Cellular Biogenesis Research Articles

Coronaviral ORF6 protein mediates inter-organelle contacts and modulates host cell lipid flux for virus production.

Lipid droplets (LDs) form inter-organelle contacts with the endoplasmic reticulum (ER) that promote their biogenesis, while LD contacts with mitochondria enhance β-oxidation of contained fatty acids. Viruses have been shown to take advantage of lipid droplets to promote viral production, but it remains unclear whether they also modulate the interactions between LDs and other organelles. Here, we showed that coronavirus ORF6 protein targets LDs and is localized to the mitochondria-LD and ER-LD contact sites, where it regulates LD biogenesis and lipolysis. At the molecular level, we find that ORF6 inserts into the LD lipid monolayer via its two amphipathic helices. ORF6 further interacts with ER membrane proteins BAP31 and USE1 to mediate ER-LDs contact formation. Additionally, ORF6 interacts with the SAM complex in the mitochondrial outer membrane to link mitochondria to LDs. In doing so, ORF6 promotes cellular lipolysis and LD biogenesis to reprogram host cell lipid flux and facilitate viral production.

A split GAL4 RUBY assay for visual in planta detection of protein-protein interactions.

Protein-protein interactions (PPIs) are a fundamental process in cellular biogenesis. Here we have developed a split GAL4 RUBY assay that enables macroscopically visual PPI detection in plant leaves in real time. Candidate interacting protein partners are fused to specific domains of the yeast GAL4 and herpes simplex virus VP16 transcription factors and transiently expressed in Nicotiana benthamina leaves by Agrobacterium infiltration. PPI, that may be either direct or indirect, results in transcriptional activation of a RUBY reporter gene leading to the production of the highly visual metabolite, betalain, in leaf tissue of living plants. Samples require no processing for in planta visual qualitative assessment, but with very simple processing steps the assay is quantitative. Its accuracy is demonstrated using a series of known interacting protein partners and mutant derivatives including transcription factors, signalling molecules and plant resistance proteins with cognate pathogen effectors. Using this assay, association between the wheat Sr27 stem rust disease resistance protein and corresponding AvrSr27 avirulence effector family produced by the rust pathogen is detected. Interaction is also observed between this resistance protein and the effector encoded by the corresponding avrSr27-3 virulence allele. However, this association appears weaker in the split GAL4 RUBY assay, which coupled with lower avrSr27-3 expression during stem rust infection, likely enables virulent races of the rust pathogen to avoid Sr27-mediated detection.

Retromer oligomerization drives SNX-BAR coat assembly and membrane constriction.

Proteins exit from endosomes through tubular carriers coated by retromer, a complex that impacts cellular signaling, lysosomal biogenesis and numerous diseases. The coat must overcome membrane tension to form tubules. We explored the dynamics and driving force of this process by reconstituting coat formation with yeast retromer and the BAR-domain sorting nexins Vps5 and Vps17 on oriented synthetic lipid tubules. This coat oligomerizes bidirectionally, forming a static tubular structure that does not exchange subunits. High concentrations of sorting nexins alone constrict membrane tubes to an invariant radius of 19 nm. At lower concentrations, oligomers of retromer must bind and interconnect the sorting nexins to drive constriction. Constricting less curved membranes into tubes, which requires more energy, coincides with an increased surface density of retromer on the sorting nexin layer. Retromer-mediated crosslinking of sorting nexins at variable densities may thus tune the energy that the coat can generate to deform the membrane. In line with this, genetic ablation of retromer oligomerization impairs endosomal protein exit in yeast and human cells.

DEAD box RNA helicases protect Antarctic Pseudomonas syringae Lz4W against oxidative stress.

DEAD box RNA helicases are involved in important cellular processes like RNA metabolism (Processing and Degradation), ribosome biogenesis and translation. Besides being crucial to the formation of cold adapted degradosomes, RNA helicases have been implicated in structural rearrangement of RNA, implying a role in alleviation of RNA secondary structure stabilization at low temperature. This study depicts the results of experiments on protective role played by DEAD box RNA helicases against nucleic acid damaging agents. RNA helicase mutants ΔrhlE, ΔsrmB, ΔcsdA, ΔdbpA and ΔrhlB cells were exposed to various DNA damaging agents (UV, Paraquat, Mitomycin C, Hydroxyurea and Hydrogen peroxide) and assessed for sensitivity to them. Our results illustrate that ∆csdA displayed sensitivity to paraquat (that causes oxidative damage) and UV radiation induced DNA damage. On the other hand, ∆srmB displays sensitivity to hydroxyurea that causes damage to the replication forks (RFs) by inhibiting ribonucleotide reductase and depleting the dNTP pool of cells. However, all five RNA helicase mutants were resistant to H2O2 mediated oxidative stress and mitomycin C induced DNA cross-links.

