26S Proteasome Regulatory Subunit Research Articles

Mutation in the 26S proteasome regulatory subunit rpn2 gene in Plasmodium falciparum confers resistance to artemisinin.

Malaria parasites increasingly develop resistance to all drugs available in the market, hampering the goal of reducing malaria burden. Herein, we evaluated the impact of a single-nucleotide variant, E738K, present in the 26S proteasome regulatory subunit rpn2 gene, identified in Plasmodium chabaudi resistant parasites. Plasmids carrying a functional rpn2 interspecies chimeric gene with 5' recombination region from P. falciparum and 3' from P. chabaudi were constructed and transfected into Dd2 P. falciparum parasites. The 738K variant parasite line presented increased parasite survival when subjected to dihydroartemisinin (DHA), as well as increased chymotrypsin-like activity and decreased accumulation of polyubiquitinated proteins. We thus conclude that the ubiquitin-proteasome pathway, including the 738K variant, play an important role in parasite response to DHA, being the first report of a mutation in a potential DHA drug target enhancing parasite survival and contributing to a significant advance in the understanding the biology of artemisinin resistance.

Controlled atmosphere storage drive proteomic change in Chinese Daohuaxiang

Although the use of a controlled atmosphere is one of the most successful storage techniques, the mechanism thereof in rice storage remains unclear. We stored aromatic rice cultivar Daohuaxiang in a package filled with 98 % N2 and 35 % CO2 for 3 months. We investigated 2-acetyl-1-pyrroline loss, enzyme activities, and proteomics changes of rice during storage. The results showed that the content of 2-acetyl-1-pyrroline was reduced by 37.40 %, 25.65 %, and 43.89 % during storage using 98 % N2, 35 % CO2 controlled atmosphere storage, and conventional storage. Controlled atmosphere storage slowed down the increase of malondialdehyde content in Daohuaxiang. The results showed that 26S proteasome regulatory particle triple-A ATPase subunit 6, superoxide dismutase, glutathione transferase, and other key proteins were upregulated during 35 % CO2 regulation. This study provided a meaningful basis for exploring the regulation strategy of aromatic rice quality and strengthening the quality control of aromatic rice industry.

Alterations in the expression pattern of RBC membrane associated proteins (RMAPs) in whole body γ-irradiated Sprague Dawley rats

Purpose Peripheral blood serum/plasma proteins are frequently studied for their potential use as radiation exposure biomarkers. Here we report RBC membrane associated proteins (RMAPs), which show alterations in expression level following whole-body γ-irradiation of rats at sub-lethal/lethal doses. Materials and Methods RBCs from peripheral blood of Sprague Dawley rats were segregated using the Ficoll-Hypaque method, and membrane fractions were hypotonically isolated at various time points (6 h, 24 h, 48 h) after γ-irradiation at 2 Gy, 5 Gy, and 7.5 Gy doses. Following purification of proteins from these fractions, two-dimensional electrophoresis (2-DE) was carried out. Treatment induced differentially expressed protein spots (≥2 fold increase/decrease) were picked up, trypsinized, and identified using LC-MS/MS analysis. Western immunoblots using protein specific antibodies were used to confirm the results. Gene ontology and interactions of these proteins were also studied. Results From a number of differentially expressed radiation-responsive 2-DE protein spots detected, eight were identified unequivocally using LC-MS/MS. Out of these, actin, cytoplasmic 1 (ACTB) showed detectable yet insignificant variation (<50%) in expression. In contrast, peroxiredoxin-2 (PRDX2) and 26S proteasome regulatory subunit RPN11 (PSMD14) were the two most prominently over-expressed proteins. Five more proteins, namely tropomyosin alpha-3 chain (TPM3), exosome component 6 (EXOSC6), isoform 4 of tropomyosin alpha-1 chain (TPM1), serum albumin (ALB), and the 55 kDa erythrocyte membrane protein (P55) showed distinct alteration in their expression at different time-points and doses. ALB, EXOSC6, and PSMD14 were the most responsive at 2 Gy, albeit at different time-points. While EXOSC6 and PSMD14 showed maximum over-expression (5-12 fold) at 6 h post-irradiation, ALB expression increased progressively (4 up to 7 fold) from 6 h to 48 h. TPM1 showed over-expression (2-3 fold) at all doses and time-points tested. TPM3 showed a dose-dependent response at all time-points studied; with no variation at 2 Gy, ∼2 fold increase at 5 Gy, and 3-6 fold at the highest dose used (7.5 Gy). The p55 protein was over-expressed (∼2.5 fold) only transiently at 24 h following the lethal (7.5 Gy) dose. Conclusion This is the first study to report γ-radiation induced alterations in the RBC membrane associated proteins. We are further evaluating the potential of these proteins as radiation biomarkers. Due to the abundance and easy use of RBCs, this approach can prove very useful for detecting ionizing radiation exposure.

