export from the nucleus to the cytoplasm after ribosome maturation. dek* is a weak mutant allele with decreased Reas1 function. In dek* cells, mature 60S ribosome subunits are reduced in the nucleus and cytoplasm, but the proportion of actively translating polyribosomes in cytosol is significantly increased. Reduced phosphorylation of eukaryotic initiation factor 2α and the increased elongation factor 1α level indicate an enhancement of general translational efficiency in dek* cells. The mutation also triggers dramatic changes in differentially transcribed genes and differentially translated RNAs. Discrepancy was observed between differentially transcribed genes and differentially translated RNAs, indicating distinct cellular responses at transcription and translation levels to the stress of defective ribosome processing. DNA replication and nucleosome assembly-related gene expression are selectively suppressed at the translational level, resulting in inhibited cell growth and proliferation in dek* cells. This study provides insight into cellular responses due to impaired ribosome biogenesis.

Comprehensive proteomic analysis of developing protein bodies in maize (Zea mays) endosperm provides novel insights into its biogenesis.

Prolamins, the major cereal seed storage proteins, are sequestered and accumulated in the lumen of the endoplasmic reticulum (ER), and are directly assembled into protein bodies (PBs). The content and composition of prolamins are the key determinants for protein quality and texture-related traits of the grain. Concomitantly, the PB-inducing fusion system provides an efficient target to produce therapeutic and industrial products in plants. However, the proteome of the native PB and the detailed mechanisms underlying its formation still need to be determined. We developed a method to isolate highly purified and intact PBs from developing maize endosperm and conducted proteomic analysis of intact PBs of zein, a class of prolamine protein found in maize. We thus identified 1756 proteins, which fall into five major categories: metabolic pathways, response to stimulus, transport, development, and growth, as well as regulation. By comparing the proteomes of crude and enriched extractions of PBs, we found substantial evidence for the following conclusions: (i) ribosomes, ER membranes, and the cytoskeleton are tightly associated with zein PBs, which form the peripheral border; (ii) zein RNAs are probably transported and localized to the PB-ER subdomain; and (iii) ER chaperones are essential for zein folding, quality control, and assembly into PBs. We futher confirmed that OPAQUE1 (O1) cannot directly interact with FLOURY1 (FL1) in yeast, suggesting that the interaction between myosins XI and DUF593-containing proteins is isoform-specific. This study provides a proteomic roadmap for dissecting zein PB biogenesis and reveals an unexpected diversity and complexity of proteins in PBs.

Identification and Characterization of Maize floury4 as a Novel Semidominant Opaque Mutant That Disrupts Protein Body Assembly.

Zeins are the major seed storage proteins in maize (Zea mays). They are synthesized on the endoplasmic reticulum (ER) and deposited into protein bodies. Failure of signal peptide cleavage from zeins can cause an opaque endosperm in the mature kernel; however, the cellular and molecular mechanisms responsible for this phenotype are not fully understood. In this study, we report the cloning and characterization of a novel, semidominant opaque mutant, floury4 (fl4). fl4 is caused by a mutated z1A 19-kD α-zein with defective signal peptide cleavage. Zein protein bodies in fl4 endosperm are misshapen and aggregated. Immunolabeling analysis indicated that fl4 participates in the assembly of zeins into protein bodies, disrupting their proper spatial distribution. ER stress is stimulated in fl4 endosperm, as illustrated by dilated rough ER and markedly up-regulated binding protein content. Further analysis confirmed that several ER stress pathways are induced in fl4 endosperm, including ER-associated degradation, the unfolded protein response, and translational suppression by the phosphorylation of eukaryotic translational initiation factor2 α-subunit. Programmed cell death is also elevated, corroborating the intensity of ER stress in fl4. These results provide new insights into cellular responses caused by storage proteins with defective signal peptides.

A Clickable NAD+ Analog-Based Assay of Poly(ADP-Ribosyl)ated Proteins.