Global Transcriptome Analyses of Cellular and Viral mRNAs during HAdV-C5 Infection Highlight New Aspects of Viral mRNA Biogenesis and Cytoplasmic Viral mRNA Accumulations.

It is well established that human adenoviruses such as species C, types 2 and 5 (HAdV-C2 and HAdV-C5), induce a nearly complete shutoff of host-cell protein synthesis in the infected cell, simultaneously directing very efficient production of viral proteins. Such preferential expression of viral over cellular genes is thought to be controlled by selective nucleocytoplasmic export and translation of viral mRNA. While detailed knowledge of the regulatory mechanisms responsible for the translation of viral mRNA is available, the viral or cellular mechanisms of mRNA biogenesis are not completely understood. To identify parameters that control the differential export of viral and cellular mRNAs, we performed global transcriptome analyses (RNAseq) and monitored temporal nucleocytoplasmic partitioning of viral and cellular mRNAs during HAdV-C5 infection of A549 cells. Our analyses confirmed previously reported features of the viral mRNA expression program, as a clear shift in viral early to late mRNA accumulation was observed upon transition from the early to the late phase of viral replication. The progression into the late phase of infection, however, did not result in abrogation of cellular mRNA export; rather, viral late mRNAs outnumbered viral early and most cellular mRNAs by several orders of magnitude during the late phase, revealing that viral late mRNAs are not selectively exported but outcompete cellular mRNA biogenesis.

NAT10: An RNA cytidine transferase regulates fatty acid metabolism in cancer cells.

Background N‐4 cytidine acetylation (ac4C) is an epitranscriptomics modification catalyzed by N‐acetyltransferase 10 (NAT10); important for cellular mRNA stability, rRNA biogenesis, cell proliferation and epithelial to mesenchymal transition (EMT). However, whether other crucial pathways are regulated by NAT10‐dependent ac4C modification in cancer cells remains unclear. Therefore, in this study, we explored the impact of NAT10 depletion in cancer cells using unbiased RNA‐seq.MethodsHigh‐throughput sequencing of knockdown NAT10 in cancer cells was conducted to identify enriched pathways. Acetylated RNA immunoprecipitation‐seq (acRIP‐seq) and RIP‐PCR were used to map and determine ac4C levels of RNA. Exogenous palmitate uptake assay was conducted to assess NAT10 knockdown cancer cells using Oil Red O staining and lipid content analysis. Gas‐chromatography–tandem mass spectroscopy (GC/MS) was used to perform untargeted lipidomics.ResultsHigh‐throughput sequencing of NAT10 knockdown in cancer cells revealed fatty acid (FA) metabolism as the top enriched pathway through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis in differentially downregulated genes. FA metabolic genes such as ELOLV6, ACSL1, ACSL3, ACSL4, ACADSB and ACAT1 were shown to be stabilised via NAT10‐dependent ac4C RNA acetylation. Additionally, NAT10 depletion was shown to significantly reduce the levels of overall lipid content, triglycerides and total cholesterol. Further, NAT10 depletion in palmitate‐loaded cancer cells showed decrease in ac4C levels across the RNA transcripts of FA metabolic genes. In untargeted lipidomics, 496 out of 2 279 lipids were statistically significant in NAT10 depleted cancer cells, of which pathways associated with FA metabolism are the most enriched.ConclusionsConclusively, our results provide novel insights into the impact of NAT10‐mediated ac4C modification as a crucial regulatory factor during FA metabolism and showed the benefit of targeting NAT10 for cancer treatment.

The Response of the Human Umbilical Vein Endothelial Cell Transcriptome to Variation in Magnesium Concentration.

