Ubiquitin-mediated Protein Degradation Research Articles

Individual components of paired typical NLR immune receptors are regulated by distinct E3 ligases.

In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins serve as intracellular immune receptors. Defence signalling by NLRs often requires the formation of NLR heteropairs. Our knowledge of the molecular mechanism regulating this process is limited. In a reverse genetic screen to identify the partner of the Arabidopsis typical NLR, SUPRESSOR OF NPR1, CONSTITUTIVE 1 (SNC1), we discovered three NLRs that are redundantly required for SNC1-mediated defence, which were named SIDEKICK SNC1 1 (SIKIC1), SIKIC2 and SIKIC3. Immunoprecipitation-mass spectrometry analyses revealed that SIKIC2 physically associates with SNC1. We also uncovered that the protein level of SIKIC2 is under the control of two previously uncharacterized redundant E3 ubiquitin ligases MUSE1 and MUSE2. As SNC1 accumulation has previously been shown to be regulated by the E3 ubiquitin ligase SCFCPR1, this report provides evidence that the homeostasis of individual components of partnered typical NLRs is subjected to differential regulation via ubiquitin-mediated protein degradation.

Abstract 4455: Fatty acid synthase-induced S6Kinase facilitates USP11-eIF4B complex formation for sustained oncogenic translation in DLBCL

Abstract Fatty acid synthase (FASN) and protein translational machinery are two emergent therapeutic targets for cancer treatment, but their mutual dependency on each other for tumor propagation especially in diffuse B-cell large lymphoma (DLBCL) is still unknown. Therefore, we investigated the functional implication of enhanced FASN activity on protein translational machinery employing a panel of 7 DLCBL cell lines and 40 clinical samples. Inhibition of FASN activity by a specific inhibitor, C75 or shRNA mediated knockdown, suppressed de novo protein synthesis in SUDHL2, TMD8, Pfeiffer and HLY-1 (ABC-origin) cells, whereas SUDHL4, SUDHL6 and Toledo (GC-origin) cells showed minimal effect after C75 treatment or FASN knockdown. Assessing the core translational machinery, the eIF4B (eukaryotic initiation factor 4B) levels were depleted in a dose dependent manner of C75 treatment or shRNA mediated knock down of FASN in ABC-DLBCLs. Mechanistically, eIF4B undergoes ubiquitin mediated protein degradation upon FASN depletion. Depleting the expression of eIF4B significantly reduced the proliferative capacity of both FASN sensitive as well resistant DLBCL cells. Screening the interacting protein partners from available databases, we observed that USP11 interacts with and deubiquitinates eIF4B increasing its overall stability. Consistently, altering the expression of USP11 or its activity by either chemical inhibition or mutation significantly alters nascent peptide synthesis. Of functional significance, either down regulating USP11 or inhibition via small molecules significantly reduced the cell proliferation with increased apoptosis of both, FASN activity sensitive and resistant DLBCL cells. USP11 expression was also elevated in DLBCL tissues as evidenced by significantly higher staining. Significantly there is a strong correlation between USP11, FASN as well as eIF4B expression in DLBCL. Elucidating the basis for FASN not regulating GC DLBCL, we observed that sustained PI3K signalling in GC-DLBCL plays a key role in FASN dependent eIF4B stability. Detailed molecular analysis established that S6Kinase phosphorylates USP11 at Ser453, which regulates its interaction with, and stability of eIF4B thereby promoting proto-oncogene expression. These results together demonstrate that FASN activated S6Kinase induced eIF4B/USP11 axis is required for cell proliferation and survival by regulating the translation of proliferative and pro-survival mRNAs. Our findings support targeting the eIF4B/USP11 axis and provide a novel therapeutic option for patients with relapsed/refractory DLBCL. Citation Format: Bandish Kapadia, Nahid Nanaji, Kavita Bhalla, Binny Bhandary, Rena Lapidus, Afshin Beheshti, Andrew Evens, Ronald Gartenhaus. Fatty acid synthase-induced S6Kinase facilitates USP11-eIF4B complex formation for sustained oncogenic translation in DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4455.

Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity

The development of nephrotoxicity largely limits the clinical use of chemotherapy. MiRNAs are able to target various genes and involved in the regulation of diverse cellular processes, including cell apoptosis and death. Our study showed that miR-181a expression was significantly increased after 5-fluorouracil (5-FU) treatment in renal mesangial cells and kidney tissue, which was associated with decreased baculoviral inhibition of apoptosis protein repeat-containing 6 (BIRC6) expression and increased apoptotic rate. Enforced miR-181a expression enhanced 5-FU-induced p53-dependent mitochondrial apoptosis, including declined Bcl-2/Bax ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase-9 and caspase-3 activation. However, inhibition of miR-181a was associated with reduced p53-mediated mitochondrial apoptosis induced by 5-FU. Moreover, miR-181a increased BIRC6 downstream gene p53 protein expression and transcriptional activity by reducing ubiquitin-mediated protein degradation. We found that miR-181a directly targeted 3′-UTR of BIRC6 mRNA and negatively regulated BIRC6 expression. In vivo study, knockdown of miR-181a with adeno-associated virus harboring miR-181a-tough decoy attenuated 5-FU-induced renal cell apoptosis, inflammation and kidney injury. In conclusion, these results demonstrate that miR-181a increases p53 protein expression and transcriptional activity by targeting BIRC6 and promotes 5-FU-induced apoptosis in mesangial cells. Inhibition of miR-181a ameliorates 5-FU-induced nephrotoxicity, suggesting that miR-181a may be a novel therapeutic target for nephrotoxicity treatment during chemotherapy.

Transcriptional and epigenetic modulation of autophagy promotes EBV oncoprotein EBNA3C induced B-cell survival

Epstein-Barr virus (EBV) oncoprotein EBNA3C is indispensable for primary B-cell transformation and maintenance of lymphoblastoid cells outgrowth. EBNA3C usurps two putative cellular pathways—cell-cycle and apoptosis, essentially through modulating ubiquitin-mediated protein-degradation or gene transcription. In cancer cells, these two pathways are interconnected with autophagy,—a survival-promoting catabolic network in which cytoplasmic material including mis/un-folded protein aggregates and damaged organelles along with intracellular pathogens are degraded and recycled in lysosomal compartments. Studies have shown that tumor viruses including EBV can manipulate autophagy as a survival strategy. Here, we demonstrate that EBNA3C elevates autophagy, which serves as a prerequisite for apoptotic inhibition and maintenance of cell growth. Using PCR based micro-array we show that EBNA3C globally accelerates autophagy gene transcription under growth limiting conditions. Reanalyzing the ENCODE ChIP-sequencing data (GSE52632 and GSE26386) followed by ChIP-PCR demonstrate that EBNA3C recruits several histone activation epigenetic marks (H3K4me1, H3K4me3, H3K9ac, and H3K27ac) for transcriptional activation of autophagy genes, notably ATG3, ATG5, and ATG7 responsible for autophagosome formation. Moreover, under growth limiting conditions EBNA3C further stimulates the autophagic response through upregulation of a number of tumor suppressor genes, notably cyclin-dependent kinase inhibitors—CDKN1B (p27Kip1) and CDKN2A (p16INK4a) and autophagy mediated cell-death modulators—DRAM1 and DAPK1. Together our data highlight a new role of an essential EBV oncoprotein in regulating autophagy cascade as a survival mechanism and offer novel-targets for potential therapeutic expansion against EBV induced B-cell lymphomas.

A Deubiquitinating Enzyme Ubp14 Is Required for Development, Stress Response, Nutrient Utilization, and Pathogenesis of Magnaporthe oryzae.