Poly(ADP-ribosyl)lation (PARylation) is a posttranslational modification that plays an important role in a variety of biological processes in both animals and plants. Identification of PARylated substrates is the key to elucidating the regulatory mechanism of PARylation. Several approaches have been developed to identify PARylated substrates over the past decade; however, a reliable and efficient method is needed to demonstrate PARylated proteins. Here, we report a simple and sensitive assay of PARylated proteins using a clickable 6-alkyne-NAD+ analog. The 6-alkyne-NAD+ is incorporated into substrate proteins in the in vitro PARylation assay. The labeled proteins are covalently captured by disulfide azide agarose beads through copper-catalyzed azide-alkyne cycloaddition (CuAAC), cleaved under reducing conditions, and analyzed by immunoblotting. The covalent bonds between the PARylated proteins and azide beads allow high stringent washing to eliminate nonspecific binding. Furthermore, the disulfide linker permits efficient cleavage and recovery of highly enriched PARylated proteins. Therefore, this approach can detect proteins that undergo PARylation at very low levels.

Increased yield of 2-O-α-d-glucopyranosyl-l-ascorbic acid synthesis by α-glucosidase using rational design that regulating the ground state of enzyme and substrate complex.

BACKGROUND: α-Glucosidase (AG) is a bifunctional enzyme, it has a capacity to synthesize 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) from l-ascorbic acid (L-AA) and low-cost maltose under mild conditions, but it can also hydrolyze AA-2G, which leads to low synthesis efficiency of AA-2G. MAIN METHODS AND MAJOR RESULTS: This study introduces a rational molecular design strategy to regulate enzymatic reactions based on inhibiting the formation of ground state of enzyme-substrate complex. Y215 was analyzed as the key amino acid site affecting the affinity of AG to AA-2G and L-AA. For the purpose of reducing the hydrolysis efficiency of AA-2G, the mutant Y215W was obtained by analyzing the molecular docking binding energy and hydrogen bond formation between AG and the substrates. Compared with the wild-type, isothermal titration calorimetry (ITC) results showed that the equilibrium dissociation constant (KD ) of the mutant for AA-2G was doubled; the Michaelis constant (Km ) for AA-2G was reduced by 1.15 times; and the yield of synthetic AA-2G was increased by 39%. CONCLUSIONS AND IMPLICATIONS: Our work also provides a new reference strategy for the molecular modification of multifunctional enzymes and other enzymes in cascade reactions system.

[Comparison of three α-glucosidases from different sources in the synthesis of L-ascorbic acid 2-glucoside].

L-ascorbic acid 2-glucoside (AA-2G) is a derivative of L-ascorbic acid (L-AA). Compared with L-AA, it has good stability and is easily decomposed by enzyme in the human body. α-Glucosidase (AG) was the first enzyme found capable of producing AA-2G. However, researches on this enzyme is still in infancy. We took AG derived from Aspergillus niger (AAG), Japanese rice (JrAG) and Rattus rattus (RAG), and compared their specific enzymatic activity and transglycosidation rate, with the aim to improve the synthesis of AA-2G by the transglycosidation of AG. The genes encoding these three different AG were cloned and expressed in engineered yeast. The conditions for the transglycosidation reaction of these three enzymes were optimized and the transglycosidation efficiency and yield of AA-2G under the optimized conditions were compared. The specific activity of AAG reached 1.0 U/mg, while the yield of AA-2G reached 153.1 mg/L with a transglycosidation rate of 0.5%. The specific activity of RAG reached 0.4 U/mg, while the yield of AA-2G reached 861.0 mg/L with a transglycosidation rate of 2.5%. JrAG showed the highest specific activity and transglycosidation rate. The enzyme specific activity of JrAG reached 1.9 U/mg, while the yield of AA-2G reached 2 577.2 mg/L with a transglycosidation rate of 7.6%, much higher than that of the other two glucosidases. JrAG may thus have potential to improve the synthesis of AA-2G.