Vascular endothelial cells have a critical role in the maintenance of cardiovascular function. Evidence suggests that endothelial function may be compromised under conditions of magnesium deficiency, which increases vulnerability to inflammation. Whole genome transcription analysis was used to explore the acute (24 h) effects of magnesium on human umbilical vascular endothelial cells (HUVEC) cultured in low (0.1 mM) or high (5 mM) concentrations. With low magnesium 2728 transcripts were differentially expressed compared to the 1 mM control cultures and 3030 were differentially expressed with high magnesium. 615 transcripts were differentially expressed under both conditions, of which only 34 showed a concentration-dependent response. Analysis indicated that cellular organisation and biogenesis and key cellular processes such as apoptosis were impacted by both low and high conditions. High magnesium also influenced protein binding functions, intracellular signal transduction, metabolic and catalytic processes. Both conditions impacted on stress-related processes, in particular the inflammatory response. Key mediators of calcium-dependent regulation of gene expression were responsive to both high and low magnesium conditions. The HUVEC transcriptome is highly sensitive to acute changes in the concentration of magnesium in culture medium. The findings of this study support the view that whilst inflammation is an important process that is responsive to magnesium, the function of the endothelium may be impacted by other magnesium-induced changes including maintenance of cellular integrity, receptor expression and metabolic functions. The high proportion of transcripts that did not show a concentration-dependent response suggests variation in magnesium may elicit indirect changes, possibly mediated by other ions.

Shared pathobiology identifies AMPK as a therapeutic target for obesity and autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common Mendelian kidney disease, affecting approximately one in 1,000 births and accounting for 5% of end-stage kidney disease in developed countries. The pathophysiology of ADPKD is strongly linked to metabolic dysregulation, which may be secondary to defective polycystin function. Overweight and obesity are highly prevalent in patients with ADPKD and constitute an independent risk factor for progression. Recent studies have highlighted reduced AMP-activated protein kinase (AMPK) activity, increased mammalian target of rapamycin (mTOR) signaling, and mitochondrial dysfunction as shared pathobiology between ADPKD and overweight/obesity. Notably, mTOR and AMPK are two diametrically opposed sensors of energy metabolism that regulate cell growth and proliferation. However, treatment with the current generation of mTOR inhibitors is poorly tolerated due to their toxicity, making clinical translation difficult. By contrast, multiple preclinical and clinical studies have shown that pharmacological activation of AMPK provides a promising approach to treat ADPKD. In this narrative review, we summarize the pleiotropic functions of AMPK as a regulator of cellular proliferation, macromolecule metabolism, and mitochondrial biogenesis, and discuss the potential for pharmacological activation of AMPK to treat ADPKD and obesity-related kidney disease.

Role of Epigenetics in Modulating Phenotypic Plasticity against Abiotic Stresses in Plants

Plants being sessile are always exposed to various environmental stresses, and to overcome these stresses, modifications at the epigenetic level can prove vital for their long-term survival. Epigenomics refers to the large-scale study of epigenetic marks on the genome, which include covalent modifications of histone tails (acetylation, methylation, phosphorylation, ubiquitination, and the small RNA machinery). Studies based on epigenetics have evolved over the years especially in understanding the mechanisms at transcriptional and posttranscriptional levels in plants against various environmental stimuli. Epigenomic changes in plants through induced methylation of specific genes that lead to changes in their expression can help to overcome various stress conditions. Recent studies suggested that epigenomics has a significant potential for crop improvement in plants. By the induction and modulation of various cellular processes like DNA methylation, histone modification, and biogenesis of noncoding RNAs, the plant genome can be activated which can help in achieving a quicker response against various plant stresses. Epigenetic modifications in plants allow them to adjust under varied environmental stresses by modulating their phenotypic plasticity and at the same time ensure the quality and yield of crops. The plasticity of the epigenome helps to adapt the plants during pre- and postdevelopmental processes. The variation in DNA methylation in different organisms exhibits variable phenotypic responses. The epigenetic changes also occur sequentially in the genome. Various studies indicated that environmentally stimulated epimutations produce variable responses especially in differentially methylated regions (DMR) that play a major role in the management of stress conditions in plants. Besides, it has been observed that environmental stresses cause specific changes in the epigenome that are closely associated with phenotypic modifications. However, the relationship between epigenetic modifications and phenotypic plasticity is still debatable. In this review, we will be discussing the role of various factors that allow epigenetic changes to modulate phenotypic plasticity against various abiotic stress in plants.

