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  This is an Open Access Journal Open Access journal
ISSN (Online) 2371-1671
Published by Canadian Science Publishing Homepage  [4 journals]
  • Concordance of MERFISH spatial transcriptomics with bulk and single-cell
           RNA sequencing

    • Authors: Liu, J; Tran, V, Vemuri, V. N. P, Byrne, A, Borja, M, Kim, Y. J, Agarwal, S, Wang, R, Awayan, K, Murti, A, Taychameekiatchai, A, Wang, B, Emanuel, G, He, J, Haliburton, J, Oliveira Pisco, A, Neff, N. F.
      Pages: e202201701 - e202201701
      Abstract: Spatial transcriptomics extends single-cell RNA sequencing (scRNA-seq) by providing spatial context for cell type identification and analysis. Imaging-based spatial technologies such as multiplexed error-robust fluorescence in situ hybridization (MERFISH) can achieve single-cell resolution, directly mapping single-cell identities to spatial positions. MERFISH produces a different data type than scRNA-seq, and a technical comparison between the two modalities is necessary to ascertain how to best integrate them. We performed MERFISH on the mouse liver and kidney and compared the resulting bulk and single-cell RNA statistics with those from the Tabula Muris Senis cell atlas and from two Visium datasets. MERFISH quantitatively reproduced the bulk RNA-seq and scRNA-seq results with improvements in overall dropout rates and sensitivity. Finally, we found that MERFISH independently resolved distinct cell types and spatial structure in both the liver and kidney. Computational integration with the Tabula Muris Senis atlas did not enhance these results. We conclude that MERFISH provides a quantitatively comparable method for single-cell gene expression and can identify cell types without the need for computational integration with scRNA-seq atlases.
      PubDate: 2022-12-16T09:11:36-08:00
      DOI: 10.26508/lsa.202201701
      Issue No: Vol. 6, No. 1 (2022)
  • ZBTB18 inhibits SREBP-dependent lipid synthesis by halting CTBPs and LSD1
           activity in glioblastoma

    • Authors: Ferrarese, R; Izzo, A, Andrieux, G, Lagies, S, Bartmuss, J. P, Masilamani, A. P, Wasilenko, A, Osti, D, Faletti, S, Schulzki, R, Yuan, S, Kling, E, Ribecco, V, Heiland, D. H, Tholen, S, Prinz, M, Pelicci, G, Kammerer, B, Boerries, M, Carro, M. S.
      Pages: e202201400 - e202201400
      Abstract: Enhanced fatty acid synthesis is a hallmark of tumors, including glioblastoma. SREBF1/2 regulate the expression of enzymes involved in fatty acid and cholesterol synthesis. Yet, little is known about the precise mechanism regulating SREBP gene expression in glioblastoma. Here, we show that a novel interaction between the co-activator/co-repressor CTBP and the tumor suppressor ZBTB18 regulates the expression of SREBP genes. In line with our findings, metabolic assays and glucose tracing analysis confirm the reduction in several phospholipid species upon ZBTB18 expression. Our study identifies CTBP1/2 and LSD1 as co-activators of SREBP genes and indicates that the functional activity of the CTBP-LSD1 complex is altered by ZBTB18. ZBTB18 binding to the SREBP gene promoters is associated with reduced LSD1 demethylase activity of H3K4me2 and H3K9me2 marks. Concomitantly, the interaction between LSD1, CTBP, and ZNF217 is increased, suggesting that ZBTB18 promotes LSD1 scaffolding function. Our results outline a new epigenetic mechanism enrolled by ZBTB18 and its co-factors to regulate fatty acid synthesis that could be targeted to treat glioblastoma patients.
      PubDate: 2022-11-22T08:48:26-08:00
      DOI: 10.26508/lsa.202201400
      Issue No: Vol. 6, No. 1 (2022)
  • Hepatic DKK1-driven steatosis is CD36 dependent

    • Authors: Yang, Z; Huang, X, Zhang, J, You, K, Xiong, Y, Fang, J, Getachew, A, Cheng, Z, Yu, X, Wang, Y, Wu, F, Wang, N, Feng, S, Lin, X, Yang, F, Chen, Y, Wei, H, Li, Y.-x.
      Pages: e202201665 - e202201665
      Abstract: Nonalcoholic fatty liver disease (NAFLD) is prevalent worldwide; about 25% of NAFLD silently progress into steatohepatitis, in which some of them may develop into fibrosis, cirrhosis and liver failure. However, few drugs are available for NAFLD, partly because of an incomplete understanding of its pathogenic mechanisms. Here, using in vivo and in vitro gain- and loss-of-function approaches, we identified up-regulated DKK1 plays a pivotal role in high-fat diet–induced NAFLD and its progression. Mechanistic analysis reveals that DKK1 enhances the capacity of hepatocytes to uptake fatty acids through the ERK-PPAR-CD36 axis. Moreover, DKK1 increased insulin resistance by activating the JNK signaling, which in turn exacerbates disorders of hepatic lipid metabolism. Our finding suggests that DKK1 may be a potential therapeutic and diagnosis candidate for NAFLD and metabolic disorder progression.
      PubDate: 2022-11-21T08:51:15-08:00
      DOI: 10.26508/lsa.202201665
      Issue No: Vol. 6, No. 1 (2022)
  • GPER1 links estrogens to centrosome amplification and chromosomal
           instability in human colon cells

    • Authors: Bühler, M; Fahrländer, J, Sauter, A, Becker, M, Wistorf, E, Steinfath, M, Stolz, A.
      Pages: e202201499 - e202201499
      Abstract: The role of the alternate G protein–coupled estrogen receptor 1 (GPER1) in colorectal cancer (CRC) development and progression is unclear, not least because of conflicting clinical and experimental evidence for pro- and anti-tumorigenic activities. Here, we show that low concentrations of the estrogenic GPER1 ligands, 17β-estradiol, bisphenol A, and diethylstilbestrol cause the generation of lagging chromosomes in normal colon and CRC cell lines, which manifest in whole chromosomal instability and aneuploidy. Mechanistically, (xeno)estrogens triggered centrosome amplification by inducing centriole overduplication that leads to transient multipolar mitotic spindles, chromosome alignment defects, and mitotic laggards. Remarkably, we could demonstrate a significant role of estrogen-activated GPER1 in centrosome amplification and increased karyotype variability. Indeed, both gene-specific knockdown and inhibition of GPER1 effectively restored normal centrosome numbers and karyotype stability in cells exposed to 17β-estradiol, bisphenol A, or diethylstilbestrol. Thus, our results reveal a novel link between estrogen-activated GPER1 and the induction of key CRC-prone lesions, supporting a pivotal role of the alternate estrogen receptor in colon neoplastic transformation and tumor progression.
      PubDate: 2022-11-16T08:50:17-08:00
      DOI: 10.26508/lsa.202201499
      Issue No: Vol. 6, No. 1 (2022)
  • Discovery of RUF6 ncRNA-interacting proteins involved in P. falciparum
           immune evasion

