Cytoplasmic Localization Research Articles

Development of selectable markers for mitochondrial transformation in yeast

Mitochondria, present in most eukaryotic organisms, are crucial for energy production and essential for cellular functions. Sequencing of the complete mitochondrial genome of Saccharomyces cerevisiae in 1998 has paved the way for mtDNA gene editing, enabling the study of mitochondrial function and potential gene therapies for mitochondrial diseases. Effective selectable markers are crucial for addressing heteroplasmic mtDNA issues after mitochondrial transformation. Antibiotic resistance (AbR) marker genes aadA1, cat, and hph confer resistance to streptomycin, chloramphenicol, and hygromycin B, respectively. This study aimed to explore the feasibility of employing these AbR markers for selecting transformed yeast cells. Additionally, the usefulness of these AbR genes as selectable markers for yeast mitochondrial transformation was assessed by fusing a mitochondrial targeting signal (MTS) to the N-terminus of these genes using overlapping PCR. The minimal inhibitory concentration (MIC) of yeast transformants expressing various AbR genes, with or without MTS fusion, was determined using the agar dilution method. Yeast transformants expressing aadA1, cat, and hph, with or without MTS fusion, displayed resistance to streptomycin (>10 mg/mL), chloramphenicol (up to 6 mg/mL), and hygromycin B (up to 4 mg/mL), respectively. MICs were similar between AbR and MTS-tagged AbR yeast transformants. To assess mitochondrial targeting, GFP was fused to the C-terminus of cat and MTS-cat gene constructs. Fluorescence microscopy confirmed MTS-tagged CAT-GFP localization to yeast mitochondria, while CAT-GFP showed cytoplasmic localization. The fluorescence microscopy results were confirmed by Western blotting. This study demonstrated that yeast transformants expressing aadA1 exhibit a significant level of streptomycin resistance (>10 mg/mL), suggesting that aadA1-mediated streptomycin resistance has the potential to serve as a selectable marker for mitochondrial transformation in yeast.

Microtubules Sequester Acetylated YAP in the Cytoplasm and Inhibit Heart Regeneration.

The Hippo pathway effector YAP (Yes-associated protein) plays an essential role in cardiomyocyte proliferation and heart regeneration. In response to physiological changes, YAP moves in and out of the nucleus. The pathophysiological mechanisms regulating YAP subcellular localization after myocardial infarction remain poorly defined. We identified YAP acetylation at site K265 by in vitro acetylation followed by mass spectrometry analysis. We used adeno-associated virus to express YAP-containing mutations that either abolished acetylation (YAP-K265R) or mimicked acetylation (YAP-K265Q) and studied how acetylation regulates YAP subcellular localization in mouse hearts. We generated a cell line with YAP-K265R mutation and investigated the protein-protein interactors by YAP immunoprecipitation followed by mass spectrometry, then validated the YAP interaction in neonatal rat ventricular myocytes. We examined colocalization of YAP and TUBA4A (tubulin α 4A) by superresolution imaging. Furthermore, we developed YAP-K265R and αMHC-MerCreMer (MCM); Yap-loxP/K265R mutant mice to examine the pathophysiological role of YAP acetylation in cardiomyocytes during cardiac regeneration. We found that YAP is acetylated at K265 by CBP (CREB-binding protein)/P300 (E1A-binding protein P300) and is deacetylated by nicotinamide phosphoribosyltransferase/nicotinamide adenine dinucleotide/sirtuins axis in cardiomyocytes. After myocardial infarction, YAP acetylation is increased, which promotes YAP cytoplasmic localization. Compared with controls, mice that were genetically engineered to express a K265R mutation that prevents YAP K256 acetylation showed improved cardiac regenerative ability and increased YAP nuclear localization. Mechanistically, YAP acetylation facilitates its interaction with TUBA4A, a component of the microtubule network that sequesters acetylated YAP in the cytoplasm. After myocardial infarction, the microtubule network increased in cardiomyocytes, resulting in the accumulation of YAP in the cytoplasm. After myocardial infarction, decreased sirtuin activity enriches YAP acetylation at K265. The growing TUBA4A network sequesters acetylated YAP within the cytoplasm, which is detrimental to cardiac regeneration.