Effects of resveratrol on renal ischemia-reperfusion injury: A systematic review and meta-analysis.

Renal ischemia-reperfusion (I/R) injury may lead to acute kidney injury, which is characterized by high morbidity and mortality rates. Resveratrol (RSV) can be extracted from Chinese herbs, and multiple animal experiments have demonstrated its potential for renal protection. This systematic review evaluates the protective effect of RSV against renal I/R injury in animal models. The PubMed, Embase, Web of Science, and Science Direct databases were searched for animal experiments related to RSV in renal I/R injury from their establishment to June 2022. In total, 19 studies were included with 249 animals (129 treated with RSV and 120 as controls). The pooled analysis revealed that RSV administration significantly decreased serum creatinine (SCr) levels (16 studies, n = 243, WMD = -58.13, 95% CI = -79.26 to -37.00, p < 0.00001) and blood urea nitrogen (BUN) levels (12 studies, n = 163, WMD = -34.37, 95% CI = -46.70 to -22.03, p < 0.00001) in the renal I/R injury model. The level of malondialdehyde (MDA), an oxidative stress index, was alleviated [7 studies, n = 106, standardized mean difference (SMD) = -6.05, 95% CI = -8.90 to -3.21, p < 0.0001] and antioxidant enzymes such as glutathione (GSH) (7 studies, n = 115, SMD = 9.25, 95% CI = 5.51-13.00, p < 0.00001) and catalase (CAT) (4 studies, n = 59, SMD = 8.69, 95% CI = 4.35-13.03, p < 0.0001) were increased after treatment of RSV. The subgroup analysis suggested that 5-10 mg/kg of RSV optimally protects against renal I/R injury as both the BUN and SCr levels were significantly decreased at this dosage. The protective effects of RSV against renal I/R injury might be attributed to multiple mechanisms, such as inhibiting oxidative stress, apoptosis, inflammation, fibrillation, and promoting autophagy. For a deeper understanding of the protective effects of RSV, experimental studies on animal models and large randomized controlled trials in humans are needed.

A four oxidative stress gene prognostic model and integrated immunity-analysis in pancreatic adenocarcinoma.

Background and aims: Pancreatic adenocarcinoma (PAAD) is highly aggressive and characterized by a poor prognosis. Oxidative stress has great impacts on the occurrence and development of tumors. However, the predictive role of oxidative stress related genes on PAAD patients' prognosis remains unclear. In this study, we aimed to construct a prognostic model for PAAD based on oxidative stress genes and to evaluate its predictive value. Methods: The Cancer Genome Atlas (TCGA) and three Gene Expression Omnibus (GEO) datasets were used to identify differentially expressed oxidative stress genes. Univariate Cox regression, Kaplan-Meier and multivariate Cox regression analysis were used to select genes and to construct a prognosis model. According to the median value of the model's risk score, patients were divided into high and low risk groups, and gene set enrichment analysis (GSEA), immune infiltration and immunotherapy effect, drug resistance and the expression of immune checkpoint related genes and synthetic driver genes of T cell proliferation were analyzed. Finally, the mRNA and protein levels of four genes in PAAD were verified by the clinical proteomic tumor analysis consortium (CPTAC) database and the immunostaining of patients' tissue. Results: 55 differentially expressed oxidative stress genes were identified, and four genes including MET, FYN, CTTN and CDK1 were selected to construct a prognosis model. GESA indicated that immune related pathways, metabolic pathways and DNA repair pathways were significantly enriched in the high risk group as compared to the low risk group. The frequency of genetic mutations was also significantly higher in high risk groups than that in low risk groups. Moreover, the infiltration level of 23 immune cells as well as the expression of immune checkpoint related and synthetic driver genes of T cell proliferation were significantly altered, with the better immunotherapy effect occurring in low risk group. In patient PAAD tissues, the mRNA and protein levels of these four genes were up-regulated. Conclusion: We have successfully constructed a four oxidative stress gene prognostic model that has important predictive value for PAAD patients, and this model might be a promising guidance for prognostic prediction and efficacy monitoring in clinical individualized therapy.