PSMD12-Mediated M1 Ubiquitination of Influenza A Virus at K102 Regulates Viral Replication.

ABSTRACTThe M1 of influenza A virus (IAV) is important for the virus life cycle, especially for the assembly and budding of viruses, which is a multistep process that requires host factors. Identifying novel host proteins that interact with M1 and understanding their functions in IAV replication are of great interest in antiviral drug development. In this study, we identified 19 host proteins in DF1 cells suspected to interact with the M1 protein of an H5N6 virus through immunoprecipitation (IP)/mass spectrometry. Among them, PSMD12, a 26S proteasome regulatory subunit, was shown to interact with influenza M1, acting as a positive host factor in IAV replication in avian and human cells. The data showed that PSMD12 promoted K63-linked ubiquitination of M1 at the K102 site. H5N6 and PR8 with an M1-K102 site mutant displayed a significantly weaker replication ability than the wild-type viruses. Mechanistically, PSMD12 promoted M1-M2 virus-like particle (VLP) release, and an M1-K102 mutation disrupted the formation of supernatant M1-M2 VLPs. An H5N6 M1-K102 site mutation or knockdown PSMD12 disrupted the budding release of the virus in chicken embryo fibroblast (CEF) cells, which was confirmed by transmission electron microscopy. Further study confirmed that M1-K102 site mutation significantly affected the virulence of H5N6 and PR8 viruses in mice. In conclusion, we report the novel host factor PSMD12 which affects the replication of influenza virus by mediating K63-linked ubiquitination of M1 at K102. These findings provide novel insight into the interactions between IAV and host cells, while suggesting an important target for anti-influenza virus drug research.IMPORTANCE M1 is proposed to play multiple biologically important roles in the life cycle of IAV, which relies largely on host factors. This study is the first one to identify that PSMD12 interacts with M1, mediates K63-linked ubiquitination of M1 at the K102 site, and thus positively regulates influenza virus proliferation. PSMD12 promoted M1-M2 VLP egress, and an M1-K102 mutation affected the M1-M2 VLP formation. Furthermore, we demonstrate the importance of this site to the morphology and budding of influenza viruses by obtaining mutant viruses, and the M1 ubiquitination regulator PSMD12 has a similar function to the M1 K102 mutation in regulating virus release and virus morphology. Additionally, we confirm the reduced virulence of H5N6 and PR8 (H1N1) viruses carrying the M1-K102 site mutation in mice. These findings provide novel insights into IAV interactions with host cells and suggest a valid and highly conserved candidate target for antiviral drug development.

Hepatitis B Virus X Protein Is Stabilized by the Deubiquitinating Enzyme VCPIP1 in a Ubiquitin-Independent Manner by Recruiting the 26S Proteasome Subunit PSMC3.

ABSTRACTHepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, and the viral X protein (HBx) is an etiological factor in HCC development. HBx is a high-turnover protein, but knowledge of the role of deubiquitinating enzymes (DUBs) in maintaining HBx homeostasis is very limited. We used a 74-DUB library-based yeast two-hybrid assay and determined that a novel DUB, valosin-containing protein-interacting protein 1 (VCPIP1), interacted with HBx. VCPIP1 and its C-terminal amino acids 863 to 1221 upregulated the HBx protein expression, with or without HBV infection. Mechanistically, VCPIP1 stabilized HBx protein through a ubiquitin-independent pathway, which was validated by the HBx ubiquitination site mutant plasmid. Coimmunoprecipitation assays demonstrated the potency of VCPIP1 in recruiting 26S proteasome regulatory subunit 6A (PSMC3) and forming a ternary complex with HBx through mutual interaction. In vitro, purified His-tagged PSMC3 protein rescued HBx degradation induced by the 20S proteasome, and in vivo VCPIP1 synergized the mechanism. Functionally, HBx specifically binding to VCPIP1 significantly enhanced the transcriptional transactivation of HBx by activating NF-κB, AP-1, and SP-1 and inhibited hepatoma cell clonogenicity in Huh7 and HepG2 cells. Moreover, we further demonstrated that overexpression of VCPIP1 significantly affected the HBV covalently closed circular DNA (cccDNA) transcription in HBV-infected HepG2-NTCP cells. Altogether, our results indicate a novel mechanism by which VCPIP1 recruits PSMC3 to bind with HBx, stabilizing it in a ubiquitin-independent manner, which might be critical for developing DUB inhibitors in the future.IMPORTANCE HBx is a multifunctional viral oncoprotein that plays an essential role in the viral life cycle and hepatocarcinogenesis. HBx degradation occurs through the ubiquitin-proteasome system (UPS). However, whether novel compartments of the DUBs in the UPS also act in regulating HBx stability is not fully understood. Here, for the first time, we defined VCPIP1 as a novel DUB for preventing HBx degradation by the 20S proteasome in a ubiquitin-independent manner. PSMC3, encoding the 26S proteasome regulatory subunit, directly stabilized HBx through physical binding instead of a common approach in protein degradation, serving as the key downstream effector of VCPIP1 on HBx. Therefore, the ternary binding pattern between VCPIP1, HBx, and PSMC3 is initiated for the first time, which eventually promotes HBx stability and its functions. Our findings provide novel insights into host-virus cross talk by targeting DUBs in the UPS.