Recent advances in AML with mutated NPM1.

Nucleophosmin 1 (NPM1) mutation is one of the most prevalent genetic mutations in adult acute myeloid leukemia (AML) and is particularly predominant in AML with a normal karyotype. NPM1 is a chaperone protein that plays various roles in several cellular processes. Wild-type NPM1 is normally localized to the nucleus, whereas mutant NPM1 proteins exhibit altered cytoplasmic localization. Clinically, AML with mutated NPM1 without FLT3-ITD is associated with a higher complete remission rate and improved overall survival. AML with mutated NPM1 is categorized as a distinct genetic entity in the World Health Organization classification of hematopoietic malignancies due to its unique clinical and biological features. However, the precise roles of NPM1 in normal hematopoiesis and in AML development remain unclear. Recent studies have revealed various clinical applications of NPM1 mutations in AML treatment, particularly in measurable residual disease analyses that target mutant NPM1 transcripts and in potential therapeutic applications of menin inhibitors and XPO-1 inhibitors for AML with mutated NPM1. Thus, NPM1 mutation is highly significant in AML classification, prognosis, response assessment, and molecular targeted therapies. Here, we review recent progress in clinical and biological aspects of AML with mutated NPM1 including molecular targeted therapy.

DEAD-box RNA helicase RH20 positively regulates RNAi-based antiviral immunity in plants by associating with SGS3/RDR6 bodies.

RNA silencing plays a crucial role in defending against viral infections in diverse eukaryotic hosts. Despite extensive studies on core components of the antiviral RNAi pathway such as DCLs, AGOs and RDRs proteins, host factors involved in antiviral RNAi remain incompletely understood. In this study, we employed the proximity labelling approach to identify the host factors required for antiviral RNAi in Nicotiana benthamiana. Using the barley stripe mosaic virus (BSMV)-encoded γb, a viral suppressor of RNA silencing (VSR), as the bait protein, we identified the DEAD-box RNA helicase RH20, a broadly conserved protein in plants and animals with a homologous human protein known as DDX5. We demonstrated the interaction between RH20 and BSMV γb. Knockdown or knockout of RH20 attenuates the accumulation of viral small interfering RNAs, leading to increased susceptibility to BSMV, while overexpression of RH20 enhances resistance to BSMV, a process requiring the cytoplasmic localization and RNA-binding activity of RH20. In addition to BSMV, RH20 also negatively regulates the infection of several other positive-sense RNA viruses, suggesting the broad-spectrum antiviral activity of RH20. Mechanistic analysis revealed the colocalization and interaction of RH20 with SGS3/RDR6, and disruption of either SGS3 or RDR6 undermines the antiviral function of RH20, suggesting RH20 as a new component of the SGS3/RDR6 bodies. As a counter-defence, BSMV γb VSR subverts the RH20-mediated antiviral defence by interfering with the RH20-SGS3 interaction. Our results uncover RH20 as a new positive regulator of antiviral RNAi and provide new potential targets for controlling plant viral diseases.

Quantitative Analysis of Rhodobacter sphaeroides Storage Organelles via Cryo-Electron Tomography and Light Microscopy.

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

Characterization of c-Jun N-terminal kinase (JNK) gene reveals involvement of immune defense against Vibrio splendidus infection in Apostichopus japonicus