Ubiquitination is an essential protein modification in eukaryotic cells, which is reversible. Deubiquitinating enzymes (DUBs) catalyze deubiquitination process to reverse ubiquitination, maintain ubiquitin homeostasis or promote protein degradation by recycling ubiquitins. In order to investigate effects of deubiquitination process in plant pathogenic fungus Magnaporthe oryzae, we generated deletion mutants of MoUBP14. Ortholog of MoUbp14 was reported to play general roles in ubiquitin-mediated protein degradation in Saccharomyces cerevisiae. The ΔMoubp14 mutant lost its pathogenicity and was severely reduced in mycelial growth, sporulation, carbon source utilization, and increased in sensitivity to distinct stresses. The mutant was blocked in penetration, which could due to defect in turgor generation. It is also blocked in invasive growth, which could due to reduction in stress tolerance and nutrient utilization. Deletion of UBP14 also led to accumulation of free polyubiquitin chains. Pulldown assay identified some proteins related to carbohydrate metabolism and stress response may putatively interact with MoUbp14, including two key rate-limiting enzymes of gluconeogenesis, MoFbp1 and MoPck1. These two proteins were degraded when the glucose was supplied to M. oryzae grown in low glucose media for a short period of time (∼12 h), and this process required MoUbp14. In summary, pleiotropic phenotypes of the deletion mutants indicated that MoUbp14 is required for different developments and pathogenicity of M. oryzae.

Structural insights into suppressor of cytokine signaling 1 protein- identification of new leads for type 2 diabetes mellitus.

The study considers the Suppressor of cytokine signaling 1 (SOCS1) protein as a novel Type 2 diabetes mellitus (T2DM) drug target. T2DM in human beings is also triggered by the over expression of SOCS proteins. The SOCS1 acts as a ubiquitin ligase (E3), degrades Insulin Receptor Substrate 1 and 2 (IRS1 and IRS2) proteins, and causes insulin resistance. Therefore, the structure of the SOCS1 protein was evaluated using homology-modeling and molecular dynamics methods and validated using standard computational protocols. The Protein-Protein docking study of SOCS1 with its natural substrates, IRS1 and IRS2, and subsequent solvent accessible surface area analysis gave insight into the binding region of the SOCS1 protein. The in silico active site prediction tools highlight the residues Val155 to Ile211 in SOCS1 being implicated in the ubiquitin mediated protein degradation of the proteins IRS1 and IRS2. Virtual screening in the active site region, using large structural databases, results in selective lead structures with 3-Pyridinol, Xanthine, and Alanine moieties as Pharmacophore. The virtual screening study shows that the residues Glu149, Gly187, Arg188, Leu191, and Ser205 of the SOCS1 are important for binding. The docking study with current anti-diabetic therapeutics shows that the drugs Glibenclamide and Glyclopyramide have a partial affinity towards SOCS1. The predicted ADMET and IC50 properties for the identified ligands are within the acceptable range with drug-like properties. The structural data of SOCS1, its active site, and the identified lead structures are expedient in the development of new T2DM therapeutics.

ITRAQ-based quantitative proteomic analysis reveals the lateral meristem developmental mechanism for branched spike development in tetraploid wheat (Triticum turgidum L.)

BackgroundSpike architecture mutants in tetraploid wheat (Triticum turgidum L., 2n = 28, AABB) have a distinct morphology, with parts of the rachis node producing lateral meristems that develop into ramified spikelete (RSs) or four-rowed spikelete (FRSs). The genetic basis of RSs and FRSs has been analyzed, but little is known about the underlying developmental mechanisms of the lateral meristem. We used isobaric tags for relative and absolute quantitation (iTRAQ) to perform a quantitative proteomic analysis of immature spikes harvested from tetraploid near-isogenic lines of wheat with normal spikelete (NSs), FRSs, and RSs and investigated the molecular mechanisms of lateral meristem differentiation and development. This work provides valuable insight into the underlying functions of the lateral meristem and how it can produce differences in the branching of tetraploid wheat spikes.ResultsUsing an iTRAQ-based shotgun quantitation approach, 104 differential abundance proteins (DAPs) with < 1% false discovery rate (FDR) and a 1.5-fold change (> 1.50 or < 0.67) were identified by comparing FRS with NS and RS with NS genotypes. To determine the functions of the proteins, 38 co-expressed DAPs from the two groups were annotated using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analytical tools. We discovered that proteins involved in “post-embryonic development” and “metabolic pathways” such as carbohydrate and nitrogen metabolism could be used to construct a developmentally associated network. Additionally, 6 out of 38 DAPs in the network were analyzed using quantitative real-time polymerase chain reaction, and the correlation coefficient between proteomics and qRT-PCR was 0.7005. These key genes and proteins were closely scrutinized and discussed.ConclusionsHere, we predicted that DAPs involved in “post-embryonic development” and “metabolic pathways” may be responsible for the spikelete architecture changes in FRS and RS. Furthermore, we discussed the potential function of several vital DAPs from GO and KEGG analyses that were closely related to histone modification, ubiquitin-mediated protein degradation, transcription factors, carbohydrate and nitrogen metabolism and heat shock proteins (HSPs). This work provides valuable insight into the underlying functions of the lateral meristem in the branching of tetraploid wheat spikes.

