DOCSLIB.ORG

Mouse Cox6a1 Knockout Project (CRISPR/Cas9)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mouse Cox6a1 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Cox6a1 Knockout Project (CRISPR/Cas9) Objective: To create a Cox6a1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cox6a1 gene (NCBI Reference Sequence: NM_007748 ; Ensembl: ENSMUSG00000041697 ) is located on Mouse chromosome 5. 3 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 3 (Transcript: ENSMUST00000040154). Exon 1~3 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Homozygous null mice exhibit impaired coordination, thinned sciatic nerves, neurogenic muscular changes and delayed motor nerve conduction velocity. Exon 1 starts from about 0.3% of the coding region. Exon 1~3 covers 100.0% of the coding region. The size of effective KO region: ~3103 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 Legends Exon of mouse Cox6a1 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of start codon is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of stop codon is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(25.65% 513) | C(24.35% 487) | T(26.4% 528) | G(23.6% 472) Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(26.85% 537) | C(23.3% 466) | T(25.1% 502) | G(24.75% 495) Note: The 2000 bp section downstream of stop codon is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr5 - 115348951 115350950 2000 browser details YourSeq 481 159 1038 2000 93.3% chr16 + 56059841 56060687 847 browser details YourSeq 437 197 1047 2000 93.5% chrX + 154415912 154416799 888 browser details YourSeq 419 173 1047 2000 92.6% chr10 + 63311988 63312721 734 browser details YourSeq 403 173 1047 2000 91.4% chr14 - 58188932 58189633 702 browser details YourSeq 400 159 1045 2000 92.1% chr4 - 62334393 62335118 726 browser details YourSeq 390 241 1047 2000 95.8% chr7 + 5417066 5417941 876 browser details YourSeq 383 155 1047 2000 93.3% chr14 - 11233957 11234769 813 browser details YourSeq 380 655 1049 2000 98.3% chr14 - 32416645 32417041 397 browser details YourSeq 380 655 1047 2000 98.5% chr9 + 91792716 91793110 395 browser details YourSeq 378 366 1047 2000 96.4% chr2 - 101050983 101051826 844 browser details YourSeq 378 655 1047 2000 98.3% chr15 - 11632181 11632575 395 browser details YourSeq 375 652 1047 2000 97.5% chr6 + 143014339 143014736 398 browser details YourSeq 374 655 1047 2000 97.8% chr12 - 48374756 48375150 395 browser details YourSeq 374 655 1047 2000 97.8% chr12 - 48376244 48376638 395 browser details YourSeq 373 653 1048 2000 97.3% chr6 + 143015831 143016228 398 browser details YourSeq 373 655 1057 2000 96.8% chr4 + 111103736 111104145 410 browser details YourSeq 373 655 1048 2000 97.5% chr17 + 19830487 19830882 396 browser details YourSeq 372 655 1047 2000 97.5% chr1 - 78301235 78301629 395 browser details YourSeq 372 655 1047 2000 97.5% chr17 + 19828995 19829389 395 Note: The 2000 bp section upstream of start codon is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr5 - 115343846 115345845 2000 browser details YourSeq 176 1 192 2000 93.7% chr19 + 3206071 3206259 189 browser details YourSeq 89 599 752 2000 89.4% chr7 - 87478348 87478501 154 browser details YourSeq 81 634 755 2000 86.0% chr10 - 69333001 69333126 126 browser details YourSeq 79 1273 1584 2000 91.6% chr2 + 128038855 128039197 343 browser details YourSeq 76 634 752 2000 84.2% chr6 + 120026817 120026934 118 browser details YourSeq 74 634 749 2000 86.9% chr1 + 86149353 86149468 116 browser details YourSeq 73 1435 1581 2000 89.6% chr2 - 131323316 131323478 163 browser details YourSeq 69 596 749 2000 91.7% chr13 - 48242316 48242569 254 browser details YourSeq 63 625 741 2000 94.6% chr13 + 7617919 7618035 117 browser details YourSeq 60 1270 1577 2000 85.8% chr16 + 54798191 54798495 305 browser details YourSeq 59 634 757 2000 94.1% chr16 - 21715392 21715530 139 browser details YourSeq 58 1342 1587 2000 91.5% chr8 - 89451810 89452077 268 browser details YourSeq 58 634 724 2000 91.6% chr11 - 105304246 105304661 416 browser details YourSeq 58 633 718 2000 91.7% chr11 - 43331345 43331441 97 browser details YourSeq 57 1433 1580 2000 89.1% chr9 + 64954412 64954577 166 browser details YourSeq 57 690 757 2000 94.0% chr5 + 52661056 52661127 72 browser details YourSeq 57 634 723 2000 95.4% chr1 + 74955126 74955222 97 browser details YourSeq 56 637 741 2000 91.2% chr11 + 116144925 116145158 234 browser details YourSeq 54 651 741 2000 88.8% chr13 - 43382326 43382423 98 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Cox6a1 cytochrome c oxidase subunit 6A1 [ Mus musculus (house mouse) ] Gene ID: 12861, updated on 12-Aug-2019 Gene summary Official Symbol Cox6a1 provided by MGI Official Full Name cytochrome c oxidase subunit 6A1 provided by MGI Primary source MGI:MGI:103099 See related Ensembl:ENSMUSG00000041697 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as VIaL Expression Ubiquitous expression in duodenum adult (RPKM 2589.4), colon adult (RPKM 2186.7) and 28 other tissues See more Orthologs human all Genomic context Location: 5 F; 5 56.06 cM See Cox6a1 in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (115345623..115348958, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (115795663..115798964, complement) Chromosome 5 - NC_000071.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Cox6a1 ENSMUSG00000041697 Description cytochrome c oxidase subunit 6A1 [Source:MGI Symbol;Acc:MGI:103099] Gene Synonyms VIaL, subunit VIaL (liver-type) Location Chromosome 5: 115,345,642-115,348,981 reverse strand. GRCm38:CM000998.2 About this gene This gene has 2 transcripts (splice variants), 225 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cox6a1-201 ENSMUST00000040154.8 571 112aa ENSMUSP00000047661.8 Protein coding CCDS19590 Q9DCW5 TSL:1 GENCODE basic APPRIS P1 Cox6a1-202 ENSMUST00000137766.1 620 No protein - Retained intron - - TSL:2 23.34 kb Forward strand 115.340Mb 115.345Mb 115.350Mb 115.355Mb Genes Triap1-201 >protein coding Gm42903-201 >lncRNA (Comprehensive set... Contigs < AC117735.8 Genes (Comprehensive set... < Gatc-201protein coding < Cox6a1-201protein coding < 4930401G09Rik-201lncRNA < Cox6a1-202retained intron Regulatory Build 115.340Mb 115.345Mb 115.350Mb 115.355Mb Reverse strand 23.34 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000040154 < Cox6a1-201protein coding Reverse strand 3.34 kb ENSMUSP00000047... Transmembrane heli... Low complexity (Seg) Superfamily Cytochrome c oxidase, subunit VIa superfamily Pfam Cytochrome c oxidase, subunit VIa PROSITE patterns Cytochrome c oxidase, subunit VIa, conserved site PIRSF Cytochrome c oxidase, subunit VIa PANTHER Cytochrome c oxidase, subunit VIa PTHR11504:SF4 Gene3D Cytochrome c oxidase, subunit VIa superfamily CDD Cytochrome c oxidase, subunit VIa All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 10 20 30 40 50 60 70 80 90 100 112 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
Load more

Recommended publications
  • Mutation in the COX4I1 Gene Is Associated with Short Stature, Poor Weight Gain and Increased Chromosomal Breaks, Simulating Fanconi Anemia
    European Journal of Human Genetics (2017) 25, 1142–1146 & 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 1018-4813/17 www.nature.com/ejhg ARTICLE Mutation in the COX4I1 gene is associated with short stature, poor weight gain and increased chromosomal breaks, simulating Fanconi anemia Bassam Abu-Libdeh*,1,4, Liza Douiev2,3,4, Sarah Amro1, Maher Shahrour1, Asaf Ta-Shma2, Chaya Miller2,3, Orly Elpeleg2 and Ann Saada*,2,3 We describe a novel autosomal recessive form of mitochondrial disease in a child with short stature, poor weight gain, and mild dysmorphic features with highly suspected Fanconi anemia due to a mutation in COX4I1 gene. Whole Exome Sequencing was performed then followed by Sanger confirmation, identified a K101N mutation in COX4I1, segregating with the disease. This nuclear gene encodes the common isoform of cytochrome c oxidase (COX) subunit 4 (COX 4-1), an integral regulatory part of COX (respiratory chain complex IV) the terminal electron acceptor of the mitochondrial respiratory chain. The patient’s fibroblasts disclosed decreased COX activity, impaired ATP production, elevated ROS production, decreased expression of COX4I1 mRNA and undetectable (COX4) protein. COX activity and ATP production were restored by lentiviral transfection with the wild-type gene. Our results demonstrate the first human mutation in the COX4I1 gene linked to diseases and confirm its role in the pathogenesis. Thus COX4I1 mutations should be considered in any patient with features suggestive of this diagnosis. European Journal of Human Genetics (2017) 25, 1142–1146; doi:10.1038/ejhg.2017.112; published online 2 August 2017 INTRODUCTION normal vaginal delivery with birth weight of 2.8 kg after an uneventful Mitochondrial cytochrome c oxidase (COX, complex IV) is the terminal pregnancy.