The c-Jun N-terminal kinase (JNK) constitutes an evolutionarily conserved family of serine/threonine protein kinases, pivotal in regulating various physiological processes in vertebrates, encompassing apoptosis and antibacterial immunity. Nevertheless, the involvement of JNK in the innate immune response remains largely unexplored in pathogen-induced echinoderms. We isolated and characterized the JNK gene from Apostichopus japonicus (AjJNK) in our investigation. The full-length cDNA sequences of AjJNK spanned 1806 bp, comprising a 1299 bp open reading frame (ORF) encoding 432 amino acids, a 274 bp 5′-untranslated region (UTR), and a 233 bp 3′-UTR. Structural analysis revealed the presence of a classical S_TKc domain (37–335 amino acids) within AjJNK and contains several putative immune-related transcription factor-binding sites, including Elk-1, NF-κB, AP-1, and STAT5. Spatial expression analysis indicated ubiquitous expression of AjJNK across all examined tissues, with the highest expression noted in coelomocytes. The mRNA, protein, and phosphorylation levels of AjJNK were obviously induced in coelomocytes upon V. splendidus challenge and lipopolysaccharide stimulation. Immunofluorescence analysis demonstrated predominant cytoplasmic localization of AjJNK in coelomocytes with subsequent nuclear translocation following the V. splendidus challenge in vivo. Moreover, siRNA-mediated knockdown of AjJNK led to a significant increase in intracellular bacterial load, as well as elevated levels of Ajcaspase 3 and coelomocyte apoptosis post V. splendidus infection. Furthermore, the phosphorylation levels of AjJNK inhibited by its specific inhibitor SP600125 and also significantly suppressed the expression of Ajcaspase 3 and coelomocyte apoptosis during pathogen infection. Collectively, these data underscored the pivotal role of AjJNK in immune defense, specifically in the regulation of coelomocyte apoptosis in V. splendidus-challenged A. japonicus.

A novel peptide encoded by circ-SLC9A6 promotes lipid dyshomeostasis through the regulation of H4K16ac-mediated CD36 transcription in NAFLD.

As the leading cause of end-stage liver disease, nonalcoholic fatty liver disease (NAFLD) is mainly induced by lipid dyshomeostasis. The translation of endogenous circular RNAs (circRNAs) is closely related to the progression of various diseases, but the involvement of circRNAs in NAFLD has not been determined. Combined high-throughput circRNA profiles were used to identify circRNAs with translational potential. The underlying molecular mechanisms were investigated by RNA sequencing, pull-down/MS and site-specific mutagenesis. In this study, we focused on circ-SLC9A6, an abnormally highly expressed circRNA in human and mouse liver tissue during NAFLD development that exacerbates metabolic dyshomeostasis in hepatocytes by encoding a novel peptide called SLC9A6-126aa in vivo and in vitro. YTHDF2-mediated degradation of m6A-modified circ-SLC9A6 was found to be essential for the regulation of SLC9A6-126aa expression. We further found that the phosphorylation of SLC9A6-126aa by AKT was crucial for its cytoplasmic localization and the maintenance of physiological homeostasis, whereas high-fat stress induced substantial translocation of unphosphorylated SLC9A6-126aa to the nucleus, resulting in a vicious cycle of lipid metabolic dysfunction. Nuclear SLC9A6-126aa promotes transcriptional activation of the target gene CD36 and enhances its occupancy of the CD36 promoter locus by regulating MOF-mediated histone H4K16 acetylation. Hepatic CD36 depletion significantly ameliorated hyperactivated MAPK signalling and lipid disturbance in SLC9A6-126aa transgenic mice. Clinically, increasing levels of SLC9A6-126aa were observed during NAFLD progression and were found to be positively correlated with the CD36 and MAPK cascades. This study revealed the role of circ-SLC9A6-derived SLC9A6-126aa in the epigenetic modification-mediated regulation of lipid metabolism. Our findings may provide promising therapeutic targets for NAFLD and new insights into the pathological mechanisms of metabolic diseases. Under normal circumstances, driven by m6A modification, YTHDF2 directly recognizes and degrades circ-SLC9A6, thereby inhibiting the translation of SLC9A6-126aa. Additionally, AKT1 phosphorylates and inhibits the nuclear translocation of SLC9A6-126aa. In NAFLD, lipid overload leads to YTHDF2 and AKT1 deficiency, ultimately increasing the expression and nuclear import of SLC9A6-126aa. Nuclear SLC9A6-126aa binds directly to the CD36 promoter and initiates CD36 transcription, which induces lipid dyshomeostasis.

The E3 ubiquitin ligase COP1 regulates salt tolerance via GIGANTEA degradation in roots.