Differences in Cellular Metabolism and Metabolic Regulation between Non‐diabetic and Diabetic Human Coronary Artery Endothelial Cells

ObjectiveTo determine the differences in cellular metabolism and metabolic regulation in non‐diabetic and diabetic human coronary artery endothelial cells (HCAEC and DM‐HCAEC) to further the understanding of the effects of metabolic syndrome on endothelial dysfunction in the setting of cardiovascular disease.MethodsBoth HCAEC (n = 5) and DM‐HCAEC (n = 5) were cultured to passage 6. The cells were then starved for 18 hours. After treatment, a Reactive Oxygen Species (ROS) Assay was performed, and cell lysates were made for proteomics analysis and western blotting.ResultsThe ROS assay demonstrated that the total ROS was significantly higher in DM‐HCAEC (p<0.0001) than in HCAEC. Proteomics analysis and western blotting showed that DM‐HCAECs had significantly altered cellular transport, RNA processing, protein biogenesis, and antioxidant pathways. Proteomics analysis demonstrated that there were about 1,500 proteins significantly upregulated or downregulated in DM‐HCAEC compared to HCAEC. Analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed a significant increase in the “vesicle‐mediated transport” pathway and significant decrease in the “mRNA metabolic process” pathway in DM‐HCAEC with false discovery rates (FDR) of 1.40 x 10‐58 and 4.90 x 10‐92, respectively. Western blot analysis showed a significant changes in phosphorylated AMPK/total AMPK, phosphorylated mTOR (serine 2481), total mTOR, and several enzymes responsible for the regulation or elimination of ROS (Figure 1). The increased levels of AMPK activation and dysregulation of mTOR may be due to the excessive ROS in DM‐HCAEC, which are consistent with the ROS assay findings and significant changes in expression of enzymes that control ROS levels. Further research is needed to determine the mechanisms of downstream effects.ConclusionsAMPK and mTOR are key regulators of energy homeostasis, cellular growth and metabolism, and are altered in diabetes in the setting of increased oxidative stress. These findings will guide further experiments to assess the effect of metabolic syndrome and diabetes on therapeutics targeted at improving the outcomes of both acute and chronic myocardial ischemia. This also underlines the need for further development of therapies or the use of current therapies to address the metabolic disturbances found in diabetes to improve clinical outcomes in cardiovascular disease.

A Pan-Cancer Analysis of the Oncogenic Role of WD Repeat Domain 74 in Multiple Tumors.

Although emerging patient-derived samples and cellular-based evidence support the relationship between WDR74 (WD Repeat Domain 74) and carcinogenesis in multiple cancers, no systematic pan-cancer analysis is available. Our preliminary research demonstrated that WDR74 is over-expressed in lung squamous cell carcinoma (LUSC) and related with worse survival. We thus investigated the potential oncogenic roles of WDR74 across 33 tumors based on the database of TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus). WDR74 is highly expressed in most cancers and correlated with poor prognosis in several cancers (all p < 0.05). Mutation analysis demonstrated that WDR74 is frequently mutated in promoter regions of lung cancer. Moreover, we found that CD8+ T-cells and the fibroblast infiltration level increased in WDR74 over-expressed cancer cells. The GO (Gene Ontology) enrichment analysis of the WDR74 pathway revealed its participation in cellular biogenesis of the RNA metabolism and its critical role in cancer initiation and progression through the tumor cell energy metabolism. Our first pan-cancer study inferred a relatively comprehensive understanding of the oncogenic roles of WDR74 across various cancers.

CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis.

The mammalian cleavage factor I (CFIm) has been implicated in alternative polyadenylation (APA) in a broad range of contexts, from cancers to learning deficits and parasite infections. To determine how the CFIm expression levels are translated into these diverse phenotypes, we carried out a multi-omics analysis of cell lines in which the CFIm25 (NUDT21) or CFIm68 (CPSF6) subunits were either repressed by siRNA-mediated knockdown or over-expressed from stably integrated constructs. We established that >800 genes undergo coherent APA in response to changes in CFIm levels, and they cluster in distinct functional classes related to protein metabolism. The activity of the ERK pathway traces the CFIm concentration, and explains some of the fluctuations in cell growth and metabolism that are observed upon CFIm perturbations. Furthermore, multiple transcripts encoding proteins from the miRNA pathway are targets of CFIm-dependent APA. This leads to an increased biogenesis and repressive activity of miRNAs at the same time as some 3′ UTRs become shorter and presumably less sensitive to miRNA-mediated repression. Our study provides a first systematic assessment of a core set of APA targets that respond coherently to changes in CFIm protein subunit levels (CFIm25/CFIm68). We describe the elicited signaling pathways downstream of CFIm, which improve our understanding of the key role of CFIm in integrating RNA processing with other cellular activities.