Cdc48a AAA‐ATPase and its association with ubiquitin‐like SAMP1 and DNA repair in Archaea

Cdc48/p97/VCP is an AAA ATPase, conserved in eukaryotes and archaea, which is demonstrated to convert chemical energy from the hydrolysis of ATP into the mechanical energy, for either unfolding protein substrates or dislodging protein substrates from complexes. The role of Cdc48 in ubiquitin mediated protein degradation has been well reported; however, its role in DNA repair is less understood so far. Evidence in eukaryotes which implicates Cdc48 in DNA repair is based on the association of Cdc48 with partners and substrate proteins, which are known to be involved in DNA damage response. In our study, we found Cdc48A of Haloferax volcanii co‐purifies with RecJ‐domain (RecJ3 and RecJ4) and RNase J1 homologs in an apparent thiol sensitive complex that binds the oxidative stress‐responsive, ubiquitin‐like SAMP1 in the presence of ATP. RecJ domain proteins are DNases reported to be involved in DNA repair pathways in bacteria. Further analysis revealed an Hfx. volcanii Δcdc48A mutant showed significant difference in survival when compared to its wildtype, on subjecting them to DNA damaging agents like phleomycin and UV. An increase (up to 3.4‐fold; p &lt; 0.05) in transcript levels of cdc48a, recj3 and rnj1 was observed after treatment with the DNA damaging agent phleomycin, suggesting the expression of these genes is correlated with DNA repair. Current focus of this study is to decipher the role of Cdc48A in DNA repair by identifying its partner and substrate proteins. In addition, we are determining the substrate modifications required for signaling the Cdc48A mediated repair pathway using classic biochemistry techniques and genetic manipulations.Support or Funding InformationThe work was supported by funds awarded to Dr. Julie A. Maupin‐Furlow through the Bilateral NSF/BIO‐BBSRC program (NSF 1642283), U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DOE DE‐FG02‐05ER15650) and the National Institutes of Health (NIH R01 GM57498).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proteomic analysis of melatonin-mediated osmotic tolerance by improving energy metabolism and autophagy in wheat (Triticum aestivum L.).

Melatonin-mediated osmotic tolerance was attributed to increased antioxidant capacity, energy metabolism, osmoregulation and autophagy in wheat (Triticum aestivum L.). Melatonin is known to play multiple roles in plant abiotic stress tolerance. However, its role in wheat has been rarely investigated. In this study, 25% polyethylene glycol 6000 (PEG 6000) was used to simulate osmotic stress, and wheat seeds and seedlings were treated with different concentrations of melatonin under PEG stress. Isobaric tag for relative and absolute quantification (iTRAQ)-based proteomic techniques were used to identify the differentially accumulated proteins from melatonin-treated and non-treated seedlings. Seeding priming with melatonin significantly increased the germination rate, coleoptile length, and primary root number of wheat under PEG stress, as well as the fresh weight, dry weight, and water content of wheat seedlings. Under PEG stress, melatonin significantly improved reactive oxygen species homeostasis, as revealed by lower H2O2 and O 2· content; and the expression of antioxidant enzymes at the transcription and translation levels was increased. Melatonin maintained seedling growth by improving photosynthetic rates and instantaneous and intrinsic water use efficiencies, as well as carbon fixation and starch synthesis at the protein level. Melatonin treatment significantly affected the expression of glycolytic proteins, including fructose-1,6-bisphosphate aldolase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and enolase, and remarkably increased the expression of the nicotinamide adenine dinucleotide transporter and nicotinamide adenine dinucleotide binding protein, thereby indirectly modulating electron transport in the respiratory chain. This indicated that melatonin improved energy production in PEG-stressed seedlings. Further, melatonin played a regulatory role in autophagy, protease expression, and ubiquitin-mediated protein degradation by significantly upregulating rab-related protein, fused signal recognition particle receptor, aspartyl protease, serine protease, ubiquitin-fold modifier 1, and ubiquitin at the mRNA or protein level. These findings suggested that melatonin might activate a metabolic cascade related to autophagy under PEG stress in wheat seedlings.