Excess soil salinity significantly impairs plant growth and development. Our previous reports demonstrated that the core circadian clock oscillator GIGANTEA (GI) negatively regulates salt stress tolerance by sequestering the SALT OVERLY SENSITIVE (SOS) 2 kinase, an essential component of the SOS pathway. Salt stress induces calcium-dependent cytoplasmic GI degradation, resulting in activation of the SOS pathway; however, the precise molecular mechanism governing GI degradation during salt stress remains enigmatic. Here, we demonstrate that salt-induced calcium signals promote the cytoplasmic partitioning of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), leading to the 26S proteasome-dependent degradation of GI exclusively in the roots. Salt stress-induced calcium signals accelerate the cytoplasmic localization of COP1 in the root cells, which targets GI for 26S proteasomal degradation. Align with this, the interaction between COP1 and GI is only observed in the roots, not the shoots, under salt-stress conditions. Notably, the gi-201 cop1-4 double mutant shows an enhanced tolerance to salt stress similar to gi-201, indicating that GI is epistatic to COP1 under salt-stress conditions. Taken together, our study provides critical insights into the molecular mechanisms governing the COP1-mediated proteasomal degradation of GI for salt stress tolerance, raising new possibilities for developing salt-tolerant crops.

"GLI1 Subcellular Localization and Overexpression as Prognostic Factors for Disease-Free Survival in Colorectal Carcinoma".

Glioma-associated oncogene homolog-1 (GLI1) is amplified in human glioblastoma, and there is growing evidence suggesting its significant role in tumor development and metastasis. Our aim was to investigate the role of the GLI-1 gene in the progression of colorectal cancer (CRC) and its correlation with various clinicopathological features. Additionally, we examined the impact of the GLI-1 gene and other factors on the prognosis of CRC. We analyzed a total of 98 confirmed CRC cases and adjacent normal tissue controls. Patients suspected of having colon cancer underwent a colonoscopy and targeted biopsy, while those with rectal cancer underwent CT scans and MRI. GLI1 expression was detected using real-time PCR assay, Western blotting, and immunohistochemistry. The GLI1 gene was observed to be overexpressed in tumor tissues at both the protein and mRNA levels (p < 0.05). In addition, GLI1 overexpression was significantly associated with various factors such as tumor invasion (T3/T4), presence of lymph nodes, lymph node metastasis (LNM), stage (III/IV), tumor site (colon), tumor size (≥ 3cm), localization (nucleocytoplasmic), strong staining intensity and recurrence (p < 0.05). The results of survival analysis showed that the patients with overexpression of GLI1 had a significantly lower DFS rate which was 21months compared to those with normal expression who had 31months (p < 0.05). Moreover, individuals with early onset disease (15months) were more likely to have cytoplasmic localization of the GLI1 gene as opposed to nucleo-cytoplasmic localization of GLI1 which presented late-onset disease( 23months) (p < 0.05). Finally, Stage and PNI (p < 0.05) were found to independently affect outcomes of CRC according to Cox regression analysis. High expression of GLI-1 in CRC is associated with adverse pathology and poor prognosis for patients. The correlation between cytoplasmic localization of GLI-1 and reduced disease-free survival holds potential for guiding prognosis and treatment. Further research is needed to develop strategies targeting GLI-1 for improved outcomes.

Deacetylation of GLUD1 maintains the survival of lung adenocarcinoma cells under glucose starvation by inhibiting autophagic cell death

Enhanced glutamine catabolism is one of the main metabolic features of cancer, providing energy and intermediate metabolites for cancer progression. However, the functions of glutamine catabolism in cancer under nutrient deprivation need to be further clarified. Here, we discovered that deacetylation of glutamate dehydrogenase 1 (GLUD1), one of the key enzymes in glutamine catabolism, maintains the survival of lung adenocarcinoma (LUAD) cells under glucose starvation by inhibiting autophagic cell death. We found that glucose starvation increased GLUD1 activity by reducing its acetylation on Lys84 and promoted its active hexamer formation. Besides, deacetylation of GLUD1 induced its cytoplasmic localization, where GLUD1 was ubiquitinated in K63-linkage by TRIM21, leading to the binding of GLUD1 with cytoplasmic glutaminase KGA. These two effects enhanced glutamine metabolism both in mitochondria and cytoplasm, increased the production of alpha-ketoglutarate (α-KG). Meanwhile, cytoplasmic GLUD1 also interacted with p62 and prevented its acetylation, leading to the inhibition of p62 body formation. All these effects blocked autophagic cell death of LUAD cells under glucose starvation. Taken together, our results reveal a novel function of GLUD1 under glucose deprivation in LUAD cells and provide new insights into the functions of glutamine catabolism during cancer progression.