Synovial fluid proteome profile of surgical versus chemical induced osteoarthritis in rabbits.

BackgroundAnimal models are significant for understanding human osteoarthritis (OA). This study compared the synovial fluid proteomics changes in surgical and chemical induced OA models.MethodsThirty rabbits either had anterior cruciate ligament transection (ACLT) procedure or injected intra-articularly with monosodium iodoacetate (MIA, 8 mg) into the right knee. The joints were anatomically assessed, and the synovial fluid proteins analyzed using two-dimensional polyacrylamide gel electrophoresis (2DGE) and MALDI TOF/TOF mass spectrometry analysis at 4, 8 and 12 weeks. The proteins’ upregulation and downregulation were compared with control healthy knees.ResultsSeven proteins (histidine-rich glycoprotein, beta-actin-like protein 2 isoform X1, retinol-binding protein-4, alpha-1-antiproteinase, gelsolin isoform, serotransferrin, immunoglobulin kappa-b4 chain-C-region) were significantly expressed by the surgical induction. They characterized cellular process (27%), organization of cellular components or biogenesis (27%), localization (27%) and biological regulation (18%), which related to synovitis, increased cellularity, and subsequently cartilage damage. Three proteins (apolipoprotein I-IV precursor, serpin peptidase inhibitor and haptoglobin precursor) were significantly modified by the chemical induction. They characterized stimulus responses (23%), immune responses (15%), biological regulations (15%), metabolism (15%), organization of cellular components or biogenesis (8%), cellular process (8%), biological adhesions (8%) and localization (8%), which related to chondrocytes glycolysis/death, neovascularization, subchondral bone necrosis/collapse and inflammation.ConclusionsThe surgical induced OA model showed a wider range of protein changes, which were most upregulated at week 12. The biological process proteins expressions showed the chemical induced joints had slower OA progression compared to surgical induced joints. The chemical induced OA joints showed early inflammatory changes, which later decreased.

The Ragulator complex serves as a substrate-specific mTORC1 scaffold in regulating the nuclear translocation of transcription factor EB

The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is activated by intracellular nutritional sufficiency and extracellular growth signals. It has been reported that mTORC1 acts as a hub that integrates these inputs to orchestrate a number of cellular responses, including translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, little is known about specific control of mTORC1 signaling downstream of this complex. Here, we demonstrate that Ragulator, a heteropentameric protein complex required for mTORC1 activation in response to amino acids, is critical for inhibiting the nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacked Ragulator but retained total mTORC1 activity. In a nutrition-sufficient state, the nuclear translocation of TFEB was markedly enhanced in the clone despite total mTORC1 kinase activity. In addition, as a cellular phenotype, the number of lysosomes was increased by tenfold in the Ragulator-deficient clone compared with that of control cells. These findings indicate that mTORC1 essentially requires the Ragulator complex for regulating the subcellular distribution of TFEB. Our findings also suggest that other scaffold proteins may be associated with mTORC1 for the specific regulation of downstream signaling.

Pyrroloquinoline Quinone Disodium (PQQ2Na) Has an NLRP Inflammasome-Induced Caspase-1 Release Influence in UVB-Irradiated but Not ATP-Treated Human Keratinocytes but Has No Influence in Increasing Skin Cell Mitochondrial Biogenesis in Either Human Keratinocytes or Fibroblasts.

IntroductionPyrroloquinoline quinone is a bacterial-derived redox factor that has been shown to have numerous benefits in humans. Recently, a model for examining the ability of normal human epidermal keratinocytes (NHEKs) to demonstrate anti-inflammatory benefits via nod-like receptor protein (NLRP)-activated caspase-1 release was reported. The question of whether PQQ2Na might have anti-inflammatory benefits that function through NLRP-activated release of active caspase-1 has not been explored. In addition, it has been reported that PQQ2Na will induce mitochondrial biogenesis in humans when taken orally. Whether or not this effect occurs in skin cells is presently unknown.MethodsThe inflammation studies followed previously published methods that demonstrated both UVB and ATP were able to upregulate the NLRP-activated release of caspase-1 in NHEKs. In addition, NHEK and normal dermal human fibroblasts (NHDF) were treated with PQQ2Na to see if the molecule might stimulate mitochondrial biogenesis measured by increased expression of cyclooxygenase-1 (COX-1) and succinate dehydrogenase complex, subunit A (SDHA).ResultsAt non-cytotoxic concentrations between 5 µg/mL and 100 µg/mL in NHEKs and between 0.1 µg/mL and 5 µg/mL in fibroblasts, the PQQ2Na had no influence on cellular mitochondrial biogenesis. In ATP-activated NHEKs at concentrations of PQQ2Na between 0.05 µg/mL and 50 µg/mL, there was no influence of PQQ2Na on release of active caspase-1. In NHEKs irradiated with 60mJ/cm2 of UVB radiation as previously described and treated with 0.05 µg/mL to 50 µg/mL of PQQ2Na, the molecule showed a dose-dependent benefit at reducing the expression of active caspase-1 in the irradiated cells.DiscussionBenefits of PQQ2Na on various skin cell types which had not been investigated previously were addressed. Surprisingly, the PPQ2Na had no apparent influence on skin cell mitochondrial biogenesis. However, the molecule has a strong suppressing influence on UVB-induced active caspase-1 release in UVB-irradiated NHEKs.