    • Authors: Diffendall, G. M; Barcons-Simon, A, Baumgarten, S, Dingli, F, Loew, D, Scherf, A.
      Pages: e202201577 - e202201577
      Abstract: Non-coding RNAs (ncRNAs) are emerging regulators of immune evasion and transmission of Plasmodium falciparum. RUF6 is an ncRNA gene family that is transcribed by RNA polymerase III but actively regulates the Pol II–transcribed var virulence gene family. Understanding how RUF6 ncRNA connects to downstream effectors is lacking. We developed an RNA-directed proteomic discovery (ChIRP-MS) protocol to identify in vivo RUF6 ncRNA–protein interactions. The RUF6 ncRNA interactome was purified with biotinylated antisense oligonucleotides. Quantitative label-free mass spectrometry identified several unique proteins linked to gene transcription including RNA Pol II subunits, nucleosome assembly proteins, and a homologue of DEAD box helicase 5 (DDX5). Affinity purification of Pf-DDX5 identified proteins originally found by our RUF6-ChIRP protocol, validating the technique’s robustness for identifying ncRNA interactomes in P. falciparum. Inducible displacement of nuclear Pf-DDX5 resulted in significant down-regulation of the active var gene. Our work identifies a RUF6 ncRNA–protein complex that interacts with RNA Pol II to sustain the var gene expression, including a helicase that may resolve G-quadruplex secondary structures in var genes to facilitate transcriptional activation and progression.
      PubDate: 2022-11-15T14:05:37-08:00
      DOI: 10.26508/lsa.202201577
      Issue No: Vol. 6, No. 1 (2022)
  • Genome-wide profiling of histone modifications in Plasmodium falciparum
           using CUT&RUN

    • Authors: Morillo, R. C; Harris, C. T, Kennedy, K, Henning, S. R, Kafsack, B. F.
      Pages: e202201778 - e202201778
      Abstract: We recently adapted a CUT&RUN protocol for genome-wide profiling of chromatin modifications in the human malaria parasite Plasmodium. Using the step-by-step protocol described below, we were able to generate high-quality profiles of multiple histone modifications using only a small fraction of the cells required for ChIP-seq. Using antibodies against two commonly profiled histone modifications, H3K4me3 and H3K9me3, we show here that CUT&RUN profiling is highly reproducible and closely recapitulates previously published ChIP-seq-based abundance profiles of histone marks. Finally, we show that CUT&RUN requires substantially lower sequencing coverage for accurate profiling compared with ChIP-seq.
      PubDate: 2022-11-15T12:55:42-08:00
      DOI: 10.26508/lsa.202201778
      Issue No: Vol. 6, No. 1 (2022)
  • Endoplasmic reticulum membranes are continuously required to maintain
           mitotic spindle size and forces

    • Authors: Araujo, M; Tavares, A, Vieira, D. V, Telley, I. A, Oliveira, R. A.
      Pages: e202201540 - e202201540
      Abstract: Membrane organelle function, localization, and proper partitioning upon cell division depend on interactions with the cytoskeleton. Whether membrane organelles also impact the function of cytoskeletal elements remains less clear. Here, we show that acute disruption of the ER around spindle poles affects mitotic spindle size and function in Drosophila syncytial embryos. Acute ER disruption was achieved through the inhibition of ER membrane fusion by the dominant-negative cytoplasmic domain of atlastin. We reveal that when centrosome-proximal ER membranes are disrupted, specifically at metaphase, mitotic spindles become smaller, despite no significant changes in microtubule dynamics. These smaller spindles are still able to mediate sister chromatid separation, yet with decreased velocity. Furthermore, by inducing mitotic exit, we found that nuclear separation and distribution are affected by ER disruption. Our results suggest that ER integrity around spindle poles is crucial for the maintenance of mitotic spindle shape and pulling forces. In addition, ER integrity also ensures nuclear spacing during syncytial divisions.
      PubDate: 2022-11-15T07:48:31-08:00
      DOI: 10.26508/lsa.202201540
      Issue No: Vol. 6, No. 1 (2022)
  • SMA-linked SMN mutants prevent phase separation properties and SMN
           interactions with FMRP family members

    • Authors: Binda, O; Juillard, F, Ducassou, J. N, Kleijwegt, C, Paris, G, Didillon, A, Baklouti, F, Corpet, A, Coute, Y, Cote, J, Lomonte, P.
      Pages: e202201429 - e202201429
      Abstract: Although recent advances in gene therapy provide hope for spinal muscular atrophy (SMA) patients, the pathology remains the leading genetic cause of infant mortality. SMA is a monogenic pathology that originates from the loss of the SMN1 gene in most cases or mutations in rare cases. Interestingly, several SMN1 mutations occur within the TUDOR methylarginine reader domain of SMN. We hypothesized that in SMN1 mutant cases, SMA may emerge from aberrant protein-protein interactions between SMN and key neuronal factors. Using a BioID proteomic approach, we have identified and validated a number of SMN-interacting proteins, including fragile X mental retardation protein (FMRP) family members (FMRFM). Importantly, SMA-linked SMNTUDOR mutant forms (SMNST) failed to interact with FMRFM. In agreement with the recent work, we define biochemically that SMN forms droplets in vitro and these droplets are stabilized by RNA, suggesting that SMN could be involved in the formation of membraneless organelles, such as Cajal nuclear bodies. Finally, we found that SMN and FMRP co-fractionate with polysomes, in an RNA-dependent manner, suggesting a potential role in localized translation in motor neurons.
      PubDate: 2022-11-14T05:57:14-08:00
      DOI: 10.26508/lsa.202201429
      Issue No: Vol. 6, No. 1 (2022)
  • Loss of autism-candidate CHD8 perturbs neural crest development and
           intestinal homeostatic balance

    • Authors: Hayot, G; Massonot, M, Keime, C, Faure, E, Golzio, C.
      Pages: e202201456 - e202201456
      Abstract: Individuals with mutations in CHD8 present with gastrointestinal complaints, yet the underlying mechanisms are understudied. Here, using a stable constitutive chd8 mutant zebrafish model, we found that the loss of chd8 leads to a reduced number of vagal neural crest cells (NCCs), enteric neural and glial progenitors, emigrating from the neural tube, and that their early migration capability was altered. At later stages, although the intestinal colonization by NCCs was complete, we found the decreased numbers of both serotonin-producing enterochromaffin cells and NCC-derived serotonergic neurons, suggesting an intestinal hyposerotonemia in the absence of chd8. Furthermore, transcriptomic analyses revealed an altered expression of key receptors and enzymes in serotonin and acetylcholine signaling pathways. The tissue examination of chd8 mutants revealed a thinner intestinal epithelium accompanied by an accumulation of neutrophils and the decreased numbers of goblet cells and eosinophils. Last, single-cell sequencing of whole intestines showed a global disruption of the immune balance with a perturbed expression of inflammatory interleukins and changes in immune cell clusters. Our findings propose a causal developmental link between chd8, NCC development, intestinal homeostasis, and autism-associated gastrointestinal complaints.
      PubDate: 2022-11-14T05:57:14-08:00
      DOI: 10.26508/lsa.202201456
      Issue No: Vol. 6, No. 1 (2022)
  • LACTB exerts tumor suppressor properties in epithelial ovarian cancer
           through regulation of Slug