Ligand-Receptor Interaction-Induced Intracellular Phase Separation: A Global Disruption Strategy for Resistance-Free Lethality of Pathogenic Bacteria.

Addressing the global challenge of bacterial resistance demands innovative approaches, among which multitargeting is a widely used strategy. Current strategies of multitargeting, typically achieved through drug combinations or single agents inherently aiming at multiple targets, face challenges such as stringent pharmacokinetic and pharmacodynamic requirements and cytotoxicity concerns. In this report, we propose a bacterial-specific global disruption approach as a vastly expanded multitargeting strategy that effectively disrupts bacterial subcellular organization. This effect is achieved through a pioneering chemical design of ligand-receptor interaction-induced aggregation of small molecules, i.e., DNA-induced aggregation of a diarginine peptidomimetic within bacterial cells. These intracellular aggregates display affinity toward various proteins and thus substantially interfere with essential bacterial functions and rupture bacterial cell membranes in an "inside-out" manner, leading to robust antibacterial activities and suppression of drug resistance. Additionally, biochemical analysis of macromolecule binding affinity, cytoplasmic localization patterns, and bacterial stress responses suggests that this bacterial-specific intracellular aggregation mechanism is fundamentally different from nonselective classic DNA or membrane binding mechanisms. These mechanistic distinctions, along with the peptidomimetic's selective permeation of bacterial membranes, contribute to its favorable biocompatibility and pharmacokinetic properties, enabling its in vivo antimicrobial efficacy in several animal models, including mice-based superficial wound models, subcutaneous abscess models, and septicemia infection models. These results highlight the great promise of ligand-receptor interaction-induced intracellular aggregation in achieving a globally disruptive multitargeting effect, thereby offering potential applications in the treatment of malignant cells, including pathogens, tumor cells, and infected tissues.

A Functional Human Glycogen Debranching Enzyme Encoded by a Synthetic Gene: Its Implications for Glycogen Storage Disease Type III Management.

Glycogen Storage Disease type III (GSD III) is a metabolic disorder resulting from a deficiency of the Glycogen Debranching Enzyme (GDE), a large monomeric protein (approximately 170 kDa) with cytoplasmic localization and two distinct enzymatic activities: 4-α-glucantransferase and amylo-α-1,6-glucosidase. Mutations in the Agl gene, with consequent deficiency in GDE, lead to the accumulation of abnormal/toxic glycogen with shorter chains (phosphorylase limit dextrin, PLD) in skeletal and/or heart muscle and/or in the liver. Currently, there is no targeted therapy, and available treatments are symptomatic, relying on specific diets. Enzyme Replacement Therapy (ERT) might represent a potential therapeutic strategy for GSD III. Moreover, the single-gene nature of GSD III, the subcellular localization of GDE, and the type of affected tissues represent ideal conditions for exploring gene therapy approaches. Toward this direction, we designed a synthetic, codon-optimized cDNA encoding the human GDE. This gene yielded high amounts of soluble, enzymatically active protein in Escherichia coli. Moreover, when transfected in Human Embryonic Kidney cells (HEK-293), it successfully encoded a functional GDE. These results suggest that our gene or protein might complement the missing function in GSD III patients, opening the door to further exploration of therapeutic approaches for this disease.

Mesenchymal Stem Cell-Derived Exosomes Mitigate Acute Murine Liver Injury via Ets-1 and Heme Oxygenase-1 Up-regulation.