Knockdown of PSMC2 contributes to suppression of cholangiocarcinoma development by regulating CDK1.

Cholangiocarcinoma (CCA) has been well known as the second most common primary tumor of hepatobiliary system. PSMC2 (proteasome 26S subunit ATPase 2) is a key member of the 19S regulatory subunit of 26S proteasome, responsible for catalyzing the unfolding and translocation of substrates into the 20S proteasome, whose role in CCA is totally unknown. In this study, the results of immunohistochemistry analysis showed the upregulation of PSMC2 in CCA tissues compared with normal tissues, which was statistically analyzed to be associated with CCA tumor grade. Subsequently, the loss-of-function study suggested that knockdown of PSMC2 significantly suppressed cell proliferation, cell migration, promoted cell apoptosis and arrested cell cycle distribution in vitro. The decreased tumorigenicity of CCA cells with PSMC2 knockdown was confirmed in vivo by using mice xenograft model. In PSMC2 knockdown cells, pro-apoptotic protein Caspase3 was upregulated; anti-apoptotic proteins such as Bcl-2 and IGF-II were downregulated; among EMT markers, E-cadherin was upregulated while N-cadherin and Vimentin were downregulated, by which may PSMC2 regulates cell apoptosis and migration. Furthermore, through RNA-seq and verification by qPCR, western blotting and co-IP assays, CDK1 was identified as the potential downstream of PSMC2 mediated regulation of CCA. PSMC2 and CDK1 showed mutual regulation effects on expression level of each other. Knockdown of PSMC2 could aggregate the influence of CDK1 knockdown on cellular functions of CCA cells. In summary, our findings suggested that PSMC2 possesses oncogene-like functions in the development and progression of CCA through regulating CDK1, which may be used as an effective therapeutic target in CCA treatment.

Genetically incorporated crosslinkers reveal NleE attenuates host autophagy dependent on PSMD10.

Autophagy acts as a pivotal innate immune response against infection. Some virulence effectors subvert the host autophagic machinery to escape the surveillance of autophagy. The mechanism by which pathogens interact with host autophagy remains mostly unclear. However, traditional strategies often have difficulty identifying host proteins that interact with effectors due to the weak, dynamic, and transient nature of these interactions. Here, we found that Enteropathogenic Escherichia coli (EPEC) regulates autophagosome formation in host cells dependent on effector NleE. The 26S Proteasome Regulatory Subunit 10 (PSMD10) was identified as a direct interaction partner of NleE in living cells by employing genetically incorporated crosslinkers. Pairwise chemical crosslinking revealed that NleE interacts with the N-terminus of PSMD10. We demonstrated that PSMD10 homodimerization is necessary for its interaction with ATG7 and promotion of autophagy, but not necessary for PSMD10 interaction with ATG12. Therefore, NleE-mediated PSMD10 in monomeric state attenuates host autophagosome formation. Our study reveals the mechanism through which EPEC attenuates host autophagy activity.

Proteins of the Ciliated Protozoan Parasite Ichthyophthirius multifiliis Identified in Common Carp Skin Mucus.