    • Authors: Cutano, V; Ferreira Mendes, J. M, Escudeiro-Lopes, S, Machado, S, Vinaixa Forner, J, Gonzales-Morena, J. M, Prevorovsky, M, Zemlianski, V, Feng, Y, Kralova Viziova, P, Hartmanova, A, Malcekova, B, Jakoube, P, Iyer, S, Keckesova, Z.
      Pages: e202201510 - e202201510
      Abstract: Epithelial–mesenchymal transition (EMT) is a cellular mechanism used by cancer cells to acquire migratory and stemness properties. In this study, we show, through in vitro, in vivo, and 3D culture experiments, that the mitochondrial protein LACTB manifests tumor suppressor properties in ovarian cancer. We show that LACTB is significantly down-regulated in epithelial ovarian cancer cells and clinical tissues. Re-expression of LACTB negatively effects the growth of cancer cells but not of non-tumorigenic cells. Mechanistically, we show that LACTB leads to differentiation of ovarian cancer cells and loss of their stemness properties, which is achieved through the inhibition of the EMT program and the LACTB-dependent down-regulation of Snail2/Slug transcription factor. This study uncovers a novel role of LACTB in ovarian cancer and proposes new ways of counteracting the oncogenic EMT program in this model system.
      PubDate: 2022-11-14T05:57:14-08:00
      DOI: 10.26508/lsa.202201510
      Issue No: Vol. 6, No. 1 (2022)
  • Protein and RNA ADP-ribosylation detection is influenced by sample
           preparation and reagents used

    • Authors: Weixler, L; Ikenga, N. J, Voorneveld, J, Aydin, G, Bolte, T. M, Momoh, J, Bütepage, M, Golzmann, A, Lüscher, B, Filippov, D. V, Zaja, R, Feijs, K. L.
      Pages: e202201455 - e202201455
      Abstract: The modification of substrates with ADP-ribose (ADPr) is important in, for example, antiviral immunity and cancer. Recently, several reagents were developed to detect ADP-ribosylation; however, it is unknown whether they recognise ADPr, specific amino acid–ADPr linkages, or ADPr with the surrounding protein backbone. We first optimised methods to prepare extracts containing ADPr–proteins and observe that depending on the amino acid modified, the modification is heatlabile. We tested the reactivity of available reagents with diverse ADP-ribosylated protein and RNA substrates and observed that not all reagents are equally suited for all substrates. Next, we determined cross-reactivity with adenylylated RNA, AMPylated proteins, and metabolites, including NADH, which are detected by some reagents. Lastly, we analysed ADP-ribosylation using confocal microscopy, where depending on the fixation method, either mitochondrion, nucleus, or nucleolus is stained. This study allows future work dissecting the function of ADP-ribosylation in cells, both on protein and on RNA substrates, as we optimised sample preparation methods and have defined the reagents suitable for specific methods and substrates.
      PubDate: 2022-11-11T10:31:44-08:00
      DOI: 10.26508/lsa.202201455
      Issue No: Vol. 6, No. 1 (2022)
  • Lipid saturation induces degradation of squalene epoxidase for sterol
           homeostasis and cell survival

    • Authors: Huang, L.-J; Chen, R.-H.
      Pages: e202201612 - e202201612
      Abstract: A fluid membrane containing a mix of unsaturated and saturated lipids is essential for life. However, it is unclear how lipid saturation might affect lipid homeostasis, membrane-associated proteins, and membrane organelles. Here, we generate temperature-sensitive mutants of the sole fatty acid desaturase gene OLE1 in the budding yeast Saccharomyces cerevisiae. Using these mutants, we show that lipid saturation triggers the endoplasmic reticulum–associated degradation (ERAD) of squalene epoxidase Erg1, a rate-limiting enzyme in sterol biosynthesis, via the E3 ligase Doa10-Ubc7 complex. We identify the P469L mutation that abolishes the lipid saturation–induced ERAD of Erg1. Overexpressed WT or stable Erg1 mutants all mislocalize into foci in the ole1 mutant, whereas the stable Erg1 causes aberrant ER and severely compromises the growth of ole1, which are recapitulated by doa10 deletion. The toxicity of the stable Erg1 and doa10 deletion is due to the accumulation of lanosterol and misfolded proteins in ole1. Our study identifies Erg1 as a novel lipid saturation–regulated ERAD target, manifesting a close link between lipid homeostasis and proteostasis that maintains sterol homeostasis under the lipid saturation condition for cell survival.
      PubDate: 2022-11-11T10:31:44-08:00
      DOI: 10.26508/lsa.202201612
      Issue No: Vol. 6, No. 1 (2022)
  • C-to-U RNA deamination is the driving force accelerating SARS-CoV-2

    • Authors: Li, Y; Hou, F, Zhou, M, Yang, X, Yin, B, Jiang, W, Xu, H.
      Pages: e202201688 - e202201688
      Abstract: Understanding the molecular mechanism underlying the rampant mutation of SARS-CoV-2 would help us control the COVID-19 pandemic. The APOBEC-mediated C-to-U deamination is a major mutation type in the SARS-CoV-2 genome. However, it is unclear whether the novel mutation rate u is higher for C-to-U than for other mutation types, and what the detailed driving force is. By analyzing the time course SARS-CoV-2 global population data, we found that C-to-U has the highest novel mutation rate u among all mutation types and that this u is still increasing with time (du/dt > 0). Novel C-to-U events, rather than other mutation types, have a preference over particular genomic regions. A less local RNA structure is correlated with a high novel C-to-U mutation rate. A cascade model nicely explains the du/dt > 0 for C-to-U deamination. In SARS-CoV-2, the RNA structure serves as the molecular basis of the extremely high and continuously accelerating C-to-U deamination rate. This mechanism is the driving force of the mutation, adaptation, and evolution of SARS-CoV-2. Our findings help us understand the dynamic evolution of the virus mutation rate.
      PubDate: 2022-11-08T07:59:43-08:00
      DOI: 10.26508/lsa.202201688
      Issue No: Vol. 6, No. 1 (2022)
  • Lack of peroxisomal catalase affects heat shock response in Caenorhabditis