Spinal Muscular Atrophy Patient iPSC-Derived Motor Neurons Display Altered Proteomes at Early Stages of Differentiation.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder characterized by loss of motor neurons (MN) in the spinal cord leading to progressive muscle atrophy and weakness. SMA is caused by mutations in the survival motor neuron 1 (SMN1) gene, resulting in reduced levels of survival motor neuron (SMN) protein. The mechanisms that link SMN deficiency to selective motor neuron dysfunction in SMA remain largely unknown. We present here, for the first time, a comprehensive quantitative TMT-10plex proteomics analysis that covers the development of induced pluripotent stem cell-derived MNs from both healthy individuals and SMA patients. We show that the proteomes of SMA samples segregate from controls already at early stages of neuronal differentiation. The altered proteomic signature in SMA MNs is associated with mRNA splicing, ribonucleoprotein biogenesis, organelle organization, cellular biogenesis, and metabolic processes. We highlight several known SMN-binding partners and evaluate their expression changes during MN differentiation. In addition, we compared our study to human and mouse in vivo proteomic studies revealing distinct and similar signatures. Altogether, our work provides a comprehensive resource of molecular events during early stages of MN differentiation, containing potentially therapeutically interesting protein expression profiles for SMA.

Autophagy and lysosomal defects in cells expressing disease‐associated tau

AbstractBackgroundIntracellular accumulation of insoluble tau is an important hallmark of Alzheimer’s disease (AD) and related tauopathies. We have previously identified in human tauopathy brain a truncated tau species (Tau35), comprising the C‐terminal half with four microtubule‐binding repeats. Minimal Tau35 expression in transgenic mice results in a progressive tauopathy phenotype including tau phosphorylation and aggregation, cognitive and behavioural abnormalities and impaired protein clearance. The autophagy‐lysosomal pathway (ALP) plays a crucial role in the clearance of protein aggregates and defects in ALP are associated with the pathogenesis of AD. We sought to explore the effect of Tau35 expression on the ALP and whether autophagy is disrupted due to lysosomal dysfunction.MethodChinese hamster ovary (CHO) cells stably expressing Tau35 (CHO‐Tau35) or full‐length human 2N4R tau (CHO‐FL) were generated. Primary cortical neurons from Tau35 transgenic and wild‐type mice were cultured for 14 days in vitro(DIV) and brain homogenates were prepared from mice aged 4 and 12 months. The effect of Tau35 on the ALP was examined using immunofluorescence and western blots.ResultNuclear translocation of transcription factor EB (TFEB), a key mediator of lysosomal biogenesis, was significantly reduced in both CHO‐FL and CHO‐Tau35 cells. However, only CHO‐Tau35 cells exhibited disrupted mammalian target of rapamycin complex 1 (mTORC1) activity and autophagic flux. Expression of ALP markers, including LC3‐I/II, LAMP1, LAMP2 and cathepsin D, were also reduced in CHO‐Tau35 cells.ConclusionOur findings suggest that N‐terminally cleaved tau damages both lysosomal clearance of cellular proteins and lysosomal biogenesis. The Tau35‐expressing cultured neurons will provide a useful tool to explore molecular mechanisms underlying tau‐induced lysosomal dysfunction, which may lead to the identification of novel therapeutic targets for dementia.