The skin mucus is the fish primary defense barrier protecting from infections via the skin epidermis. In a previous study, we have investigated the proteome of common carp (Cyprinus carpio) skin mucus at two different time points (1 and 9 days) post-exposure to Ichthyophthirius multifiliis. Applying a nano-LC ESI MS/MS technique, we have earlier revealed that the abundance of 44 skin mucus proteins has been differentially regulated including proteins associated with host immune responses and wound healing. Herein, in skin mucus samples, we identified six proteins of I. multifiliis associated with the skin mucus in common carp. Alpha and beta tubulins were detected in addition to the elongation factor alpha, 26S proteasome regulatory subunit, 26S protease regulatory subunit 6B, and heat shock protein 90. The identified proteins are likely involved in motility, virulence, and general stress during parasite growth and development after parasite attachment and invasion. Two KEGG pathways, phagosome and proteasome, were identified among these parasite proteins, mirroring the proteolytic and phagocytic activities of this parasite during host invasion, growth, and development, which represent a plausible host invasion strategy of this parasite. The results obtained from this study can support revealing molecular aspects of the interplay between carp and I. multifiliis and may help us understand the I. multifiliis invasion strategy at the skin mucus barrier. The data may advance the development of novel drugs, vaccines, and diagnostics suitable for the management and prevention of ichthyophthiriosis in fish.

Silencing of PSMC2 inhibits development and metastasis of prostate cancer through regulating proliferation, apoptosis and migration

BackgroundProstate cancer is the most common malignant tumor of male genitourinary system, molecular mechanism of which is still not clear. PSMC2 (proteasome 26S subunit ATPase 2) is a key member of the 19S regulatory subunit of 26S proteasome, whose relationship with prostate cancer is rarely studied.MethodsHere, expression of PSMC2 in tumor tissues or cells of prostate cancer was detected by qPCR, western blotting and immunohistochemical analysis. The effects of PSMC2 knockdown on cell proliferation, colony formation, cell migration, cell cycle and apoptosis were assessed by Celigo cell counting assay, colony formation assay, wound-healing assay, Transwell assay and flow cytometry, respectively. The influence of PSMC2 knockdown on tumor growth in vivo was evaluated by mice xenograft models.ResultsThe results demonstrated that PSMC2 was upregulated in tumor tissues of prostate cancer and its high expression was significantly associated with advanced Gleason grade and higher Gleason score. Knockdown of PSMC2 could inhibited cell proliferation, colony formation and cell migration of prostate cancer cells, while promoting cell apoptosis and cell cycle arrest. The suppression of tumor growth in vivo by PSMC2 knockdown was also showed by using mice xenograft models. Moreover, the regulation of prostate cancer by PSMC2 may be mediated by Akt/Cyclin D1/CDK6 signaling pathway.ConclusionsTherefore, our studies suggested that PSMC2 may act as a tumor promotor in the development and progression of prostate cancer, and could be considered as a novel therapeutic target for prostate cancer treatment.

The 26S Proteasome Regulatory Subunit GmPSMD Promotes Resistance to Phytophthora sojae in Soybean.

Phytophthora root rot, caused by Phytophthora sojae is a destructive disease of soybean (Glycine max) worldwide. We previously confirmed that the bHLH transcription factor GmPIB1 (P. sojae-inducible bHLH transcription factor) reduces accumulation of reactive oxygen species (ROS) in cells by inhibiting expression of the peroxidase-related gene GmSPOD thus improving the resistance of hairy roots to P. sojae. To identify proteins interacting with GmPIB1 and assess their participation in the defense response to P. sojae, we obtained transgenic soybean hairy roots overexpressing GmPIB1 by Agrobacterium rhizogenes mediated transformation and examined GmPIB1 protein–protein interactions using immunoprecipitation combined with mass spectrometry. We identified 392 proteins likely interacting with GmPIB1 and selected 20 candidate genes, and only 26S proteasome regulatory subunit GmPSMD (Genbank accession no. XP_014631720) interacted with GmPIB1 in luciferase complementation and pull-down experiments and yeast two-hybrid assays. Overexpression of GmPSMD (GmPSMD-OE) in soybean hairy roots remarkably improved resistance to P. sojae and RNA interference of GmPSMD (GmPSMD -RNAi) increased susceptibility. In addition, accumulation of total ROS and hydrogen peroxide (H2O2) in GmPSMD-OE transgenic soybean hairy roots were remarkably lower than those of the control after P. sojae infection. Moreover, in GmPSMD-RNAi transgenic soybean hairy roots, H2O2 and the accumulation of total ROS exceeded those of the control. There was no obvious difference in superoxide anion (O2–) content between control and transgenic hairy roots. Antioxidant enzymes include peroxidase (POD), glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT) are responsible for ROS scavenging in soybean. The activities of these antioxidant enzymes were remarkably higher in GmPSMD-OE transgenic soybean hairy roots than those in control, but were reduced in GmPSMD-RNAi transgenic soybean hairy roots. Moreover, the activity of 26S proteasome in GmPSMD-OE and GmPIB1-OE transgenic soybean hairy roots was significantly higher than that in control and was significantly lower in PSMD-RNAi soybean hairy roots after P. sojae infection. These data suggest that GmPSMD might reduce the production of ROS by improving the activity of antioxidant enzymes such as POD, SOD, GPX, CAT, and GmPSMD plays a significant role in the response of soybean to P. sojae. Our study reveals a valuable mechanism for regulation of the pathogen response by the 26S proteasome in soybean.