    [Show full text]
  • Structure of the Intact 14-Subunit Human Cytochrome C Oxidase
    www.nature.com/cr www.cell-research.com ARTICLE Structure of the intact 14-subunit human cytochrome c oxidase Shuai Zong 1, Meng Wu1, Jinke Gu1, Tianya Liu1, Runyu Guo1 and Maojun Yang1,2 Respiration is one of the most basic features of living organisms, and the electron transport chain complexes are probably the most complicated protein system in mitochondria. Complex-IV is the terminal enzyme of the electron transport chain, existing either as randomly scattered complexes or as a component of supercomplexes. NDUFA4 was previously assumed as a subunit of complex-I, but recent biochemical data suggested it may be a subunit of complex-IV. However, no structural evidence supporting this notion was available till now. Here we obtained the 3.3 Å resolution structure of complex-IV derived from the human supercomplex I1III2IV1 and assigned the NDUFA4 subunit into complex-IV. Intriguingly, NDUFA4 lies exactly at the dimeric interface observed in previously reported crystal structures of complex-IV homodimer which would preclude complex- IV dimerization. Combining previous structural and biochemical data shown by us and other groups, we propose that the intact complex-IV is a monomer containing 14 subunits. Cell Research (2018) 28:1026–1034; https://doi.org/10.1038/s41422-018-0071-1 INTRODUCTION states under physiological conditions, either being assembled into Mitochondria are critical to many cellular activities. Respiration is supercomplexes or freely scattered on mitochondrial inner the central function of mitochondria, and is exquisitely regulated membrane.36 NDUFA4 was originally considered as a subunit of in multiple ways in response to varying cell conditions.1–3 The Complex-I37 but was proposed to belong to Complex-IV recently.38 assembly of respiratory chain complexes, Complex I–IV (CI, NADH: However, the precise location of NDUFA4 in CIV remains unknown, ubiquinone oxidoreductase; CII, succinate:ubiquinone oxidoreduc- and thus this notion still lacks structural support.
    [Show full text]
  • A High-Resolution Genome-Wide CRISPR/Cas9 Viability Screen
    RESEARCH ARTICLE crossm A High-Resolution Genome-Wide CRISPR/Cas9 Viability Screen Reveals Structural Features and Contextual Diversity of the Human Cell-Essential Proteome Downloaded from Thierry Bertomeu,a Jasmin Coulombe-Huntington,a Andrew Chatr-aryamontri,a Karine G. Bourdages,a Etienne Coyaud,b Brian Raught,b Yu Xia,c Mike Tyersa aInstitute for Research in Immunology and Cancer, Department of Medicine, University of Montreal, Montreal, Quebec, Canada bPrincess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada cDepartment of Bioengineering, McGill University, Montreal, Quebec, Canada ABSTRACT To interrogate genes essential for cell growth, proliferation and survival http://mcb.asm.org/ in human cells, we carried out a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 screen in a B-cell lymphoma line using a custom extended-knockout (EKO) library of 278,754 single-guide RNAs (sgRNAs) that tar- geted 19,084 RefSeq genes, 20,852 alternatively spliced exons, and 3,872 hypotheti- cal genes. A new statistical analysis tool called robust analytics and normalization for knockout screens (RANKS) identified 2,280 essential genes, 234 of which were unique. Individual essential genes were validated experimentally and linked to ribo- some biogenesis and stress responses. Essential genes exhibited a bimodal distribu- on December 15, 2018 by guest tion across 10 different cell lines, consistent with a continuous variation in essential- ity as a function of cell type. Genes essential in more lines had more severe fitness defects and encoded the evolutionarily conserved structural cores of protein com- plexes, whereas genes essential in fewer lines formed context-specific modules and encoded subunits at the periphery of essential complexes.