    • Authors: Musa, M; Dionisio, P. A, Casqueiro, R, Milosevic, I, Raimundo, N, Krisko, A.
      Pages: e202201737 - e202201737
      Abstract: Exact mechanisms of heat shock–induced lifespan extension, although documented across species, are still not well understood. Here, we show that fully functional peroxisomes, specifically peroxisomal catalase, are needed for the activation of canonical heat shock response and heat-induced hormesis in Caenorhabditis elegans. Although during heat shock, the HSP-70 chaperone is strongly up-regulated in the WT and in the absence of peroxisomal catalase (ctl-2(ua90)II), the small heat shock proteins display modestly increased expression in the mutant. Nuclear foci formation of HSF-1 is reduced in the ctl-2(ua90)II mutant. In addition, heat-induced lifespan extension, observed in the WT, is absent in the ctl-2(ua90)II strain. Activation of the antioxidant response and pentose phosphate pathway are the most prominent changes observed during heat shock in the WT worm but not in the ctl-2(ua90)II mutant. Involvement of peroxisomes in the cell-wide cellular response to transient heat shock reported here gives new insight into the role of organelle communication in the organism’s stress response.
      PubDate: 2022-11-08T07:59:43-08:00
      DOI: 10.26508/lsa.202201737
      Issue No: Vol. 6, No. 1 (2022)
  • The potential of a universal influenza virus-like particle vaccine
           expressing a chimeric cytokine

    • Authors: Nerome, K; Imagawa, T, Sugita, S, Arasaki, Y, Maegawa, K, Kawasaki, K, Tanaka, T, Watanabe, S, Nishimura, H, Suzuki, T, Kuroda, K, Kosugi, I, Kajiura, Z.
      Pages: e202201548 - e202201548
      Abstract: The efficacy of the current influenza vaccines is frequently reduced because of antigenic drift, a trade-off of developing improved vaccines with broad cross-protective activity against influenza A viruses. In this study, we have successfully constructed a chimeric cytokine (CC) comprising the M2 protein, influenza A neuraminidase stalk, and interleukin-12. We produced virus-like particles (VLPs) containing CC and influenza hemagglutinin (HA) proteins using a baculovirus system in Eri silkworm pupae. The protective efficacy of the CCHA-VLP vaccine was evaluated in mice. The CCFkH5HA-VLP vaccine increased the survival rates of BALB/c mice, infected with a lethal dose of PRH1 and HKH5 viruses, to 80% and 100%, respectively. The results suggested that CCHA-VLP successfully induced potent cross-reactive protective immunity against infection with homologous and heterologous subtypes of the influenza A virus. This is the first study to design a CC-containing HA-VLP vaccine and validate its protective efficacy.
      PubDate: 2022-11-07T06:07:39-08:00
      DOI: 10.26508/lsa.202201548
      Issue No: Vol. 6, No. 1 (2022)
  • Myeloma immunoglobulin rearrangement and translocation detection through
           targeted capture sequencing

    • Authors: Chow, S; Kis, O, Mulder, D. T, Danesh, A, Bruce, J, Wang, T. T, Reece, D, Bhalis, N, Neri, P, Sabatini, P. J, Keats, J, Trudel, S, Pugh, T. J.
      Pages: e202201543 - e202201543
      Abstract: Multiple myeloma is a plasma cell neoplasm characterized by clonal immunoglobulin V(D)J signatures and oncogenic immunoglobulin gene translocations. Additional subclonal genomic changes are acquired with myeloma progression and therapeutic selection. PCR-based methods to detect V(D)J rearrangements can have biases introduced by highly multiplexed reactions and primers undermined by somatic hypermutation, and are not readily extended to include mutation detection. Here, we report a hybrid-capture approach (CapIG-seq) targeting the 3' and 5' ends of the V and J segments of all immunoglobulin loci that enable the efficient detection of V(D)J rearrangements. We also included baits for oncogenic translocations and mutation detection. We demonstrate complete concordance with matched whole-genome sequencing and/or PCR clonotyping of 24 cell lines and report the clonal sequences for 41 uncharacterized cell lines. We also demonstrate the application to patient specimens, including 29 bone marrow and 39 cell-free DNA samples. CapIG-seq shows concordance between bone marrow and cfDNA blood samples (both contemporaneous and follow-up) with regard to the somatic variant, V(D)J, and translocation detection. CapIG-seq is a novel, efficient approach to examining genomic alterations in myeloma.
      PubDate: 2022-11-03T13:15:20-07:00
      DOI: 10.26508/lsa.202201543
      Issue No: Vol. 6, No. 1 (2022)
  • Tanshinone functions as a coenzyme that confers gain of function of NQO1
           to suppress ferroptosis

    • Authors: Wang, T.-X; Duan, K.-L, Huang, Z.-X, Xue, Z.-A, Liang, J.-Y, Dang, Y, Zhang, A, Xiong, Y, Ding, C, Guan, K.-L, Yuan, H.-X.
      Pages: e202201667 - e202201667
      Abstract: Ferroptosis is triggered by the breakdown of cellular iron-dependent redox homeostasis and the abnormal accumulation of lipid ROS. Cells have evolved defense mechanisms to prevent lipid ROS accumulation and ferroptosis. Using a library of more than 4,000 bioactive compounds, we show that tanshinone from Salvia miltiorrhiza (Danshen) has very potent inhibitory activity against ferroptosis. Mechanistically, we found that tanshinone functions as a coenzyme for NAD(P)H:quinone oxidoreductase 1 (NQO1), which detoxifies lipid peroxyl radicals and inhibits ferroptosis both in vitro and in vivo. Although NQO1 is recognized as an oxidative stress response gene, it does not appear to have a direct role in ferroptosis inhibition in the absence of tanshinone. Here, we demonstrate a gain of function of NQO1 induced by tanshinone, which is a novel mechanism for ferroptosis inhibition. Using mouse models of acute liver injury and ischemia/reperfusion heart injury, we observed that tanshinone displays protective effects in both the liver and the heart in a manner dependent on NQO1. Our results link the clinical use of tanshinone to its activity in ferroptosis inhibition.
      PubDate: 2022-11-01T07:20:57-07:00
      DOI: 10.26508/lsa.202201667
      Issue No: Vol. 6, No. 1 (2022)
  • In vivo probing of SECIS-dependent selenocysteine translation in Archaea

    • Authors: Peiter, N; Rother, M.
      Pages: e202201676 - e202201676
      Abstract: Cotranslational insertion of selenocysteine (Sec) proceeds by recoding UGA to a sense codon. This recoding is governed by the Sec insertion sequence (SECIS) element, an RNA structure on the mRNA, but size, location, structure determinants, and mechanism differ for Bacteria, Eukarya, and Archaea. For Archaea, the structure–function relation of the SECIS is poorly understood, as only rather laborious experimental approaches are established. Furthermore, these methods do not allow for quantitative probing of Sec insertion. In order to overcome these limitations, we engineered bacterial β-lactamase into an archaeal selenoprotein, thereby establishing a reporter system, which correlates enzyme activity to Sec insertion. Using this system, in vivo Sec insertion depending on the availability of selenium and the presence of a SECIS element was assessed in Methanococcus maripaludis. Furthermore, a minimal SECIS element required for Sec insertion in M. maripaludis was defined and a conserved structural motif shown to be essential for function. Besides developing a convenient tool for selenium research, converting a bacterial enzyme into an archaeal selenoprotein provides proof of concept that novel selenoproteins can be engineered in Archaea.
      PubDate: 2022-10-31T06:28:53-07:00
      DOI: 10.26508/lsa.202201676
      Issue No: Vol. 6, No. 1 (2022)
  • CRISPRi screening reveals regulators of tau pathology shared between
           exosomal and vesicle-free tau