MicroRNAs expression profiles in Schistosoma japonicum of different sex 14 and 28 days post-infection.

Different miRNAs are involved in the life cycles of Schistosoma japonicum. The aim of this study was to examine the expression profile of miRNAs in individual S. japonicum of different sex before and after pairing (18 and 24 dpi). The majority of differential expressed miRNAs were highly abundant at 14 dpi, except for sja-miR-125b and sja-miR-3505, in both male and female. Moreover, it was estimated that sja-miR-125b and sja-miR-3505 might be related to laying eggs. sja-miR-2a-5p and sja-miR-3484-5p were expressed at 14 dpi in males and were significantly clustered in DNA topoisomerase III, Rap guanine nucleotide exchange factor 1 and L-serine/L-threonine ammonia-lyase. Target genes of sja-miR-2d-5p, sja-miR-31- 5p and sja-miR-125a, which were expressed at 14 dpi in males but particularly females, were clustered in kelch-like protein 12, fructose-bisphosphate aldolase, class I, and heat shock protein 90 kDa beta. Predicted target genes of sja-miR-3483-3p (expressed at 28 dpi in females but not in males) were clustered in 26S proteasome regulatory subunit N1, ATPdependent RNA helicase DDX17. Predicted target genes of sja-miR-219-5p, which were differentially expressed at 28 dpi in females but particularly males, were clustered in DNA excision repair protein ERCC-6, protein phosphatase 1D, and ATPase family AAA domaincontaining protein 3A/B. Moreover, at 28 dpi, eight miRNAs were significantly up-regulated in females compared to males. The predicted target genes of these miRNAs were significantly clustered in heat shock protein 90 kDa beta, 26S proteasome regulatory subunit N1, and protein arginine N-methyltransferase 1. To sum up, differentially expressed miRNAs may have an essential role and provide necessary information on clarifying this trematode's growth, development, maturation, and infection ability to mammalian hosts in its complex life cycle, and may be helpful for developing new drug targets and vaccine candidates for schistosomiasis.

Identification of valid reference genes for quantitative RT-PCR in Caragana microphylla under salt and drought stresses.

Caragana microphylla is a leguminosae plant and grows mainly in semi-arid areas of northwest China and Mongolia. However, the lack of studies on C. microphylla reference genes limits the accurate understanding of the molecular biology mechanisms in this crop under abiotic stresses. In this study, we selected nine candidate genes from salt-treated C. microphylla transcriptome data and evaluated their stability by using geNorm, NormFinder, BestKeeper, and RefFinder in salt and drought conditions. In addition, the relative expressions of Delta 1-pyrroline-5-carboxylate synthase 2 (P5CS2) and Catalase 2 (CAT2) were examined to confirm the stability of the candidate reference genes. As a results, glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2) and 26S proteasome regulatory subunit (RPN5) were the most stable in both salt and drought treatments. The relative expression of P5CS2 and CAT2 also showed more stable levels in normalization by GAPC2 and RPN5 than the most unstable gene, Ubiquitin 4 (UBQ4). Therefore, it is believed that these candidate reference genes selected and validated in our study could be used to study the molecular biological study of response to salt and drought stress in C. microphylla.