Ubiquitin-related genes are differentially expressed in isogenic lines contrasting for pericarp cell size and grain weight in hexaploid wheat

BackgroundThere is an urgent need to increase global crop production. Identifying and combining specific genes controlling distinct biological processes holds the potential to enhance crop yields. Transcriptomics is a powerful tool to gain insights into the complex gene regulatory networks that underlie such traits, but relies on the availability of a high-quality reference sequence and accurate gene models. Previously, we identified a grain weight QTL on wheat chromosome 5A (5A QTL) which acts during early grain development to increase grain length through cell expansion in the pericarp. In this study, we performed RNA-sequencing on near isogenic lines (NILs) segregating for the 5A QTL and used the latest gene models to identify differentially regulated genes and pathways that potentially influence pericarp cell size and grain weight in wheat.ResultsWe sampled grains at 4 and 8 days post anthesis and found that genes associated with metabolism, biosynthesis, proteolysis and the defence response are upregulated during this stage of grain development in both NILs. We identified a specific set of 112 transcripts differentially expressed (DE) between 5A NILs at either time point, including eight potential candidates for the causal 5A gene and its downstream targets. The 112 DE transcripts had functional annotations including non-coding RNA, transposon-associated, cell-cycle control, ubiquitin-related, heat-shock, transcription and histone-related. Many of the genes identified belong to families that have been previously associated with seed/grain development in other species. Notably, we identified DE transcripts at almost all steps of the pathway associated with ubiquitin-mediated protein degradation. In the promoters of a subset of DE transcripts we identified enrichment of binding sites associated with C2H2, MYB/SANT, YABBY, AT-HOOK and Trihelix transcription factor families.ConclusionsIn this study, we identified DE transcripts with a diverse range of predicted biological functions, reflecting the complex nature of the pathways that control early grain development. Few of these are the direct orthologues of grain size genes in other species and none have been previously characterised in wheat. Further functional characterisation of these candidates and how they interact will provide novel insights into the control of grain size in cereals.

Oryza sativa salt-induced RING E3 ligase 2 (OsSIRP2) acts as a positive regulator of transketolase in plant response to salinity and osmotic stress.

A rice gene (OsSIRP2) encoding the RING Ub E3 ligase was highly induced under salinity stress and physically interacted with a transketolase (OsTKL1). Overexpression of OsSIRP2 conferred salinity and osmotic stress tolerance in plants. The RING E3 ligases play a vital role in post transitional modification through ubiquitination-mediated protein degradation that mediate plants responses during abiotic stresses and signal transduction. In this study, we report an Oryza sativa salt induced Really Interesting New Gene (RING) finger protein 2 gene (OsSIRP2) and elucidate its role under salinity and osmotic stress. The transcript levels of OsSIRP2 in rice leaves were induced in response to different abiotic stresses, such as salt, drought, heat, and abscisic acid (ABA) exposure. In vitro ubiquitination revealed that the OsSIRP2 protein formed poly-ubiquitin products, whereas a single amino acid substitution in OsSIRP2 (OsSIRP2C149A) in the RING domain did not form ubiquitinated substrates, supporting the hypothesis that E3 ligase activity requires the functional RING domain. Using the yeast two-hybrid (Y2H) assay, O. sativa transketolase 1 (OsTKL1) was identified as an interacting partner. OsSIRP2 was localized in the nucleus, whereas its interacting partner (OsTKL1) was localized in the cytosol and plastids in the rice protoplasts. Fluorescence signals between OsSIRP2 and OsTKL1 were observed in the cytosol. The pull-down assay confirmed the physical interaction between OsSIRP2 and OsTKL1. In vitro ubiquitination assay and in vitro protein degradation assay revealed that OsSIRP2 ubiquitinates OsTKL1 and enhances the degradation of OsTKL1 through the 26S proteasomal pathway. Heterogeneous overexpression of OsSIRP2 resulted in conferring tolerance against salinity and osmotic stress. Overall, our findings suggest that OsSIRP2 may be associated with plant responses to abiotic stresses and act as a positive regulator of salt and osmotic stress tolerance.