    • Authors: Polanco, J. C; Akimov, Y, Fernandes, A, Briner, A, Hand, G. R, van Roijen, M, Balistreri, G, Götz, J.
      Pages: e202201689 - e202201689
      Abstract: The aggregation of the microtubule-associated protein tau is a defining feature of Alzheimer’s disease and other tauopathies. Tau pathology is believed to be driven by free tau aggregates and tau carried within exosome-like extracellular vesicles, both of which propagate trans-synaptically and induce tau pathology in recipient neurons by a corrupting process of seeding. Here, we performed a genome-wide CRISPRi screen in tau biosensor cells and identified cellular regulators shared by both mechanisms of tau seeding. We identified ANKLE2, BANF1, NUSAP1, EIF1AD, and VPS18 as the top validated regulators that restrict tau aggregation initiated by both exosomal and vesicle-free tau seeds. None of our validated hits affected the uptake of either form of tau seeds, supporting the notion that they operate through a cell-autonomous mechanism downstream of the seed uptake. Lastly, validation studies with human brain tissue also revealed that several of the identified protein hits are down-regulated in the brains of Alzheimer’s patients, suggesting that their decreased activity may be required for the emergence or progression of tau pathology in the human brain.
      PubDate: 2022-10-31T06:28:53-07:00
      DOI: 10.26508/lsa.202201689
      Issue No: Vol. 6, No. 1 (2022)
  • Miro GTPase domains regulate the assembly of the mitochondrial
           motor-adaptor complex

    • Authors: Davis, K; Basu, H, Izquierdo-Villalba, I, Shurberg, E, Schwarz, T. L.
      Pages: e202201406 - e202201406
      Abstract: Mitochondrial transport relies on a motor–adaptor complex containing Miro1, a mitochondrial outer membrane protein with two GTPase domains, and TRAK1/2, kinesin-1, and dynein. Using a peroxisome-directed Miro1, we quantified the ability of GTPase mutations to influence the peroxisomal recruitment of complex components. Miro1 whose N-GTPase is locked in the GDP state does not recruit TRAK1/2, kinesin, or P135 to peroxisomes, whereas the GTP state does. Similarly, the expression of the MiroGAP VopE dislodges TRAK1 from mitochondria. Miro1 C-GTPase mutations have little influence on complex recruitment. Although Miro2 is thought to support mitochondrial motility, peroxisome-directed Miro2 did not recruit the other complex components regardless of the state of its GTPase domains. Neurons expressing peroxisomal Miro1 with the GTP-state form of the N-GTPase had markedly increased peroxisomal transport to growth cones, whereas the GDP-state caused their retention in the soma. Thus, the N-GTPase domain of Miro1 is critical for regulating Miro1’s interaction with the other components of the motor–adaptor complex and thereby for regulating mitochondrial motility.
      PubDate: 2022-10-27T14:57:51-07:00
      DOI: 10.26508/lsa.202201406
      Issue No: Vol. 6, No. 1 (2022)
  • circEXOC5 promotes acute lung injury through the PTBP1/Skp2/Runx2 axis to
           activate autophagy

    • Authors: Gao, P; Wu, B, Ding, Y, Yin, B, Gu, H.
      Pages: e202201468 - e202201468
      Abstract: To understand the pathogenesis of acute lung injury (ALI), we focused on circEXOC5, a significantly up-regulated circular RNA in ALI. Using the in vivo cecal ligation and puncture (CLP)–induced ALI mouse model and in vitro LPS-challenged mouse pulmonary microvascular endothelial cell (MPVEC) model, we examined the impacts of knockdown circEXOC5 on lung injury, inflammation, and autophagy. The regulation between circEXOC5, polypyrimidine tract-binding protein 1 (PTBP1), S-phase kinase-associated protein 2 (Skp2), and Runt-related transcription factor 2 (Runx2) was investigated by combining RNA immunoprecipitation, qRT–PCR, mRNA stability, and ubiquitination assays. The significance of PTBP1 in circEXOC5-induced ALI phenotypes was examined both in vitro and in vivo. circEXOC5 was up-regulated and associated with increased inflammation and activated autophagy in cecal ligation and puncture–induced ALI lung tissues and LPS-challenged MPVECs. Through the interaction with PTBP1, circEXOC5 accelerated Skp2 mRNA decay, an E3 ubiquitin ligase for Runx2, and therefore increased Runx2 expression. Functionally, overexpressing PTBP1 reversed shcircEXOC5-inhibited ALI, inflammation, or autophagy. The signaling cascade circEXOC5/PTBP1/Skp2/Runx2, by essentially regulating inflammation and autophagy in MPVECs, aggravates sepsis-induced ALI.
      PubDate: 2022-10-27T14:57:51-07:00
      DOI: 10.26508/lsa.202201468
      Issue No: Vol. 6, No. 1 (2022)
  • Palmitate impairs circadian transcriptomics in muscle cells through
           histone modification of enhancers

    • Authors: Pillon, N. J; Sardon Puig, L, Altıntas, A, Kamble, P. G, Casani-Galdon, S, Gabriel, B. M, Barres, R, Conesa, A, Chibalin, A. V, Näslund, E, Krook, A, Zierath, J. R.
      Pages: e202201598 - e202201598
      Abstract: Obesity and elevated circulating lipids may impair metabolism by disrupting the molecular circadian clock. We tested the hypothesis that lipid overload may interact with the circadian clock and alter the rhythmicity of gene expression through epigenomic mechanisms in skeletal muscle. Palmitate reprogrammed the circadian transcriptome in myotubes without altering the rhythmic mRNA expression of core clock genes. Genes with enhanced cycling in response to palmitate were associated with post-translational modification of histones. The cycling of histone 3 lysine 27 acetylation (H3K27ac), a marker of active gene enhancers, was modified by palmitate treatment. Chromatin immunoprecipitation and sequencing confirmed that palmitate exposure altered the cycling of DNA regions associated with H3K27ac. The overlap between mRNA and DNA regions associated with H3K27ac and the pharmacological inhibition of histone acetyltransferases revealed novel cycling genes associated with lipid exposure of primary human myotubes. Palmitate exposure disrupts transcriptomic rhythmicity and modifies enhancers through changes in histone H3K27 acetylation in a circadian manner. Thus, histone acetylation is responsive to lipid overload and may redirect the circadian chromatin landscape, leading to the reprogramming of circadian genes and pathways involved in lipid biosynthesis in skeletal muscle.
      PubDate: 2022-10-27T14:57:51-07:00
      DOI: 10.26508/lsa.202201598
      Issue No: Vol. 6, No. 1 (2022)
  • Actin-binding domain of Rng2 sparsely bound on F-actin strongly inhibits
           actin movement on myosin II