Identification of salt-responsive genes using transcriptome analysis in Dunaliella viridis

Dunaliella viridis is one of the most salt-tolerant eukaryotes and a model organism to study plant adaptation to salinity. To explore the molecular mechanisms mediating salt stress response in D. viridis, transcriptome analysis was performed by high-throughput sequencing. A total of 2242 differentially expressed genes (DEGs) were obtained, including 1563 upregulated and 679 downregulated, in plants after 6 h of salt stress (S_6h) compared with the control (Con); 394 DEGs, including 159 upregulated and 235 downregulated, in plants after 1 h of salt stress (S_1h) compared with the control; and 1379 DEGs, including 471 upregulated and 908 downregulated, in S_6h compared with S_1h. GO and KEGG analyses revealed that these DEGs were enriched in biological processes such as metabolism, genetic information transmission, signal transduction, and material transport. Further gene expression analysis and functional annotation showed that genes involved in photosynthesis were induced under salt stress. Ornithine decarboxylase and S-adenosylmethionine decarboxylase involved in polyamine synthesis and metabolism were significantly upregulated. Synthesis of molecules involved in cell cycle regulation such as G1-S phase transition protein, cyclin, and cyclin-dependent kinase was induced under salt stress. Ubiquitin, ubiquitin conjugatase, and 26S proteasome regulatory subunit were significantly different in S_6h vs._S_1h (P < 0.01). We also found that calmodulin (CaM), calcium-calmodulin dependent protein kinase (CaMK), calcium-calmodulin dependent protein kinase kinase (CaMKK), and cGMP-dependent protein kinase genes were significantly upregulated. These results indicate that calcium messenger pathway and cGMP signaling pathway play important roles in salt stress response in D. viridis. Quantitative real-time PCR (qRT-PCR) was used to validate the high-throughput sequencing, and the expression patterns of ten randomly selected genes under salt stress at different time points were further analyzed.

Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes

The intracellular accumulation of aggregated misfolded proteins is a cytopathological hallmark of neurodegenerative diseases. However, the functional relationship between protein misfolding or aggregation and the cellular proteostasis network that monitors and maintains proteome health is poorly understood. Previous studies have associated translational suppression and transcriptional remodeling with the appearance of protein aggregates, but whether these responses are induced by aggregates or their misfolded monomeric or oligomeric precursors remains unclear. Because aggregation in cells is rapid, nonlinear, and asynchronous, it has not been possible to deconvolve these kinetically linked processes to determine the earliest cellular responses to misfolded proteins. Upon removal of the synthetic, biologically inert ligand shield-1 (S1), AgDD, an engineered variant FK506-binding protein (FKBP1A), rapidly (t½ ∼5 min) unfolds and self-associates, forming detergent-insoluble, microscopic cytoplasmic aggregates. Using global diglycine-capture (K-GG) proteomics, we found here that this solubility transition is associated with immediate increases in ubiquitylation of AgDD itself, along with that of endogenous proteins that are components of the ribosome and the 26S proteasome. We also found that the earliest cellular responses to acute S1 removal include recruitment of ubiquitin protein ligase E3C (UBE3C) to the 26S proteasome and ubiquitylation of two key proteasomal ubiquitin receptors, 26S proteasome regulatory subunit RPN10 (RPN10) and Rpn13 homolog (RPN13 or ADRM1). We conclude that these proteasomal responses are due to AgDD protein misfolding and not to the presence of detergent-insoluble aggregates.

Comparative proteomic analysis of Ulva prolifera response to high temperature stress

BackgroundUlva prolifera belongs to green macroalgae and is the dominant species of green tide. It is distributed worldwide and is therefore subject to high-temperature stress during the growth process. However, the adaptation mechanisms of the response of U. prolifera to high temperatures have not been clearly investigated yet.MethodsIn this study, isobaric tags for relative and absolute quantitation (iTRAQ) labelling was applied in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) to conduct comparative proteomic analysis of the response of U. prolifera to high-temperature stress and to elucidate the involvement of this response in adaptation mechanisms. Differentially expressed proteins (DEPs) of U. prolifera under high temperature (denote UpHT) compared with the control (UpC) were identified. Bioinformatic analyses including GO analysis, pathway analysis, and pathway enrichment analysis was performed to analyse the key metabolic pathways that underlie the thermal tolerance mechanism through protein networks. Quantitative real-time PCR and western blot were performed to validate selected proteins.ResultsIn the present study, 1223 DEPs were identified under high temperature compared with the control, which included 790 up-regulated and 433 down-regulated proteins. The high-temperature stimulus mainly induced the expression of glutathione S-transferase, heat shock protein, ascorbate peroxidase, manganese superoxide dismutase, ubiquitin-related protein, lhcSR, rubisco activase, serine/threonine protein kinase 2, adenylate kinase, Ca2+-dependent protein kinase (CDPK), disease resistance protein EDS1, metacaspase type II, NDPK2a, 26S proteasome regulatory subunit, ubiquinone oxidoreductase, ATP synthase subunit, SnRK2s, and cytochrome P450. The down-regulated proteins were photosynthesis-related proteins, glutathione reductase, catalase-peroxidase, thioredoxin, thioredoxin peroxidase, PP2C, and carbon fixation-related proteins. Furthermore, biological index analysis indicated that protein content and SOD activity decreased; the value of Fv/Fm dropped to the lowest point after culture for 96 h. However, APX activity and MDA content increased under high temperature.ConclusionThe present study implied an increase in proteins that were associated with the stress response, oxidative phosphorylation, the cytokinin signal transduction pathway, the abscisic acid signal transduction pathway, and the glutathione metabolism pathway. Proteins that were associated with photosynthesis, carbon fixation in photosynthesis organisms, and the photosynthesis antenna protein pathway were decreased. These pathways played a pivotal role in high temperature regulation. These novel proteins provide a good starting point for further research into their functions using genetic or other approaches. These findings significantly improve the understanding of the molecular mechanisms involved in the tolerance of algae to high-temperature stress.