Overexpression of the maize E3 ubiquitin ligase gene ZmAIRP4 enhances drought stress tolerance in Arabidopsis

Ubiquitin-mediated protein degradation plays a crucial role in enabling plants to effectively and efficiently cope with environmental stresses. The E3 ligases have emerged as a central component of the ubiquitination pathway and modulate plant response to abiotic stresses. However, few such studies have been reported in maize. In this study, a C3HC4-type RING finger E3 ligase in maize, ZmAIRP4 (Zea mays Abscisic acid [ABA]-Insensitive RING Protein 4), which is an ortholog of AtAIRP4, was isolated by reverse transcription polymerase chain reaction with specific primers, and its functions in tolerance to drought stress were described. ZmAIRP4 was upregulated by ABA, polyethylene glycol and sodium chloride. In vitro ubiquitination assays and subcellular localization indicated that ZmAIRP4 was an active E3 ligase predominantly localized in the cytoplasm and nucleus. Compared to wild type, ZmAIRP4-overexpressing Arabidopsis plants were hypersensitive to ABA during early seedling development, and showed enhanced drought tolerance. Moreover, the transcript levels of several drought-related downstream genes in transgenic plants were dramatically increased compared with wild type plants. Our results suggested that E3 ligase ZmAIRP4 is a positive regulator in the drought tolerance response pathway.

Sumoylation, Phosphorylation, and Acetylation Fine-Tune the Turnover of Plant Immunity Components Mediated by Ubiquitination.

Ubiquitination-mediated protein degradation plays a crucial role in the turnover of immune proteins through rapid alteration of protein levels. Specifically, the over-accumulation of immune proteins and consequent activation of immune responses in uninfected cells is prevented through degradation. Protein post-translational modifications can influence and affect ubiquitination. There is accumulating evidence that suggests sumoylation, phosphorylation, and acetylation differentially affect the stability of immune-related proteins, so that control over the accumulation or degradation of proteins is fine-tuned. In this paper, we review the function and mechanism of sumoylation, phosphorylation, acetylation, and ubiquitination in plant disease resistance responses, focusing on how ubiquitination reacts with sumoylation, phosphorylation, and acetylation to regulate plant disease resistance signaling pathways. Future research directions are suggested in order to provide ideas for signaling pathway studies, and to advance the implementation of disease resistance proteins in economically important crops.

Efficient ASK-assisted system for expression and purification of plant F-box proteins.

Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins.

The Toxoplasma Centrocone Houses Cell Cycle Regulatory Factors.

ABSTRACTOur knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures, and we named this factor ECR1 for essential for chromosome replication 1. ECR1 was discovered by complementation of a temperature-sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40°C similar to a ts mutant carrying a defect in topoisomerase. ECR1 is a 52-kDa protein containing divergent RING and TRAF-Sina-like zinc binding domains that are dynamically expressed in the tachyzoite cell cycle. ECR1 first appears in the unique spindle compartment of the Apicomplexa (centrocone) of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughter parasites separate from the mother parasite, ECR1 is downregulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitin-mediated protein degradation machinery and the minichromosome maintenance complex, and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the cyclin-dependent kinase (CDK)-related kinase, T. gondii Crk5 (TgCrk5), which displays a similar cell cycle expression and localization during tachyzoite replication. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates that this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle.

Global gene expression analysis of the response of physic nut (Jatropha curcas L.) to medium- and long-term nitrogen deficiency.