    [Show full text]
  • Role of Cytochrome C Oxidase Nuclear-Encoded Subunits in Health and Disease
    Physiol. Res. 69: 947-965, 2020 https://doi.org/10.33549/physiolres.934446 REVIEW Role of Cytochrome c Oxidase Nuclear-Encoded Subunits in Health and Disease Kristýna ČUNÁTOVÁ1, David PAJUELO REGUERA1, Josef HOUŠTĚK1, Tomáš MRÁČEK1, Petr PECINA1 1Department of Bioenergetics, Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic Received February 2, 2020 Accepted September 13, 2020 Epub Ahead of Print November 2, 2020 Summary [email protected] and Tomáš Mráček, Department of Cytochrome c oxidase (COX), the terminal enzyme of Bioenergetics, Institute of Physiology CAS, Vídeňská 1083, 142 mitochondrial electron transport chain, couples electron transport 20 Prague 4, Czech Republic. E-mail: [email protected] to oxygen with generation of proton gradient indispensable for the production of vast majority of ATP molecules in mammalian Cytochrome c oxidase cells. The review summarizes current knowledge of COX structure and function of nuclear-encoded COX subunits, which may Energy demands of mammalian cells are mainly modulate enzyme activity according to various conditions. covered by ATP synthesis carried out by oxidative Moreover, some nuclear-encoded subunits possess tissue-specific phosphorylation apparatus (OXPHOS) located in the and development-specific isoforms, possibly enabling fine-tuning central bioenergetic organelle, mitochondria. OXPHOS is of COX function in individual tissues. The importance of nuclear- composed of five multi-subunit complexes embedded in encoded subunits is emphasized by recently discovered the inner mitochondrial membrane (IMM). Electron pathogenic mutations in patients with severe mitopathies. In transport from reduced substrates of complexes I and II to addition, proteins substoichiometrically associated with COX were cytochrome c oxidase (COX, complex IV, CIV) is found to contribute to COX activity regulation and stabilization of achieved by increasing redox potential of individual the respiratory supercomplexes.
    [Show full text]
  • Human Mitochondrial Pathologies of the Respiratory Chain and ATP Synthase: Contributions from Studies of Saccharomyces Cerevisiae
    life Review Human Mitochondrial Pathologies of the Respiratory Chain and ATP Synthase: Contributions from Studies of Saccharomyces cerevisiae Leticia V. R. Franco 1,2,* , Luca Bremner 1 and Mario H. Barros 2 1 Department of Biological Sciences, Columbia University, New York, NY 10027, USA; [email protected] 2 Department of Microbiology,Institute of Biomedical Sciences, Universidade de Sao Paulo, Sao Paulo 05508-900, Brazil; [email protected] * Correspondence: [email protected] Received: 27 October 2020; Accepted: 19 November 2020; Published: 23 November 2020 Abstract: The ease with which the unicellular yeast Saccharomyces cerevisiae can be manipulated genetically and biochemically has established this organism as a good model for the study of human mitochondrial diseases. The combined use of biochemical and molecular genetic tools has been instrumental in elucidating the functions of numerous yeast nuclear gene products with human homologs that affect a large number of metabolic and biological processes, including those housed in mitochondria. These include structural and catalytic subunits of enzymes and protein factors that impinge on the biogenesis of the respiratory chain. This article will review what is currently known about the genetics and clinical phenotypes of mitochondrial diseases of the respiratory chain and ATP synthase, with special emphasis on the contribution of information gained from pet mutants with mutations in nuclear genes that impair mitochondrial respiration. Our intent is to provide the yeast mitochondrial specialist with basic knowledge of human mitochondrial pathologies and the human specialist with information on how genes that directly and indirectly affect respiration were identified and characterized in yeast. Keywords: mitochondrial diseases; respiratory chain; yeast; Saccharomyces cerevisiae; pet mutants 1.
