Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

Asparagine‐linked glycosylation is a common post‐translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site‐specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH‐MS to enable automated measurement of site‐specific occupancy at many glycosylation sites. Deglycosylation with peptide‐endoglycosidase H, leaving a remnant N‐acetylglucosamine on asparagines previously carrying high‐mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide‐N‐glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site‐specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site‐specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.     

四种豆科作物的光合生理和生长发育对弱光的响应

为了确定豆科作物作为林果行间套种作物的适宜性, 于2014年通过田间试验和盆栽试验对4种豆科作物的耐阴能力及其光合生理进行了研究。测定全光和弱光处理(全光的48%)下豆科作物在初花期的叶片光合特征参数、光合色素含量与核酮糖-1,5-二磷酸羧化酶(RuBPCase)活性, 监测其生长发育对弱光的响应。结果表明: 弱光使供试作物的最大净光合速率、光饱和点、光补偿点、表观量子效率和暗呼吸速率不同程度地向耐阴植物变化; 弱光还使供试作物的净光合速率、气孔导度、蒸腾速率、瞬时水分利用效率和RuBPCase活性不同程度地下降, 而胞间CO₂浓度显著上升; 遮阴后, 乌豇豆(Vigna cylindrica)和绿豆(Vigna radiata)的叶绿素(Chl) a和Chl b含量显著增加, Chl a/b值显著降低, 大猪屎豆(Crotalaria assamica)和望江南(Senna occidentalis)的光合色素含量也有不同程度的变化; 弱光使供试作物茎变细, 侧枝数减少, 生物量和干物质积累效率降低, 根冠比降低, 根瘤量减少, 叶片变小变薄, 叶片数减少, 叶面积指数降低; 弱光胁迫下, 大猪屎豆不开花, 望江南只开花不结实, 而绿豆和乌豇豆开花数减少, 花期缩短, 种子产量显著降低。根据供试作物的光合特性和生长发育对弱光的响应, 得出它们的耐阴能力排序是: 乌豇豆>绿豆>望江南>大猪屎豆, 其中乌豇豆和绿豆适宜与林果套种, 而望江南和大猪屎豆不适宜套种。     

Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine,Arginine, and Lysine Residues

Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein.     

Construction and Immunogenicity Evaluation of Recombinant Influenza A Viruses Containing Chimeric Hemagglutinin Genes Derived from Genetically Divergent Influenza A H1N1 Subtype Viruses

Background and Objectives

Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes.

Methods and Results

Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8_LAIV-129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8_LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates.

Conclusion

This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines.     

Traits mediate drought effects on wood carbon fluxes

CO₂ fluxes from wood decomposition represent an important source of carbon from forest ecosystems to the atmosphere, which are determined by both wood traits and climate influencing the metabolic rates of decomposers. Previous studies have quantified the effects of moisture and temperature on wood decomposition, but these effects were not separated from the potential influence of wood traits. Indeed, it is not well understood how traits and climate interact to influence wood CO₂ fluxes. Here, we examined the responses of CO₂ fluxes from dead wood with different traits (angiosperm and gymnosperm) to 0%, 35%, and 70% rainfall reduction across seasonal temperature gradients. Our results showed that drought significantly decreased wood CO₂ fluxes, but its effects varied with both taxonomical group and drought intensity. Drought‐induced reduction in wood CO₂ fluxes was larger in angiosperms than gymnosperms for the 35% rainfall reduction treatment, but there was no significant difference between these groups for the 70% reduction treatment. This is because wood nitrogen density and carbon quality were significantly higher in angiosperms than gymnosperms, yielding a higher moisture sensitivity of wood decomposition. These findings were demonstrated by a significant positive interaction effect between wood nitrogen and moisture on CO₂ fluxes in a structural equation model. Additionally, we ascertained that a constant temperature sensitivity of CO₂ fluxes was independent of wood traits and consistent with previous estimates for extracellular enzyme kinetics. Our results highlight the key role of wood traits in regulating drought responses of wood carbon fluxes. Given that both climate and forest management might extensively modify taxonomic compositions in the future, it is critical for carbon cycle models to account for such interactions between wood traits and climate in driving dynamics of wood decomposition.     

Adoptive transfers of CD4+CD25+ Tregs partially alleviate mouse premature ovarian insufficiency

This study was designed to investigate the protective effect of CD4⁺CD25⁺ regulatory T cells (Tregs) against zona pellucida glycoprotein 3 peptide (pZP3) immunization‐induced premature ovarian insufficiency (POI) in mice. A mouse POI model was induced by two subcutaneous injections of pZP3 (50 nmol/L). Mice in the pZP3‐Treg group were intraperitoneally injected with 5 × 10⁵ CD4⁺CD25⁺ Tregs after the POI model was established. Sex hormone levels, follicle numbers, apoptotic events, and the Akt/FOXO3a signaling pathway molecules in the ovaries were assessed. Compared with control group, the weight of ovaries in both pZP3 group and pZP3‐Treg group was decreased and no difference was found between them. The number of follicles in the Treg transferred mice, like in pZP3 group, was significantly reduced compared to the control group, but showed a modest improvement when compared the pZP3 group alone. Significantly lower serum concentrations of follicle‐stimulating hormone, luteinizing hormone, and anti‐zona pellucida antibodies (AZPAbs) were found, while the concentrations of estradiol and anti‐Mullerian hormone increased. In mechanism, Treg cell transfer to ZP3 treated mice restored the levels of Caspase3 to control levels, and partially restored Bax, however, had no effect on Bcl‐2. Moreover, Treg cell transfer to ZP3 treated mice partially restored the levels of Akt and FOXO3a, and partially restored the ratios of p‐Akt/Akt and p‐FOXO3a/FOXO3a. In conclusion, Treg cells improved some aspects of ZP3‐induced POI which may be mediate by suppressing ovarian cells apoptosis and involving the Akt/FOXO3a signaling pathway. Therefore, Treg cells may be protective against autoimmune POI.     

Tau inhibits PKA by nuclear proteasome‐dependent PKAR2α elevation with suppressed CREB/GluA1 phosphorylation

Intraneuronal accumulation of wild‐type tau plays a key role in Alzheimer's disease, while the mechanisms underlying tauopathy and memory impairment remain unclear. Here, we report that overexpressing full‐length wild‐type human tau (hTau) in mouse hippocampus induces learning and memory deficits with remarkably reduced levels of multiple synapse‐ and memory‐associated proteins. Overexpressing hTau inhibits the activity of protein kinase A (PKA) and decreases the phosphorylation level of cAMP‐response element binding protein (CREB), GluA1, and TrkB with reduced BDNF mRNA and protein levels both in vitro and in vivo. Simultaneously, overexpressing hTau increased PKAR2α (an inhibitory subunit of PKA) in nuclear fraction and inactivated proteasome activity. With an increased association of PKAR2α with PA28γ (a nuclear proteasome activator), the formation of PA28γ‐20S proteasome complex remarkably decreased in the nuclear fraction, followed by a reduced interaction of PKAR2α with 20S proteasome. Both downregulating PKAR2α by shRNA and upregulating proteasome by expressing PA28γ rescued hTau‐induced PKA inhibition and CREB dephosphorylation, and upregulating PKA improved hTau‐induced cognitive deficits in mice. Together, these data reveal that intracellular tau accumulation induces synapse and memory impairments by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling, and deficit of PA28γ‐20S proteasome complex formation contributes to PKAR2α elevation and PKA inhibition.