Mesenchymal stem cells (MSCs)-derived exosomes have been previously demonstrated to promote tissue regeneration in various animal disease models. This study investigated the protective effect of exosome treatment in carbon tetrachloride (CCl4)-induced acute liver injury and delineated possible underlying mechanism. Exosomes collected from conditioned media of previously characterized human umbilical cord-derived MSCs were intravenously administered into male CD-1 mice with CCl4-induced acute liver injury. Biochemical, histological and molecular parameters were used to evaluate the severity of liver injury. A rat hepatocyte cell line, Clone-9, was used to validate the molecular changes by exosome treatment. Exosome treatment significantly suppressed plasma levels of AST, ALT, and pro-inflammatory cytokines, including IL-6 and TNF-α, in the mice with CCl4-induced acute liver injury. Histological morphometry revealed a significant reduction in the necropoptic area in the injured livers following exosome therapy. Consistently, western blot analysis indicated marked elevations in hepatic expression of PCNA, c-Met, Ets-1, and HO-1 proteins after exosome treatment. Besides, the phosphorylation level of signaling mediator JNK was significantly increased, and that of p38 was restored by exosome therapy. Immunohistochemistry double staining confirmed nuclear Ets-1 expression and cytoplasmic localization of c-Met and HO-1 proteins. In vitro studies demonstrated that exosome treatment increased the proliferation of Clone-9 hepatocytes and protected them from CCl4-induced cytotoxicity. Kinase inhibition experiment indicated that the exosome-driven hepatoprotection might be mediated through the JNK pathway. Exosome therapy activates the JNK signaling activation pathway as well as up-regulates Ets-1 and HO-1 expression, thereby protecting hepatocytes against hepatotoxin-induced cell death.

Oligomerization regulates the interaction of Gemin5 with members of the SMN complex and the translation machinery

RNA-binding proteins are multifunctional molecules impacting on multiple steps of gene regulation. Gemin5 was initially identified as a member of the survival of motor neurons (SMN) complex. The protein is organized in structural and functional domains, including a WD40 repeats domain at the N-terminal region, a tetratricopeptide repeat (TPR) dimerization module at the central region, and a non-canonical RNA-binding site at the C-terminal end. The TPR module allows the recruitment of the endogenous Gemin5 protein in living cells and the assembly of a dimer in vitro. However, the biological relevance of Gemin5 oligomerization is not known. Here we interrogated the Gemin5 interactome focusing on oligomerization-dependent or independent regions. We show that the interactors associated with oligomerization-proficient domains were primarily annotated to ribosome, splicing, translation regulation, SMN complex, and RNA stability. The presence of distinct Gemin5 protein regions in polysomes highlighted differences in translation regulation based on their oligomerization capacity. Furthermore, the association with native ribosomes and negative regulation of translation was strictly dependent on both the WD40 repeats domain and the TPR dimerization moiety, while binding with the majority of the interacting proteins, including SMN, Gemin2, and Gemin4, was determined by the dimerization module. The loss of oligomerization did not perturb the predominant cytoplasmic localization of Gemin5, reinforcing the cytoplasmic functions of this essential protein. Our work highlights a distinctive role of the Gemin5 domains for its functions in the interaction with members of the SMN complex, ribosome association, and RBP interactome.

HGG-25. IDENTIFYING YB1 AND NPM1 AS PART OF THE MUTANT HISTONE 3 PROTEIN INTERACTOME IN H3 MUTATED PHGG

Abstract Paediatric high-grade gliomas (pHGG) are the leading cause of childhood cancer deaths, accounting for over 40% of deaths in the UK. pHGG tumours harbour histone H3 mutations, defining 50% of pHGG cases. Studies show prominent mutations as being H3.3K27M and H3.3G34R/V. Our previous proteomic research on H3 intercatomes, identified unique and common interacting binding partners of H3-G34R and H3-G34V mutant proteins relative to wild type H3. Among these proteins we have focused on YB-1 and Nucleophosmin 1 (NPM1). YB-1, is multifunctional transcription factor and a well-characterised oncogenic protein. NPM1 is a molecular chaperone and frequently overexpressed in cancers. Our aim is to analyse the role of YB-1 and NPM1 in H3 mutated pHGG, in order to identify targeted therapeutic options for these difficult to treat tumours. H3 mutated glioma cell lines, H3 wild type pHGG cells and control cells were cultured and then subjected to comparative immunofluorescence microscopy, and western blot analyses to investigate YB-1 and NPM1 expression. Results showed statistically significant altered protein expression and localisation of YB-1 and NPM1 in H3 mutated pHGG in comparison to wild type and control cells. YB-1 expression was increased in H3 mutated cells and NPM1 showed increased cytoplasmic localization in H3 mutated cells in comparison to H3 WT glioma and control cells. Results of the study support that YB-1 and NPM1 are important interacting proteins in H3G34-mutated pHGGs and can be explored as potential therapeutic targets in H3 mutated pHGG.