    [Show full text]
  • Electron Transport Chain Activity Is a Predictor and Target for Venetoclax Sensitivity in Multiple Myeloma
    ARTICLE https://doi.org/10.1038/s41467-020-15051-z OPEN Electron transport chain activity is a predictor and target for venetoclax sensitivity in multiple myeloma Richa Bajpai1,7, Aditi Sharma 1,7, Abhinav Achreja2,3, Claudia L. Edgar1, Changyong Wei1, Arusha A. Siddiqa1, Vikas A. Gupta1, Shannon M. Matulis1, Samuel K. McBrayer 4, Anjali Mittal3,5, Manali Rupji 6, Benjamin G. Barwick 1, Sagar Lonial1, Ajay K. Nooka 1, Lawrence H. Boise 1, Deepak Nagrath2,3,5 & ✉ Mala Shanmugam 1 1234567890():,; The BCL-2 antagonist venetoclax is highly effective in multiple myeloma (MM) patients exhibiting the 11;14 translocation, the mechanistic basis of which is unknown. In evaluating cellular energetics and metabolism of t(11;14) and non-t(11;14) MM, we determine that venetoclax-sensitive myeloma has reduced mitochondrial respiration. Consistent with this, low electron transport chain (ETC) Complex I and Complex II activities correlate with venetoclax sensitivity. Inhibition of Complex I, using IACS-010759, an orally bioavailable Complex I inhibitor in clinical trials, as well as succinate ubiquinone reductase (SQR) activity of Complex II, using thenoyltrifluoroacetone (TTFA) or introduction of SDHC R72C mutant, independently sensitize resistant MM to venetoclax. We demonstrate that ETC inhibition increases BCL-2 dependence and the ‘primed’ state via the ATF4-BIM/NOXA axis. Further, SQR activity correlates with venetoclax sensitivity in patient samples irrespective of t(11;14) status. Use of SQR activity in a functional-biomarker informed manner may better select for MM patients responsive to venetoclax therapy. 1 Department of Hematology and Medical Oncology, Winship Cancer Institute, School of Medicine, Emory University, Atlanta, GA, USA.
    [Show full text]
  • Observation of Novel COX20 Mutations Related to Autosomal Recessive Axonal Neuropathy and Static Encephalopathy
    Human Genetics (2019) 138:749–756 https://doi.org/10.1007/s00439-019-02026-4 ORIGINAL INVESTIGATION Observation of novel COX20 mutations related to autosomal recessive axonal neuropathy and static encephalopathy Hongliang Xu1 · Tuo Ji1 · Yajun Lian1 · Shuya Wang2 · Xin Chen1 · Shuang Li1 · Yuhui Yin3 · Xiubing Dong4 Received: 21 February 2019 / Accepted: 7 May 2019 / Published online: 11 May 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Cytochrome c oxidase 20 (COX20)/FAM36A encodes a conserved protein that is important for the assembly of COX, com- plex IV of the mitochondrial respiratory chain. A homozygous mutation (p.Thr52Pro) in COX20 gene has been previously described to cause muscle hypotonia and ataxia. In this study, we describe two patients from a non-consanguineous family exhibiting autosomal recessive sensory-dominant axonal neuropathy and static encephalopathy. The whole-exome sequencing analysis revealed that both patients harbored compound heterozygous mutations (p.Lys14Arg and p.Trp74Cys) of COX20 gene. The pathogenicity of the variants was further supported by morphological alternations of mitochondria observed in sural nerve and decreased COX20 protein level of peripheral blood leucocytes derived from the patients. In conclusion, COX20 might be considered as a candidate gene for the complex inherited disease. This observation broadens the clinical and genetic spectrum of COX20-related disease. However, due to the limitation of a single-family study, additional cases and studies are defnitely needed to further confrm the association. Introduction a frequent cause of oxidative phosphorylation disorders in humans. Patients sufering from COX-related mitochondrial Cytochrome c oxidase 20 (COX20, also known as FAM36A) diseases present with heterogeneous clinical phenotypes encodes a conserved protein that is important for the assem- ranging from encephalomyopathy, hypertrophic cardio- bly of COX, complex IV of the mitochondrial respiratory myopathy and liver disease to Leigh’s syndrome (Bourens chain (Bourens et al.
