Lowering Endogenous Cathepsin D Abundance Results in Reactive Oxygen Species Accumulation and Cell Senescence.

Cathepsin D is reportedly to be closely associated with tumor development, migration, and invasion, but its pathological mechanism is not fully elucidated. We aimed to evaluate phenotypic changes and molecular events in response to cathepsin D knockdown. Lowering endogenous cathepsin D abundance (CR) induced senescence in HeLa cells, leading to reduced rate of cell proliferation and impaired tumorigenesis in a mouse model. Quantitative proteomics revealed that compared with control cells (EV), the abundances of several typical lysosomal proteases were decreased in the lysosomal fraction in CR cells. We further showed that cathepsin D knockdown caused increased permeability of lysosomal membrane and reactive oxygen species accumulation in CR cells, and the scavenging of reactive oxygen species by antioxidant was able to rescue cell senescence. Despite the increased reactive oxygen species, the proteomic data suggested a global reduction of redox-related proteins in CR cells. Subsequent analysis indicated that the transcriptional activity of nuclear factor erythroid-related factor 2 (Nrf2), which regulates the expression of groups of antioxidant enzymes, was down-regulated by cathepsin D knockdown. Importantly, Nrf2 overexpression significantly reduced cell senescence. Although transient oxidative stress promoted the accumulation of Nrf2 in the nucleus, we showed that the Nrf2 protein exited nucleus if oxidative stress persisted. In addition, when cathepsin D was transiently knocked down, the cathepsin-related events followed a sequential order, including lysosomal leakage during the early stage, followed by oxidative stress augmentation, and ultimately Nrf2 down-regulation and senescence. Our results suggest the roles of cathepsin D in cancer cells in maintaining lysosomal integrity, redox balance, and Nrf2 activity, thus promoting tumorigenesis. The MS Data are available via ProteomeXchange with identifier PXD002844.

Ubiquitination of inositol-requiring enzyme 1 (IRE1) by the E3 ligase CHIP mediates the IRE1/TRAF2/JNK pathway.

Deciphering the inositol-requiring enzyme 1 (IRE1) signaling pathway is fundamentally important for understanding the unfolded protein response (UPR). The ubiquitination of proteins residing on the endoplasmic reticulum (ER) membrane has been reported to be involved in the UPR, although the mechanism has yet to be fully elucidated. Using immunoprecipitation and mass spectrometry, IRE1 was identified as a substrate of the E3 ligase CHIP (carboxyl terminus of HSC70-interacting protein) in HEK293 cells under geldanamycin-induced ER stress. Two residues of IRE1, Lys(545) and Lys(828), were targeted for Lys(63)-linked ubiquitination. Moreover, in CHIP knockdown cells, IRE1 phosphorylation and the IRE1-TRAF2 interaction were nearly abolished under ER stress, which may be due to lacking ubiquitination of IRE1 on Lys(545) and Lys(828), respectively. The cellular responses were evaluated, and the data indicated that CHIP-regulated IRE1/TRAF2/JNK signaling antagonized the senescence process. Therefore, our findings suggest that CHIP-mediated ubiquitination of IRE1 contributes to the dynamic regulation of the UPR.

Evaluation of barley genotypes for drought adaptability: based on stress indices and comprehensive evaluation as criteria.

The prevalence of drought events worldwide emphasizes the importance of screening and cultivating drought-adapted crops. In this study, 206 germplasm resources were used as materials, dry weight as target trait, and two genotyping methods as criteria to evaluate drought adaptability at the seedling establishment stage. The results showed a significant decrease in average dry weight of the tested germplasm resources (from 746.90 mg to 285.40 mg) and rich variation in the responses of dry weight among each genotype to drought (CV=61.14%). In traditional evaluation method, drought resistance coefficient (DC), geometric mean productivity index (GMP), mean productivity index (MP), stress susceptibility index (SSI), stress tolerance index (STI), and tolerance index (TOL) also exhibited diversity in tested genotypes (CV>30%). However, these indices showed varying degrees of explanation for dry weight under stress and non-stress environments and failed to differentiate drought adaptability among genotypes clearly. In new evaluation method, four stress indices were developed to quantify barley seedling production and stability capacities. Compared to traditional stress indices, the stress production index (SI) explained dry weight more comprehensively under stress conditions (R2 = 0.98), while the ideal production index (II) explained dry weight better under non-stress conditions (R2 = 0.89). Furthermore, the potential index (PI) and elasticity index (EI) eliminated disparities in traditional stress indices and comprehensively clarified the contribution of elasticity and potential to production capacity under drought stress. Ultimately, through grading evaluation and cluster analysis, the tested germplasm resources were effectively categorized, and 11 genotypes were identified as suitable for cultivation in arid areas. Overall, the comprehensive evaluation method based on the newly developed stress indices surpasses the traditional method in screening drought adaptability of crops and serves as a vital tool for identifying high-stability and high-production capacities genotypes in various environments, which is expected to provide practical guidance for barley planting and breeding in arid areas.