Jatropha curcas L. is an important biofuel plant with excellent tolerance of barren environments. However, studies on the regulatory mechanisms that operate in this plant in response to nitrogen (N) shortage are scarce. In this study, genome-wide transcriptional profiles of the roots and leaves of 8-week old physic nut seedlings were analyzed after 2 and 16 days of N starvation. Enrichment results showed that genes associated with N metabolism, processing and regulation of RNA, and transport predominated among those showing alterations in expression. Genes encoding transporter families underwent major changes in expression in both roots and leaves; in particular, those with roles in ammonia, amino acid and peptide transport were generally up-regulated after long-term starvation, while AQUAPORIN genes, whose products function in osmoregulation, were down-regulated. We also found that ASPARA−GINASE B1 and SARCOSINE OXIDASE genes were up-regulated in roots and leaves after 2 and 16 d N starvation. Genes associated with ubiquitination-mediated protein degradation were significantly up-regulated. In addition, genes in the JA biosynthesis pathway were strongly activated while expression of those in GA signaling was inhibited in leaves. We showed that four major classes of genes, those with roles in N uptake, N reutilization, C/N ratio balance, and cell structure and synthesis, were particularly influenced by long-term N limitation. Our discoveries may offer clues to the molecular mechanisms that regulate N reallocation and reutilization so as to maintain or increase plant performance even under adverse environmental conditions.

USP21 regulates Hippo pathway activity by mediating MARK protein turnover.

The Hippo pathway, which acts to repress the activity of YAP and TAZ trancriptional co-activators, serve as a barrier for oncogenic transformation. Unlike other oncoproteins, YAP and TAZ are rarely activated by mutations or amplified in cancer. However, elevated YAP/TAZ activity is frequently observed in cancer and often correlates with worse survival. The activity and stability of Hippo pathway components, including YAP/TAZ, AMOT and LATS1/2, are regulated by ubiquitin-mediated protein degradation. Aberrant expression of ubiquitin ligase complexes that regulate the turnover of Hippo components and deubiquitylating enzymes that counteract these ubiquitin ligases have been implicated in human cancer. Here we identify the USP21 deubiquitylating enzyme as a novel regulator of Hippo pathway activity. We provide evidence that USP21 regulates YAP/TAZ activity by controlling the stability of MARK kinases, which promote Hippo signaling. Low expression of USP21 in early stage renal clear cell carcinoma suggests that USP21 may be a useful biomarker.

Abstract 2126: CA102N, a conjugate of hyaluronic acid (ha) and Nimuselide derivative (H-Nim) interferes with PI3K/Akt/mTOR signaling pathway in colorectal cancer (CRC) cells and inhibits tumor growth in vivo

Abstract Aberrant PI3K/Akt/mTOR signaling pathway is common in several human cancers, including CRC. The pathway regulates many major cellular processes and promotes tumorigenesis and inhibition of apoptosis. Targeting the PI3K/Akt/mTOR network could be important in resistance mechanisms in cancer. CA102N, is a conjugate of Hyaluronic Acid (HA) and H-Nim, a nimuselide (COX-2 inhibitor) derivative, currently under investigation for preclinical development in CRC. Studies have suggested that the anti-proliferative effects of COX-2 inhibitors could be attributed to modulation of the PI3K/AKT signaling pathway; therefore, in this study we aimed to examine the overall molecular mechanism of the antitumor activity of CA102N and evaluate its impact on the PI3K pathway proteins. Cell based studies indicated that in addition to cell cycle arrest; the antitumor activity of CA102N is also related to apoptosis. Administration of CA102N to nude mice bearing established HT29 subcutaneous tumor xenografts caused significant tumor growth inhibition with no signs of systemic toxicity. Immunohistochemical analyses of xenograft tumors demonstrated inhibition of VEGF and CD31, suggesting a role for CA102N in angiogenesis. Disruption of angiogenesis and apoptosis was correlated with a decrease in VEGF and the antiapoptotic protein BCL-2. Suppression of PI3K downstream signaling was a key observation in HT29 cells in vitro and in xenograft tumors treated with CA102N. As compared to untreated vehicle control, a significant decrease in phosphorylation of AKT, mTOR, p70S6K, PRAS40, 4EBP1 and FOXO1 was detected by Western blot analyses. The reduction observed may be caused by both specific protein dephosphorylation/deactivation and by ubiquitin-mediated proteasomal degradation of pathway proteins. These results suggest that the antitumor activity of CA102N may be at least partly related to the modulation of the PI3K/Akt pathway proteins. CA102N represents a promising anticancer agent with minimal toxicity, the insights provided into the mechanisms of its anticancer activity may be the basis for advancing this candidate into clinic. Citation Format: Eskouhie H. Tchaparian, Louis Lin. CA102N, a conjugate of hyaluronic acid (ha) and Nimuselide derivative (H-Nim) interferes with PI3K/Akt/mTOR signaling pathway in colorectal cancer (CRC) cells and inhibits tumor growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2126. doi:10.1158/1538-7445.AM2017-2126