Cloning, expression, purification and characterization of an aflatoxin-converting enzyme from Armillaria tabescens.

OBJECTIVE: Aflatoxin B1 (AFB1) is extremely mutagenic, toxic and a potent carcinogen both to humans and livestock. Aflatoxin-oxidase (AFO) was an aflatoxin-converting enzyme previously purified by us from Armillaria tabescens. In order to know better about the molecular characterization of this distinct enzyme, we expressed, purified and characterized the His6 tag fused aflatoxin-oxidase. METHODS: Based on sequences of peptides fragments of AFO previously obtained by Electrophoresis-Electrospray Ionization tandem mass spectrometry (ESI-MS/MS), we cloned the cDNA of AFO using Switching Mechanism At 5' end of the RNA Transcript (SMART) Rapid Amplification of cDNA Ends (RACE) technology and expressed this gene as a fusion protein in Pichia pastoris by using pPIC9-afo as vector. We purified the fusion enzyme using nickel affinity chromatography. We identified the recombinant aflatoxin-oxidase (rAFO) by both western blot and peptide mass fingerprinting (PMF). Moreover, we characterized several enzymatic properties of the rAFO using AFB1 as the substrate including Km value, optimum temperature, optimum pH, thermal stability and pH stability. RESULTS: The AFO gene is 2321 bp long with a coding region of 2088 bp encoding 695 amino acids. Peptide mass fingerprinting (PMF) identification showed a 63.2% coverage of the molecule compared to the theoretical tryptic cleavage of the rAFO. The recombinant aflatoxin oxidase was purified 5.99-folds using nickel affinity chromatography. It has a specific activity of 234 U/mg. Kinetics studies showed that the rAFO converted AFB1 with the Km value of 3.93 +/- 0.20 x 10(-6) mol/L under its optimal conditions of pH 6.0 and 30 degrees C. Thermostability investigation revealed that the rAFO had a half-life of 90 min at 30 degrees C, and pH stability results suggested that the rAFO was relatively stable when pH ranged from 5.5 to 7.5. CONCLUSION: It appears to be the first successful production of the recombinant aflatoxin oxidase (rAFO) with AFB1-converting ability from Armillaria tabescens. The purified rAFO with preferably AFB1-converting activity confirms that this recombinant aflatoxin oxidase is now ready for further studying.

[Production of high-purity recombinant human vascular endothelial growth factor (rhVEGF165) by Pichia pastoris].

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.

Coordinated regulation of plant immunity by poly(ADP-ribosyl)ation and K63-linked ubiquitination.

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification reversibly catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs) and plays a key role in multiple cellular processes. The molecular mechanisms by which PARylation regulates innate immunity remain largely unknown in eukaryotes. Here we show that Arabidopsis UBC13A and UBC13B, the major drivers of lysine 63 (K63)-linked polyubiquitination, directly interact with PARPs/PARGs. Activation of pathogen-associated molecular pattern (PAMP)-triggered immunity promotes these interactions and enhances PARylation of UBC13. Both parp1 parp2 and ubc13a ubc13b mutants are compromised in immune responses with increased accumulation of total pathogenesis-related (PR) proteins but decreased accumulation of secreted PR proteins. Protein disulfide-isomerases (PDIs), essential components of endoplasmic reticulum quality control (ERQC) that ensure proper folding and maturation of proteins destined for secretion, complex with PARPs/PARGs and are PARylated upon PAMP perception. Significantly, PARylation of UBC13 regulates K63-linked ubiquitination of PDIs, which may further promote their disulfide isomerase activities for correct protein folding and subsequent secretion. Taken together, these results indicate that plant immunity is coordinately regulated by PARylation and K63-linked ubiquitination.