The Drosophila TGF-beta/Activin-like ligands Dawdle and Myoglianin appear to modulate adult lifespan through regulation of 26S proteasome function in adult muscle.

ABSTRACTThe Drosophila Activin signaling pathway employs at least three separate ligands – Activin-β (Actβ), Dawdle (Daw), and Myoglianin (Myo) – to regulate several general aspects of fruit fly larval development, including cell proliferation, neuronal remodeling, and metabolism. Here we provide experimental evidence indicating that both Daw and Myo are anti-ageing factors in adult fruit flies. Knockdown of Myo or Daw in adult fruit flies reduced mean lifespan, while overexpression of either ligand in adult muscle tissues but not in adipose tissues enhanced mean lifespan. An examination of ubiquitinated protein aggregates in adult muscles revealed a strong inverse correlation between Myo- or Daw-initiated Activin signaling and the amount of ubiquitinated protein aggregates. We show that this correlation has important functional implications by demonstrating that the lifespan extension effect caused by overexpression of wild-type Daw or Myo in adult muscle tissues can be completely abrogated by knockdown of a 26S proteasome regulatory subunit Rpn1 in adult fly muscle, and that the prolonged lifespan caused by overexpression of Daw or Myo in adult muscle could be due to enhanced protein levels of the key subunits of 26S proteasome. Overall, our data suggest that Activin signaling initiated by Myo and Daw in adult Drosophila muscles influences lifespan, in part, by modulation of protein homeostasis through either direct or indirect regulation of the 26S proteasome levels. Since Myo is closely related to the vertebrate muscle mass regulator Myostatin (GDF8) and the Myostatin paralog GDF11, our observations may offer a new experimental model for probing the roles of GDF11/8 in ageing regulation in vertebrates.This article has an associated First Person interview with the first author of the paper.

A Novel QTL for Powdery Mildew Resistance in Nordic Spring Barley (Hordeum vulgare L. ssp. vulgare) Revealed by Genome-Wide Association Study.

The powdery mildew fungus, Blumeria graminis f. sp. hordei is a worldwide threat to barley (Hordeum vulgare L. ssp. vulgare) production. One way to control the disease is by the development and deployment of resistant cultivars. A genome-wide association study was performed in a Nordic spring barley panel consisting of 169 genotypes, to identify marker-trait associations significant for powdery mildew. Powdery mildew was scored during three years (2012–2014) in four different locations within the Nordic region. There were strong correlations between data from all locations and years. In total four QTLs were identified, one located on chromosome 4H in the same region as the previously identified mlo locus and three on chromosome 6H. Out of these three QTLs identified on chromosome 6H, two are in the same region as previously reported QTLs for powdery mildew resistance, whereas one QTL appears to be novel. The top NCBI BLASTn hit of the SNP markers within the novel QTL predicted the responsible gene to be the 26S proteasome regulatory subunit, RPN1, which is required for innate immunity and powdery mildew-induced cell death in Arabidopsis. The results from this study have revealed SNP marker candidates that can be exploited for use in marker-assisted selection and stacking of genes for powdery mildew resistance in barley.