Andrographolide reduced VEGFA expression in hepatoma cancer cells by inactivating HIF-1α: The involvement of JNK and MTA1/HDCA

Andrographolide (Andro) is the main active compound in medicinal herb Andrographis paniculata Nees (Acanthaceae). Vascular endothelial growth factor A (VEGFA), a key pro-angiogenic factor, contributes greatly to tumor growth. The purpose of this study is to observe the inhibition of Andro on VEGFA expression in hepatoma cancer cells and its engaged mechanism. Andro decreased mRNA and protein expression of VEGFA in hepatoma Hep3B and HepG2 cells. Andro also decreased hypoxia-inducible factor 1-alpha (HIF-1α) protein expression and its subsequent nuclear translocation. Further results showed that Andro induced the polyubiquitination of HIF-1α protein, and proteasome inhibitor MG132 reversed Andro-induced decrease in the expression of HIF-1α protein and VEGFA mRNA and protein. Andro reduced the expression of metastasis-associated protein 1 (MTA1) and histone deacetylase 1 (HDAC1) in hepatoma cancer cells. SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), reversed Andro-induced decrease in the expression of HIF-1α and VEGFA, but not MTA1 and HDAC1. Andro (10 mg/kg) inhibited tumor growth in mice implanted with hepatoma Hep3B cells in vivo, and reduced the expression of CD31, VEGFA and HIF-1α in tumor tissues. In conclusion, Andro inhibited hepatoma tumor growth by reducing HIF-1α expression and its-mediated VEGFA expression via inducing ubiquitination-mediated HIF-1α protein degradation, and JNK and MTA1/HDAC1 may be involved in this process. Natural product Andro has huge potential in hepatoma cancer treatment.

LSD1 demethylates HIF1α to inhibit hydroxylation and ubiquitin-mediated degradation in tumor angiogenesis.

Lysine-specific demethylase 1 (LSD1), which has been considered as a potential therapeutic target in human cancer, has been known to regulate many biological functions through its non-histone substrates. Although LSD1-induced hypoxia-inducible factor alpha (HIF1α) demethylation has recently been proposed, the effect of LSD1 on the relationship between HIF1α post-translational modifications (PTMs) and HIF1α-induced tumor angiogenesis remains to be elucidated. Here, we identify a new methylation site of the HIF1α protein antagonized by LSD1 and the interplay between HIF1α protein methylation and other PTMs in regulating tumor angiogenesis. LSD1 demethylates HIF1α at lysine (K) 391, which protects HIF1α against ubiquitin-mediated protein degradation. LSD1 also directly suppresses PHD2-induced HIF1α hydroxylation, which has a mutually dependent interplay with Set9-mediated HIF1α methylation. Moreover, the HIF1α acetylation that occurs in a HIF1α methylation-dependent manner is inhibited by the LSD1/NuRD complex. HIF1α stabilized by LSD1 cooperates with CBP and MTA1 to enhance vascular endothelial growth factor (VEGF)-induced tumor angiogenesis. Thus, LSD1 is a key regulator of HIF1α/VEGF-mediated tumor angiogenesis by antagonizing the crosstalk between PTMs involving HIF1α protein degradation.