    • Authors: Hayakawa, Y; Takaine, M, Ngo, K. X, Imai, T, Yamada, M. D, Behjat, A. B, Umeda, K, Hirose, K, Yurtsever, A, Kodera, N, Tokuraku, K, Numata, O, Fukuma, T, Ando, T, Nakano, K, Uyeda, T. Q.
      Pages: e202201469 - e202201469
      Abstract: We report a case in which sub-stoichiometric binding of an actin-binding protein has profound structural and functional consequences, providing an insight into the fundamental properties of actin regulation. Rng2 is an IQGAP contained in contractile rings in the fission yeast Schizosaccharomyces pombe. Here, we used high-speed atomic force microscopy and electron microscopy and found that sub-stoichiometric binding of the calponin-homology actin-binding domain of Rng2 (Rng2CHD) induces global structural changes in skeletal muscle actin filaments, including shortening of the filament helical pitch. Sub-stoichiometric binding of Rng2CHD also reduced the affinity between actin filaments and muscle myosin II carrying ADP and strongly inhibited the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II–coated surfaces, Rng2CHD stopped the actin movements at a binding ratio of 11%. Rng2CHD also inhibited actin movements on myosin II of the amoeba Dictyostelium, but in this case, by detaching actin filaments from myosin II–coated surfaces. Thus, sparsely bound Rng2CHD induces apparently cooperative structural changes in actin filaments and inhibits force generation by actomyosin II.
      PubDate: 2022-10-26T08:01:30-07:00
      DOI: 10.26508/lsa.202201469
      Issue No: Vol. 6, No. 1 (2022)
  • Profiling subcellular localization of nuclear-encoded mitochondrial gene
           products in zebrafish

    • Authors: Uszczynska-Ratajczak, B; Sugunan, S, Kwiatkowska, M, Migdal, M, Carbonell-Sala, S, Sokol, A, Winata, C. L, Chacinska, A.
      Pages: e202201514 - e202201514
      Abstract: Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a–/– mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to use localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.
      PubDate: 2022-10-25T05:59:07-07:00
      DOI: 10.26508/lsa.202201514
      Issue No: Vol. 6, No. 1 (2022)
  • A high-content endogenous GLUT4 trafficking assay reveals new aspects of
           adipocyte biology

    • Authors: Diaz-Vegas, A; Norris, D. M, Jall-Rogg, S, Cooke, K. C, Conway, O. J, Shun-Shion, A. S, Duan, X, Potter, M, van Gerwen, J, Baird, H. J, Humphrey, S. J, James, D. E, Fazakerley, D. J, Burchfield, J. G.
      Pages: e202201585 - e202201585
      Abstract: Insulin-induced GLUT4 translocation to the plasma membrane in muscle and adipocytes is crucial for whole-body glucose homeostasis. Currently, GLUT4 trafficking assays rely on overexpression of tagged GLUT4. Here we describe a high-content imaging platform for studying endogenous GLUT4 translocation in intact adipocytes. This method enables high fidelity analysis of GLUT4 responses to specific perturbations, multiplexing of other trafficking proteins and other features including lipid droplet morphology. Using this multiplexed approach we showed that Vps45 and Rab14 are selective regulators of GLUT4, but Trarg1, Stx6, Stx16, Tbc1d4 and Rab10 knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some overlap upon insulin-stimulation. In addition, we identified Kif13A, a Rab10 binding molecular motor, as a novel regulator of GLUT4 traffic. Finally, comparison of endogenous to overexpressed GLUT4 highlights that the endogenous GLUT4 methodology has an enhanced sensitivity to genetic perturbations and emphasises the advantage of studying endogenous protein trafficking for drug discovery and genetic analysis of insulin action in relevant cell types.
      PubDate: 2022-10-25T05:59:07-07:00
      DOI: 10.26508/lsa.202201585
      Issue No: Vol. 6, No. 1 (2022)
  • Stepwise progression of {beta}-selection during T cell development
           involves histone deacetylation

    • Authors: Chann, A. S; Charnley, M, Newton, L. M, Newbold, A, Wiede, F, Tiganis, T, Humbert, P. O, Johnstone, R. W, Russell, S. M.
      Pages: e202201645 - e202201645
      Abstract: During T cell development, the first step in creating a unique T cell receptor (TCR) is genetic recombination of the TCRβ chain. The quality of the new TCRβ is assessed at the β-selection checkpoint. Most cells fail this checkpoint and die, but the coordination of fate at the β-selection checkpoint is not yet understood. We shed new light on fate determination during β-selection using a selective inhibitor of histone deacetylase 6, ACY1215. ACY1215 disrupted the β-selection checkpoint. Characterising the basis for this disruption revealed a new, pivotal stage in β-selection, bookended by up-regulation of TCR co-receptors, CD28 and CD2, respectively. Within this "DN3bPre" stage, CD5 and Lef1 are up-regulated to reflect pre-TCR signalling, and their expression correlates with proliferation. These findings suggest a refined model of β-selection in which a coordinated increase in expression of pre-TCR, CD28, CD5 and Lef1 allows for modulating TCR signalling strength and culminates in the expression of CD2 to enable exit from the β-selection checkpoint.
      PubDate: 2022-10-25T05:59:07-07:00
      DOI: 10.26508/lsa.202201645
      Issue No: Vol. 6, No. 1 (2022)
  • Dlk1-Dio3 cluster miRNAs regulate mitochondrial functions in the
           dystrophic muscle in Duchenne muscular dystrophy

    • Authors: Vu Hong, A; Bourg, N, Sanatine, P, Poupiot, J, Charton, K, Gicquel, E, Massourides, E, Spinazzi, M, Richard, I, Israeli, D.
      Pages: e202201506 - e202201506
      Abstract: Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by impaired expression of dystrophin. Whereas mitochondrial dysfunction is thought to play an important role in DMD, the mechanism of this dysfunction remains to be clarified. Here we demonstrate that in DMD and other muscular dystrophies, a large number of Dlk1-Dio3 clustered miRNAs (DD-miRNAs) are coordinately up-regulated in regenerating myofibers and in the serum. To characterize the biological effect of this dysregulation, 14 DD-miRNAs were simultaneously overexpressed in vivo in mouse muscle. Transcriptomic analysis revealed highly similar changes between the muscle ectopically overexpressing 14 DD-miRNAs and the mdx diaphragm, with naturally up-regulated DD-miRNAs. Among the commonly dysregulated pathway we found repressed mitochondrial metabolism, and oxidative phosphorylation (OxPhos) in particular. Knocking down the DD-miRNAs in iPS-derived skeletal myotubes resulted in increased OxPhos activities. The data suggest that (1) DD-miRNAs are important mediators of dystrophic changes in DMD muscle, (2) mitochondrial metabolism and OxPhos in particular are targeted in DMD by coordinately up-regulated DD-miRNAs. These findings provide insight into the mechanism of mitochondrial dysfunction in muscular dystrophy.
      PubDate: 2022-10-20T11:21:31-07:00
      DOI: 10.26508/lsa.202201506
      Issue No: Vol. 6, No. 1 (2022)
  • MDF is a conserved splicing factor and modulates cell division and stress
           response in Arabidopsis