Proteomic Analysis of Avian Influenza A (H7N9) Patients within a Family Cluster.

BACKGROUND: To date, there is limited information on the progression of human infections of avian influenza virus A (H7N9). This study investigated differential blood protein profiling of a H7N9-infected family cluster to find a slice of crucial proteins concerning disease attack and virus clearance. MATERIALS AND METHODS: Plasma samples from one family cluster (including one index case and one asymptomatic case) were collected at four time points. The protein profiles were identified by isobaric tagging for relative and absolute quantification-based quantitative differential LC/MS/MS, and their functional annotations were analyzed by PANTHER and STRING tools. RESULTS: A total of 1257 nonredundant proteins were identified from 3027 unique peptides. Three differential protein profiles for each subject were generated by comparing relative protein abundance between samples of each of the first three time points and the last time point. Gene ontology analysis indicated that differential protein profiles for the two cases were mainly enriched in the biological processes of response to stimulus, immunity, blood coagulation, lipid transport, and cell adhesion. Two groups of proteins with an upward or downward expression change according to the postinfection time points were detected for each case. STRING analysis further indicated that the hubs in the network of these time-dependent proteins were mostly apolipoproteins. CONCLUSIONS: Significant perturbation of the response upon viral infection occurred immediately after confirmation of H7N9 virus infection. The differential protein profiles shed further light on distinguishing the index case from the asymptomatic one. Furthermore, apolipoproteins may play an important role in the progression of the disease.

mTORC1 alters the expression of glycolytic genes by regulating KPNA2 abundances.

UNLABELLED: Mammalian target of rapamycin complex 1 (mTORC1) plays important roles in regulating cell growth and proliferation, and the aberrant activation of mTORC1 has been observed in many human diseases. However, the proteins regulated by mTORC1 activation and their roles in mTORC1 downstream functions are still poorly understood. Using proteomic analysis, we found that proteins regulated by mTORC1 in MEFs could be categorized into eight functional groups including protein nuclear import and glycolysis. The positive regulation of Karyopherin subunit alpha-2 (KPNA2), an importer protein involved in protein nuclear import, by mTORC1 was verified in several other mouse and human cell lines. The regulation occurred at the transcriptional level, rather than at the level of S6K1- and 4E-BP1-dependent protein synthesis. KPNA2 knockdown partially blocked upregulation of glycolytic genes by mTORC1 activation, indicating that mTORC1 activation enhanced expression of glycolytic genes by increasing KPNA2 abundances. Furthermore, KPNA2 knockdown had no effects on the expression and subcellular localization of HIF1α, a transcription factor involved in regulating glycolytic genes downstream of mTORC1. In conclusion, our results proved that KPNA2 regulated the expression of glycolytic genes downstream of mTORC1 in a HIF1α-independent manner. SIGNIFICANCE: Identifying mTORC1-regulated proteins through proteomic method is a feasible way to study the downstream functions of mTORC1. In this study, we identified many mTORC1-regulated proteins using proteomic analysis by overlapping two different high vs low/no mTORC1 activity comparisons, TSC2(-/-) vs WT MEFs and TSC2(-/-) with/without rapamycin treatment. We found the abundances of many enzymes in glycolysis pathway and several proteins involved in protein nuclear import were positively regulated by mTORC1. More importantly, we first discovered that mTORC1 positively regulated the importer protein KPNA2, which participated in glycolysis regulation downstream of mTORC1 in a HIF1α-independent manner, indicating that mTORC1 regulates glycolysis through multiple ways.