Poldip2 is an Oxygen-sensitive Mitochondrial Protein that Controls Oxidative/glycolytic Metabolism Balance and Proteasome Activity

The polymerase delta interacting protein 2 (Poldip2) is a nuclear-encoded mitochondrial protein of unknown function. We recently reported that Poldip2 is repressed under hypoxia and that its deficiency results in repressed mitochondrial function and increased glycolytic activity. However, the mechanisms responsible for this metabolic reprograming and its consequences are unknown. In this study, we show that Poldip2 controls the lipoylation and consequently the activity of the pyruvate and α-ketoglutarate dehydrogenase complexes by a mechanism that involves Clp-protease complex-mediated degradation of Acyl-CoA Synthetase Medium-Chain Family Member 1 (ACSM1), a lipoate-activating enzyme required for the utilization of lipoic acid derived from the salvage pathway. With regards to the increased glycolytic activity, we found that Poldip2 deficiency inhibits PHDs inducing HIF-1α-dependent upregulation of Hexokinase 2. As a result, we observed an increase in the flux through the Hexosamine biosynthetic pathway (HBP), a glucose metabolic pathway that produces uridine diphosphate N-acetylglucosamine, the sugar donor for the O-GlcNAc transferase (OGT). The OGT-catalyzed transfer of O-GlcNAc is a post-translational modification implicated in cell signaling. Interestingly we found that Poldip2 deficiency induces the inhibition of the ubiquitin proteasome system (UPS) by OGT-dependent O-GlcNAcylation of the 19S regulatory unit of the 26S proteasome regulatory subunit 4 (PRS4). Additionally, we demonstrated that Poldip2-mediated inhibition of the UPS is required for the hypoxia-induced expression of connective tissue growth factor (CTGF), a cytokine that participates in multiple pathologies including fibrotic diseases and cancer. In all, we demonstrate that Poldip2 is an oxygen sensitive protein that regulates the activity of key enzymes of the Krebs cycle by coupling proteasome activity to mitochondrial function and fibrotic responses under hypoxia.

Increased Ubqln2 expression causes neuron death in transgenic rats.

Pathogenic mutation of ubiquilin 2 (UBQLN2) causes neurodegeneration in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. How UBQLN2 mutations cause the diseases is not clear. While over-expression of UBQLN2 with pathogenic mutation causes neuron death in rodent models, deletion of the Ubqln2 in rats has no effect on neuronal function. Previous findings in animal models suggest that UBQLN2 mutations cause the diseases mainly through a gain rather than a loss of functions. To examine whether the toxic gain in UBQLN2 mutation is related to the enhancement of UBQLN2 functions, we created new transgenic rats over-expressing wild-type human UBQLN2. Considering that human UBQLN2 may not function properly in the rat genome, we also created transgenic rats over-expressing rat's own Ubqln2. When over-expressed in rats, both human and rat wild-type Ubqln2 caused neuronal death and spatial learning deficits, the pathologies that were indistinguishable from those observed in mutant UBQLN2 transgenic rats. Over-expressed wild-type UBQLN2 formed protein inclusions attracting the autophagy substrate sequestosome-1 and the proteasome component 26S proteasome regulatory subunit 7. These findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that the enhancement of UBQLN2 functions is involved in UBQLN2 pathogenesis. Pathogenic mutation in ubiquilin 2 (UBQLN2) causes neurodegeneration in ALS and FTLD. Studies in rodent models suggest a gain of toxic function in mutant UBQLN2. We created new transgenic rats as a relevant model and examined whether enhancing wild-type UBQLN2 expression is implicated in the pathogenesis of mutant UBQLN2. We observed that over-expression of human or rat wild-type Ubqln2 caused protein aggregation and neuronal death in transgenic rats. Our findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that uncontrolled enhancement of UBQLN2 function is involved in UBQLN2 pathogenesis. Read the Editorial Highlight for this article on page 159.

Proteasome activation is a mechanism for pyrazolone small molecules displaying therapeutic potential in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action. Probes were confirmed to be neuroprotective in PC12-SOD1G93A cells. PC12-SOD1G93A cell lysates were used for protein pull-down, affinity purification, and subsequent proteomic analysis using LC-MS/MS. Proteomics identified the 26S proteasome regulatory subunit 4 (PSMC1), 26S proteasome regulatory subunit 6B (PSMC4), and T-complex protein 1 (TCP-1) as putative protein targets. Coincubation with appropriate competitors confirmed the authenticity of the proteomics results. Activation of the proteasome by pyrazolones was demonstrated in the absence of exogenous proteasome inhibitor and by restoration of cellular protein degradation of a fluorogenic proteasome substrate in PC12-SOD1G93A cells. Importantly, supplementary studies indicated that these molecules do not induce a heat shock response. We propose that pyrazolones represent a rare class of molecules that enhance proteasomal activation in the absence of a heat shock response and may have therapeutic potential in ALS.