    • Authors: de Luxan-Hernandez, C; Lohmann, J, Tranque, E, Chumova, J, Binarova, P, Salinas, J, Weingartner, M.
      Pages: e202201507 - e202201507
      Abstract: The coordination of cell division with stress response is essential for maintaining genome stability in plant meristems. Proteins involved in pre-mRNA splicing are important for these processes in animal and human cells. Based on its homology to the splicing factor SART1, which is implicated in the control of cell division and genome stability in human cells, we analyzed if MDF has similar functions in plants. We found that MDF associates with U4/U6.U5 tri-snRNP proteins and is essential for correct splicing of 2,037 transcripts. Loss of MDF function leads to cell division defects and cell death in meristems and was associated with up-regulation of stress-induced genes and down-regulation of mitotic regulators. In addition, the mdf-1 mutant is hypersensitive to DNA damage treatment supporting its role in coordinating stress response with cell division. Our analysis of a dephosphomutant of MDF suggested how its protein activity might be controlled. Our work uncovers the conserved function of a plant splicing factor and provides novel insight into the interplay of pre-mRNA processing and genome stability in plants.
      PubDate: 2022-10-20T11:21:31-07:00
      DOI: 10.26508/lsa.202201507
      Issue No: Vol. 6, No. 1 (2022)
  • An inducible amphipathic {alpha}-helix mediates subcellular targeting and
           membrane binding of RPE65

    • Authors: Uppal, S; Liu, T, Galvan, E, Gomez, F, Tittley, T, Poliakov, E, Gentleman, S, Redmond, T. M.
      Pages: e202201546 - e202201546
      Abstract: RPE65 retinol isomerase is an indispensable player in the visual cycle between the vertebrate retina and RPE. Although membrane association is critical for RPE65 function, its mechanism is not clear. Residues 107–125 are believed to interact with membranes but are unresolved in all RPE65 crystal structures, whereas palmitoylation at C112 also plays a role. We report the mechanism of membrane recognition and binding by RPE65. Binding of aa107–125 synthetic peptide with membrane-mimicking micellar surfaces induces transition from unstructured loop to amphipathic α-helical (AH) structure but this transition is automatic in the C112-palmitoylated peptide. We demonstrate that the AH significantly affects palmitoylation level, membrane association, and isomerization activity of RPE65. Furthermore, aa107–125 functions as a membrane sensor and the AH as a membrane-targeting motif. Molecular dynamic simulations clearly show AH-membrane insertion, supporting our experimental findings. Collectively, these studies allow us to propose a working model for RPE65-membrane binding, and to provide a novel role for cysteine palmitoylation.
      PubDate: 2022-10-20T06:16:33-07:00
      DOI: 10.26508/lsa.202201546
      Issue No: Vol. 6, No. 1 (2022)
  • Tandemly repeated NBPF HOR copies (Olduvai triplets): Possible impact on
           human brain evolution

    • Authors: Gluncic, M; Vlahovic, I, Rosandic, M, Paar, V.
      Pages: e202101306 - e202101306
      Abstract: Previously it was found that the neuroblastoma breakpoint family (NBPF) gene repeat units of ~1.6 kb have an important role in human brain evolution and function. The higher order organization of these repeat units has been discovered by both methods, the higher order repeat (HOR)-searching method and the HLS searching method. Using the HOR searching method with global repeat map algorithm, here we identified the tandemly organized NBPF HORs in the human and nonhuman primate NCBI reference genomes. We identified 50 tandemly organized canonical 3mer NBPF HOR copies (Olduvai triplets), but none in nonhuman primates chimpanzee, gorilla, orangutan, and Rhesus macaque. This discontinuous jump in tandemly organized HOR copy number is in sharp contrast to the known gradual increase in the number of Olduvai domains (NBPF monomers) from nonhuman primates to human, especially from ~138 in chimpanzee to ~300 in human genome. Using the same global repeat map algorithm method we have also determined the 3mer tandems of canonical 3mer HOR copies in 20 randomly chosen human genomes (10 male and 10 female). In all cases, we found the same 3mer HOR copy numbers as in the case of the reference human genome, with no mutation. On the other hand, some point mutations with respect to reference genome are found for some NBPF monomers which are not tandemly organized in canonical HORs.
      PubDate: 2022-10-19T08:19:45-07:00
      DOI: 10.26508/lsa.202101306
      Issue No: Vol. 6, No. 1 (2022)
  • Replication-associated inversions are the dominant form of bacterial
           chromosome structural variation

    • Authors: DIorio, M; Dewar, K.
      Pages: e202201434 - e202201434
      Abstract: The structural arrangements of bacterial chromosomes vary widely between closely related species and can result in significant phenotypic outcomes. The appearance of large-scale chromosomal inversions that are symmetric relative to markers for the origin of replication (OriC) has been previously observed; however, the overall prevalence of replication-associated structural rearrangements (RASRs) in bacteria and their causal mechanisms are currently unknown. Here, we systematically identify the locations of RASRs in species with multiple complete-sequenced genomes and investigate potential mediating biological mechanisms. We found that 247 of 313 species contained sequences with at least one large (>50 Kb) inversion in their sequence comparisons, and the aggregated inversion distances away from symmetry were normally distributed with a mean of zero. Many inversions that were offset from dnaA were found to be centered on a different marker for the OriC. Instances of flanking repeats provide evidence that breaks formed during the replication process could be repaired to opposing positions. We also found a strong relationship between the later stages of replication and the range in distance variation from symmetry.
      PubDate: 2022-10-19T08:19:45-07:00
      DOI: 10.26508/lsa.202201434
      Issue No: Vol. 6, No. 1 (2022)
  • A prediction model for COVID-19 liver dysfunction in patients with normal
           hepatic biochemical parameters