    [Show full text]
  • Transcriptomic and Proteomic Landscape of Mitochondrial
    TOOLS AND RESOURCES Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals Inge Ku¨ hl1,2†*, Maria Miranda1†, Ilian Atanassov3, Irina Kuznetsova4,5, Yvonne Hinze3, Arnaud Mourier6, Aleksandra Filipovska4,5, Nils-Go¨ ran Larsson1,7* 1Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany; 2Department of Cell Biology, Institute of Integrative Biology of the Cell (I2BC) UMR9198, CEA, CNRS, Univ. Paris-Sud, Universite´ Paris-Saclay, Gif- sur-Yvette, France; 3Proteomics Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany; 4Harry Perkins Institute of Medical Research, The University of Western Australia, Nedlands, Australia; 5School of Molecular Sciences, The University of Western Australia, Crawley, Australia; 6The Centre National de la Recherche Scientifique, Institut de Biochimie et Ge´ne´tique Cellulaires, Universite´ de Bordeaux, Bordeaux, France; 7Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Abstract Dysfunction of the oxidative phosphorylation (OXPHOS) system is a major cause of human disease and the cellular consequences are highly complex. Here, we present comparative *For correspondence: analyses of mitochondrial proteomes, cellular transcriptomes and targeted metabolomics of five [email protected] knockout mouse strains deficient in essential factors required for mitochondrial DNA gene (IKu¨ ); expression, leading to OXPHOS dysfunction. Moreover,
    [Show full text]
  • Conserved in Situ Arrangement of Complex I and III2 in Mitochondrial Respiratory Chain Supercomplexes of Mammals, Yeast, and Plants
    Conserved in situ arrangement of complex I and III2 in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants Karen M. Daviesa,1, Thorsten B. Bluma,2, and Werner Kühlbrandta,3 aDepartment of Structural Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany Edited by Richard Henderson, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom, and approved February 13, 2018 (received for review November 30, 2017) We used electron cryo-tomography and subtomogram averaging 16, 17), but is likely to occur both as a monomer and a dimer in to investigate the structure of complex I and its supramolecular the membrane (18, 19). assemblies in the inner mitochondrial membrane of mammals, fungi, Supramolecular assemblies, or supercomplexes, of respiratory and plants. Tomographic volumes containing complex I were aver- chain complexes were first identified by blue-native gel electro- aged at ∼4 nm resolution. Principal component analysis indicated that phoresis (BN-PAGE) of detergent-solubilized inner mitochon- ∼ 60% of complex I formed a supercomplex with dimeric complex III, drial membranes (20). Supercomplexes containing complexes I, III2, while ∼40% were not associated with other respiratory chain com- and IV are sometimes referred to as respirasomes. Negative-stain plexes. The mutual arrangement of complex I and III2 was essentially electron microscopy of samples extracted from gel bands yielded conserved in all supercomplexes investigated. In addition, up to two initial 3D maps at low resolution (21–23). The structures of copies of monomeric complex IV were associated with the complex supercomplexes I1III2 and I1III2IV isolated from bovine, porcine, I1III2 assembly in bovine heart and the yeast Yarrowia lipolytica, but and ovine mitochondria have been determined by single-particle their positions varied.
    [Show full text]
  • Mice Deficient in the Respiratory Chain Gene Cox6a2 Are Protected Against High-Fat Diet-Induced Obesity and Insulin Resistance
    Mice Deficient in the Respiratory Chain Gene Cox6a2 Are Protected against High-Fat Diet-Induced Obesity and Insulin Resistance Roel Quintens1¤a, Sarvjeet Singh2, Katleen Lemaire1, Katrien De Bock3,4, Mikaela Granvik1, Anica Schraenen1, Irene Olga Cornelia Maria Vroegrijk5, Veronica Costa6, Pieter Van Noten7, Dennis Lambrechts8, Stefan Lehnert1, Leentje Van Lommel1, Lieven Thorrez1¤b, Geoffroy De Faudeur1, Johannes Anthonius Romijn5, John Michael Shelton2, Luca Scorrano6, Henri Roger Lijnen9, Peter Jacobus Voshol5, Peter Carmeliet3,4, Pradeep Puthenveetil Abraham Mammen2, Frans Schuit1* 1 Gene Expression Unit, Department of Molecular and Cellular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium, 2 Division of Cardiology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 3 Vesalius Research Center, Katholieke Universiteit Leuven, Leuven, Belgium, 4 Vesalius Research Center, Vlaams Instituut voor Biotechnologie (VIB), Leuven, Belgium, 5 Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands, 6 Department of Cell Physiology and Metabolism, University of Geneva, Geneve, Switzerland, 7 Physical Activity and Health Laboratory, Biomedical Kinesiology Department, Katholieke Universiteit Leuven, Leuven, Belgium, 8 Department of Metallurgy and Materials Engineering, KU Leuven, Leuven, Belgium, 9 Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Leuven, Belgium Abstract Oxidative phosphorylation in mitochondria is responsible for 90% of ATP synthesis in most cells. This essential housekeeping function is mediated by nuclear and mitochondrial genes encoding subunits of complex I to V of the respiratory chain. Although complex IV is the best studied of these complexes, the exact function of the striated muscle- specific subunit COX6A2 is still poorly understood. In this study, we show that Cox6a2-deficient mice are protected against high-fat diet-induced obesity, insulin resistance and glucose intolerance.