REST-dependent downregulation of von Hippel-Lindau tumor suppressor promotes autophagy in SHH-medulloblastoma

The RE1 silencing transcription factor (REST) is a driver of sonic hedgehog (SHH) medulloblastoma genesis. Our previous studies showed that REST enhances cell proliferation, metastasis and vascular growth and blocks neuronal differentiation to drive progression of SHH medulloblastoma tumors. Here, we demonstrate that REST promotes autophagy, a pathway that is found to be significantly enriched in human medulloblastoma tumors relative to normal cerebella. In SHH medulloblastoma tumor xenografts, REST elevation is strongly correlated with increased expression of the hypoxia-inducible factor 1-alpha (HIF1α)—a positive regulator of autophagy, and with reduced expression of the von Hippel-Lindau (VHL) tumor suppressor protein – a component of an E3 ligase complex that ubiquitinates HIF1α. Human SHH-medulloblastoma tumors with higher REST expression exhibit nuclear localization of HIF1α, in contrast to its cytoplasmic localization in low-REST tumors. In vitro, REST knockdown promotes an increase in VHL levels and a decrease in cytoplasmic HIF1α protein levels, and autophagy flux. In contrast, REST elevation causes a decline in VHL levels, as well as its interaction with HIF1α, resulting in a reduction in HIF1α ubiquitination and an increase in autophagy flux. These data suggest that REST elevation promotes autophagy in SHH medulloblastoma cells by modulating HIF1α ubiquitination and stability in a VHL-dependent manner. Thus, our study is one of the first to connect VHL to REST-dependent control of autophagy in a subset of medulloblastomas.

A viral E3 ubiquitin ligase produced by herpes simplex virus 1 inhibits the NLRP1 inflammasome.

Guard proteins initiate defense mechanisms upon sensing pathogen-encoded virulence factors. Successful viral pathogens likely inhibit guard protein activity, but these interactions have been largely undefined. Here, we demonstrate that the human pathogen herpes simplex virus 1 (HSV-1) stimulates and inhibits an antiviral pathway initiated by NLRP1, a guard protein that induces inflammasome formation and pyroptotic cell death when activated. Notably, HSV-1 infection of human keratinocytes promotes posttranslational modifications to NLRP1, consistent with MAPK-dependent NLRP1 activation, but does not result in downstream inflammasome formation. We identify infected cell protein 0 (ICP0) as the critical HSV-1 protein that is necessary and sufficient for inhibition of the NLRP1 pathway. Mechanistically, ICP0's cytoplasmic localization and function as an E3 ubiquitin ligase prevents proteasomal degradation of the auto-inhibitory NT-NLRP1 fragment, thereby preventing inflammasome formation. Further, we demonstrate that inhibiting this inflammasome is important for promoting HSV-1 replication. Thus, we have established a mechanism by which HSV-1 overcomes a guard-mediated antiviral defense strategy in humans.

Poly (ADP-ribose) polymerase 1 promotes HuR/ELAVL1 cytoplasmic localization and inflammatory gene expression by regulating p38 MAPK activity

Post-transcriptional regulation of cytokine/chemokine mRNA turnover is critical for immune processes and contributes to the mammalian cellular response to diverse inflammatory stimuli. The ubiquitous RNA-binding protein human antigen R (HuR) is an integral regulator of inflammation-associated mRNA fate. HuR function is regulated by various post-translational modifications that alter its subcellular localization and ability to stabilize target mRNAs. Both poly (ADP-ribose) polymerase 1 (PARP1) and p38 mitogen-activated protein kinases (MAPKs) have been reported to regulate the biological function of HuR, but their specific regulatory and crosstalk mechanisms remain unclear. In this study, we show that PARP1 acts via p38 to synergistically promote cytoplasmic accumulation of HuR and stabilization of inflammation-associated mRNAs in cells under inflammatory conditions. Specifically, p38 binds to auto-poly ADP-ribosylated (PARylated) PARP1 resulting in the covalent PARylation of p38 by PARP1, thereby promoting the retention and activity of p38 in the nucleus. In addition, PARylation of HuR facilitates the phosphorylation of HuR at the serine 197 site mediated by p38, which then increases the translocation of HuR to the cytoplasm, ultimately stabilizing the inflammation-associated mRNA expression at the post-transcriptional level.