    • Authors: Bao, J; Liu, S, Liang, X, Wang, C, Cao, L, Li, Z, Wei, F, Fu, A, Shi, Y, Shen, B, Zhu, X, Zhao, Y, Liu, H, Miao, L, Wang, Y, Liang, S, Wu, L, Huang, J, Guo, T, Liu, F.
      Pages: e202201576 - e202201576
      Abstract: Coronavirus disease 2019 (COVID-19) patients with liver dysfunction (LD) have a higher chance of developing severe and critical disease. The routine hepatic biochemical parameters ALT, AST, GGT, and TBIL have limitations in reflecting COVID-19–related LD. In this study, we performed proteomic analysis on 397 serum samples from 98 COVID-19 patients to identify new biomarkers for LD. We then established 19 simple machine learning models using proteomic measurements and clinical variables to predict LD in a development cohort of 74 COVID-19 patients with normal hepatic biochemical parameters. The model based on the biomarker ANGL3 and sex (AS) exhibited the best discrimination (time-dependent AUCs: 0.60–0.80), calibration, and net benefit in the development cohort, and the accuracy of this model was 69.0–73.8% in an independent cohort. The AS model exhibits great potential in supporting optimization of therapeutic strategies for COVID-19 patients with a high risk of LD. This model is publicly available at https://xixihospital-liufang.shinyapps.io/DynNomapp/.
      PubDate: 2022-10-19T08:19:45-07:00
      DOI: 10.26508/lsa.202201576
      Issue No: Vol. 6, No. 1 (2022)
  • The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial
           protein import

    • Authors: Rödl, S; den Brave, F, Räschle, M, Kizmaz, B, Lenhard, S, Groh, C, Becker, H, Zimmermann, J, Morgan, B, Richling, E, Becker, T, Herrmann, J. M.
      Pages: e202201526 - e202201526
      Abstract: Mitochondria play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here, we show that the yeast cytosolic ubiquitin conjugase Ubc8 plays a crucial role in the remodeling process when cells transition from respiratory to fermentative conditions. Ubc8 is a conserved and well-studied component of the catabolite control system that is known to regulate the stability of gluconeogenic enzymes. Unexpectedly, we found that Ubc8 also promotes the assembly of the translocase of the outer membrane of mitochondria (TOM) and increases the levels of its cytosol-exposed receptor subunit Tom22. Ubc8 deficiency results in compromised protein import into mitochondria and reduced steady-state levels of mitochondrial proteins. Our observations show that Ubc8, which is controlled by the prevailing metabolic conditions, promotes the switch from glucose synthesis to glucose usage in the cytosol and induces the biogenesis of the mitochondrial TOM machinery to improve mitochondrial protein import during phases of metabolic transition.
      PubDate: 2022-10-17T11:17:39-07:00
      DOI: 10.26508/lsa.202201526
      Issue No: Vol. 6, No. 1 (2022)
  • Low expression of EXOSC2 protects against clinical COVID-19 and impedes
           SARS-CoV-2 replication

    • Authors: Moll, T; Odon, V, Harvey, C, Collins, M. O, Peden, A, Franklin, J, Graves, E, Marshall, J. N, dos Santos Souza, C, Zhang, S, Castelli, L, Hautbergue, G, Azzouz, M, Gordon, D, Krogan, N, Ferraiuolo, L, Snyder, M. P, Shaw, P. J, Rehwinkel, J, Cooper-Knock, J.
      Pages: e202201449 - e202201449
      Abstract: New therapeutic targets are a valuable resource for treatment of SARS-CoV-2 viral infection. Genome-wide association studies have identified risk loci associated with COVID-19, but many loci are associated with comorbidities and are not specific to host–virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of the 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins. Aggregating COVID-19 genome-wide association studies statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19. EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. EXOSC2 is a component of the RNA exosome, and here, LC-MS/MS analysis of protein pulldowns demonstrated interaction between the SARS-CoV-2 RNA polymerase and most of the human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression and impeded SARS-CoV-2 replication without impacting cellular viability. Targeted depletion of EXOSC2 may be a safe and effective strategy to protect against clinical COVID-19.
      PubDate: 2022-10-14T09:43:05-07:00
      DOI: 10.26508/lsa.202201449
      Issue No: Vol. 6, No. 1 (2022)
  • A dominant negative mitofusin causes mitochondrial perinuclear clusters
           because of aberrant tethering

    • Authors: Sloat, S. R; Hoppins, S.
      Pages: e202101305 - e202101305
      Abstract: In vertebrates, mitochondrial outer membrane fusion is mediated by two mitofusin paralogs, Mfn1 and Mfn2, conserved dynamin superfamily proteins. Here, we characterize a variant of mitofusin reported in patients with CMT2A where a serine is replaced with a proline (Mfn2-S350P and the equivalent in Mfn1, S329P). This serine is in a hinge domain (Hinge 2) that connects the globular GTPase domain to the adjacent extended helical bundle. We find that expression of this variant results in prolific and stable mitochondrial tethering that also blocks mitochondrial fusion by endogenous wild-type mitofusin. The formation of mitochondrial perinuclear clusters by this CMT2A variant requires normal GTPase domain function and formation of a mitofusin complex across two membranes. We propose that conformational dynamics mediated by Hinge 2 and regulated by GTP hydrolysis are disrupted by the substitution of proline at S329/S350 and this prevents progression from tethering to membrane fusion. Thus, our data are consistent with a model for mitofusin-mediated membrane fusion where Hinge 2 supports a power stroke to progress from the tethering complex to membrane fusion.
      PubDate: 2022-10-13T10:14:39-07:00
      DOI: 10.26508/lsa.202101305
      Issue No: Vol. 6, No. 1 (2022)
  • Exploratory meta-analysis of hypoxic transcriptomes using a precise
           transcript reference sequence set

    • Authors: Ono, Y; Bono, H.
      Pages: e202201518 - e202201518
      Abstract: Gene expression studies are intrinsically biased, with many studies influenced by concomitant information such as gene–disease associations. This limitation can be overcome using a data-driven analysis approach without relying on ancillary information. The FANTOM CAGE–Associated Transcriptome project provides a comprehensive meta-assembly of the human transcriptome using coding and noncoding genes. Hypoxia strongly influences gene expression; in addition, noncoding RNA (ncRNA) metabolism is down-regulated in response to hypoxic stimuli. We evaluated the differential response of various transcripts to hypoxia by determining their hypoxia responsiveness scores. Enrichment analysis revealed that several genes associated with ncRNA metabolism, particularly those involved in ribosomal RNA processing, were down-regulated in response to hypoxia. Previously published information from the FANTOM CAGE–Associated Transcriptome project was suitable for meta-analysis of the transcriptome sequencing data from both coding and ncRNAs and to evaluate the hypoxia responsiveness of target transcripts and relationship between sense–antisense transcripts from the same locus. Our results may facilitate functional annotation of various transcripts including ncRNAs, allowing for both sense and antisense and coding and noncoding evaluations.
      PubDate: 2022-10-10T11:44:11-07:00
      DOI: 10.26508/lsa.202201518
      Issue No: Vol. 6, No. 1 (2022)
School of Mathematical and Computer Sciences
Heriot-Watt University
Edinburgh, EH14 4AS, UK
Email: journaltocs@hw.ac.uk
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School of Mathematical and Computer Sciences
Heriot-Watt University
Edinburgh, EH14 4AS, UK
Email: journaltocs@hw.ac.uk
Tel: +00 44 (0)131 4513762

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