Defects in the autophagy lysosomal pathway in a cell model of disease‐associated tau

Intracellular accumulation of insoluble tau is an important hallmark of Alzheimer's disease (AD) and related tauopathies. We have previously identified in human tauopathy brain a truncated tau species (Tau35), comprising the C-terminal half with four microtubule-binding repeats. Minimal Tau35 expression in transgenic mice results in a progressive tauopathy phenotype including tau phosphorylation and aggregation, cognitive and behavioural abnormalities and impaired protein clearance. The autophagy-lysosomal pathway (ALP) plays a crucial role in the clearance of protein aggregates and defects in ALP are associated with the pathogenesis of AD. We sought to explore the effect of Tau35 expression on the ALP and whether autophagy is disrupted due to lysosomal dysfunction. Chinese hamster ovary (CHO) cells stably expressing Tau35 (CHO-Tau35) or full-length human 2N4R tau (CHO-FL) were generated. Primary cortical neurons from Tau35 transgenic and wild-type mice were cultured for 14 days in vitro(DIV) and brain homogenates were prepared from mice aged 4 and 12 months. The effect of Tau35 on the ALP was examined using immunofluorescence and western blots. Nuclear translocation of transcription factor EB (TFEB), a key mediator of lysosomal biogenesis, was significantly reduced in both CHO-FL and CHO-Tau35 cells. However, only CHO-Tau35 cells exhibited disrupted mammalian target of rapamycin complex 1 (mTORC1) activity and autophagic flux. Expression of ALP markers, including LC3-I/II, LAMP1, LAMP2 and cathepsin D, were also reduced in CHO-Tau35 cells and in post-symptomatic Tau35 transgenic mice. Our findings suggest that N-terminally cleaved tau damages both lysosomal clearance of cellular proteins and lysosomal biogenesis. The Tau35-expressing cultured neurons will provide a useful tool to explore molecular mechanisms underlying tau-induced lysosomal dysfunction, which may lead to the identification of novel therapeutic targets for dementia.

The orf virus (ORFV) protein OV20.0 interacts with the microprocessor complex subunit DGCR8 to regulate miRNA biogenesis and ORFV infection.

Cellular double-stranded RNA-binding proteins (DRBPs) play important roles in the regulation of innate immune responses and microRNA (miRNA) biogenesis. The current study aimed to understand whether OV20.0, a DRBP of orf virus (ORFV), is involved in cellular RNA biogenesis via association with host DRBPs. We found that OV20.0 interacts with DiGeorge syndrome critical region 8 (DGCR8), a subunit of the miRNA processor complex, and binds to primary- and precursor-miRNA. Additionally, OV20.0 regulates DGCR8 expression in multiple ways, including through interaction with the DGCR8 protein and binding to DGCR8 mRNA. Lastly, our data show that DGCR8 plays an antiviral role against ORFV infection, whereas it is beneficial for influenza virus propagation, indicating that the underlying mechanisms could be diverse among different viruses.

Chronic Activation of AMPK Induces Mitochondrial Biogenesis through Differential Phosphorylation and Abundance of Mitochondrial Proteins in Dictyostelium discoideum.

Mitochondrial biogenesis is a highly controlled process that depends on diverse signalling pathways responding to cellular and environmental signals. AMP-activated protein kinase (AMPK) is a critical metabolic enzyme that acts at a central control point in cellular energy homeostasis. Numerous studies have revealed the crucial roles of AMPK in the regulation of mitochondrial biogenesis; however, molecular mechanisms underlying this process are still largely unknown. Previously, we have shown that, in cellular slime mould Dictyostelium discoideum, the overexpression of the catalytic α subunit of AMPK led to enhanced mitochondrial biogenesis, which was accompanied by reduced cell growth and aberrant development. Here, we applied mass spectrometry-based proteomics of Dictyostelium mitochondria to determine the impact of chronically active AMPKα on the phosphorylation state and abundance of mitochondrial proteins and to identify potential protein targets leading to the biogenesis of mitochondria. Our results demonstrate that enhanced mitochondrial biogenesis is associated with variations in the phosphorylation levels and abundance of proteins related to energy metabolism, protein synthesis, transport, inner membrane biogenesis, and cellular signalling. The observed changes are accompanied by elevated mitochondrial respiratory activity in the AMPK overexpression strain. Our work is the first study reporting on the global phosphoproteome profiling of D. discoideum mitochondria and its changes as a response to constitutively active AMPK. We also propose an interplay between the AMPK and mTORC1 signalling pathways in controlling the cellular growth and biogenesis of mitochondria in Dictyostelium as a model organism.