    [Show full text]
  • RNA-Seq and GSEA Identifies Suppression of Ligand-Gated
    www.nature.com/scientificreports OPEN RNA‑seq and GSEA identifes suppression of ligand‑gated chloride efux channels as the major gene pathway contributing to form deprivation myopia Loretta Giummarra Vocale1,4*, Sheila Crewther1, Nina Riddell1, Nathan E. Hall1,2, Melanie Murphy1 & David Crewther1,3 Currently there is no consensus regarding the aetiology of the excessive ocular volume that characterizes high myopia. Thus, we aimed to test whether the gene pathways identifed by gene set enrichment analysis of RNA‑seq transcriptomics refutes the predictions of the Retinal Ion Driven Efux (RIDE) hypothesis when applied to the induction of form‑deprivation myopia (FDM) and subsequent recovery (post‑occluder removal). We found that the induction of profound FDM led to signifcant suppression in the ligand‑gated chloride ion channel transport pathway via suppression of glycine, GABAA and GABAC ionotropic receptors. Post‑occluder removal for short term recovery from FDM of 6 h and 24 h, induced signifcant upregulation of the gene families linked to cone receptor phototransduction, mitochondrial energy, and complement pathways. These fndings support a model of form deprivation myopia as a Cl− ion driven adaptive fuid response to the modulation of the visual signal cascade by form deprivation that in turn afects the resultant ionic environment of the outer and inner retinal tissues, axial and vitreal elongation as predicted by the RIDE model. Occluder removal and return to normal light conditions led to return to more normal upregulation of phototransduction, slowed growth rate, refractive recovery and apparent return towards physiological homeostasis. Myopia (short-sightedness) is the most common visual disorder worldwide and the greatest risk factor for severe ophthalmic diseases in older individuals especially those with high (-5D) refractive errors1.
    [Show full text]
  • Functions of Cytochrome C Oxidase Assembly Factors
    International Journal of Molecular Sciences Review Functions of Cytochrome c Oxidase Assembly Factors Shane A. Watson and Gavin P. McStay * Department of Biological Sciences, Faculty of School of Life Sciences and Education, Staffordshire University, Science Centre, Leek Road, Stoke-on-Trent ST4 2DF, UK; [email protected]ffs.ac.uk * Correspondence: gavin.mcstay@staffs.ac.uk; Tel.: +44-01782-295741 Received: 17 September 2020; Accepted: 23 September 2020; Published: 30 September 2020 Abstract: Cytochrome c oxidase is the terminal complex of eukaryotic oxidative phosphorylation in mitochondria. This process couples the reduction of electron carriers during metabolism to the reduction of molecular oxygen to water and translocation of protons from the internal mitochondrial matrix to the inter-membrane space. The electrochemical gradient formed is used to generate chemical energy in the form of adenosine triphosphate to power vital cellular processes. Cytochrome c oxidase and most oxidative phosphorylation complexes are the product of the nuclear and mitochondrial genomes. This poses a series of topological and temporal steps that must be completed to ensure efficient assembly of the functional enzyme. Many assembly factors have evolved to perform these steps for insertion of protein into the inner mitochondrial membrane, maturation of the polypeptide, incorporation of co-factors and prosthetic groups and to regulate this process. Much of the information about each of these assembly factors has been gleaned from use of the single cell eukaryote Saccharomyces cerevisiae and also mutations responsible for human disease. This review will focus on the assembly factors of cytochrome c oxidase to highlight some of the outstanding questions in the assembly of this vital enzyme complex.
    [Show full text]