Tumour-specific phosphorylation of serine 419 drives alpha-enolase (ENO1) nuclear export in triple negative breast cancer progression

BackgroundThe glycolytic enzyme alpha-enolase is a known biomarker of many cancers and involved in tumorigenic functions unrelated to its key role in glycolysis. Here, we show that expression of alpha-enolase correlates with subcellular localisation and tumorigenic status in the MCF10 triple negative breast cancer isogenic tumour progression model, where non-tumour cells show diffuse nucleocytoplasmic localisation of alpha-enolase, whereas tumorigenic cells show a predominantly cytoplasmic localisation. Alpha-enolase nucleocytoplasmic localisation may be regulated by tumour cell-specific phosphorylation at S419, previously reported in pancreatic cancer.ResultsHere we show ENO1 phosphorylation can also be observed in triple negative breast cancer patient samples and MCF10 tumour progression cell models. Furthermore, prevention of alpha-enolase-S419 phosphorylation by point mutation or a casein kinase-1 specific inhibitor D4476, induced tumour-specific nuclear accumulation of alpha-enolase, implicating S419 phosphorylation and casein kinase-1 in regulating subcellular localisation in tumour cell-specific fashion. Strikingly, alpha-enolase nuclear accumulation was induced in tumour cells by treatment with the specific exportin-1-mediated nuclear export inhibitor Leptomycin B. This suggests that S419 phosphorylation in tumour cells regulates alpha-enolase subcellular localisation by inducing its exportin-1-mediated nuclear export. Finally, as a first step to analyse the functional consequences of increased cytoplasmic alpha-enolase in tumour cells, we determined the alpha-enolase interactome in the absence/presence of D4476 treatment, with results suggesting clear differences with respect to interaction with cytoskeleton regulating proteins.ConclusionsThe results suggest for the first time that tumour-specific S419 phosphorylation may contribute integrally to alpha-enolase cytoplasmic localisation, to facilitate alpha-enolase’s role in modulating cytoskeletal organisation in triple negative breast cancer. This new information may be used for development of triple negative breast cancer specific therapeutics that target alpha-enolase.

Terpene synthases GhTPS6 and GhTPS47 participate in resistance to Verticillium dahliae in upland cotton

Terpene synthases (TPSs) are enzymes responsible for catalyzing the production of diverse terpenes, the largest class of secondary metabolites in plants. Here, we identified 107 TPS gene loci encompassing 92 full-length TPS genes in upland cotton (Gossypium hirsutum L.). Phylogenetic analysis showed they were divided into six subfamilies. Segmental duplication and tandem duplication events contributed greatly to the expansion of TPS gene family, particularly the TPS-a and TPS-b subfamilies. Expression profile analysis screened out that GhTPSs may mediate the interaction between cotton and Verticillium dahliae. Three-dimensional structures and subcellular localizations of the two selected GhTPSs, GhTPS6 and GhTPS47, which belong to the TPS-a subfamily, demonstrated similarity in protein structures and nucleus and cytoplasm localization. Virus-induced gene silencing (VIGS) of the two GhTPSs yielded plants characterized by increased wilting and chlorosis, more severe vascular browning, and higher disease index than control plants. Additionally, knockdown of GhTPS6 and GhTPS47 led to the down-regulation of cotton terpene synthesis following V. dahliae infection, indicating that these two genes may positively regulate resistance to V. dahliae through the modulation of disease-resistant terpene biosynthesis. Overall, our study represents a comprehensive analysis of the G. hirsutum TPS gene family, revealing their potential roles in defense responses against Verticillium wilt.