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Virtanen, Anders
Publications (10 of 23) Show all publications
Liljeruhm, J., Funk, S. K., Tietscher, S., Edlund, A. D., Jamal, S., Yuen, P., . . . Forster, A. C. (2018). Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. Journal of Biological Engineering, 12, Article ID 8.
Open this publication in new window or tab >>Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology
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2018 (English)In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 12, article id 8Article in journal (Refereed) Published
Abstract [en]

Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.

Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.

Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.

Place, publisher, year, edition, pages
BIOMED CENTRAL LTD, 2018
Keywords
Chromoprotein, Fluorescent protein, Coral, Escherichia coli, Genetic marker, Reporter gene, Integration, Fitness cost, BioBrick, iGEM
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-356454 (URN)10.1186/s13036-018-0100-0 (DOI)000432246200001 ()29760772 (PubMedID)
Funder
VINNOVASwedish Research Council, 349-2006-267Swedish Research Council, 2011-5787Swedish Research Council, 2016-1Swedish Research Council, 2017-04148Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-07-30 Created: 2018-07-30 Last updated: 2018-07-30Bibliographically approved
Nosrati, M., Solbak, S., Nordesjö, O., Nissbeck, M., Dourado, D. F. A., Andersson, K. G., . . . Flores, S. C. (2017). Insights from engineering the Affibody-Fc interaction with a computational-experimental method. Protein Engineering Design & Selection, 30(9), 593-601
Open this publication in new window or tab >>Insights from engineering the Affibody-Fc interaction with a computational-experimental method
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2017 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 30, no 9, p. 593-601Article in journal (Refereed) Published
Abstract [en]

The interaction between the Staphylococcal Protein A (SpA) domain B (the basis of the Affibody) molecule and the Fc of IgG is key to the use of Affibodies in affinity chromatography and in potential therapies against certain inflammatory diseases. Despite its importance and four-decade history, to our knowledge this interaction has never been affinity matured. We elucidate reasons why single-substitutions in the SpA which improve affinity to Fc may be very rare, and also discover substitutions which potentially serve several engineering purposes. We used a variation of FoldX to predict changes in protein-protein-binding affinity, and produce a list of 41 single-amino acid substitutions on the SpA molecule, of which four are near wild type (wt) and five are at most a factor of four from wt affinity. The nine substitutions include one which removes lysine, and several others which change charge. Subtle modulations in affinity may be useful for modifying column elution conditions. The method is applicable to other protein-protein systems, providing molecular insights with lower workload than existing experimental techniques.

Keywords
Staphylococcal Protein A, affinity, computational prediction, protein-protein interactions, surface plasmon resonance
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-337576 (URN)10.1093/protein/gzx023 (DOI)000413767000004 ()28472513 (PubMedID)
Funder
Swedish Research Council, D0571301
Available from: 2018-01-02 Created: 2018-01-02 Last updated: 2018-02-16Bibliographically approved
Niedzwiecka, A., Nilsson, P., Worch, R., Stepinski, J., Darzynkiewicz, E. & Virtanen, A. (2016). Molecular recognition of mRNA 5' cap by 3' poly(A)-specific ribonuclease (PARN) differs from interactions known for other cap-binding proteins. Biochimica et Biophysica Acta - Proteins and Proteomics, 1864(4), 331-345
Open this publication in new window or tab >>Molecular recognition of mRNA 5' cap by 3' poly(A)-specific ribonuclease (PARN) differs from interactions known for other cap-binding proteins
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2016 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1864, no 4, p. 331-345Article in journal (Refereed) Published
Abstract [en]

The mRNA 5' cap structure plays a pivotal role in coordination of eukaryotic translation and mRNA degradation. Poly(A)-specific ribonuclease (PARN) is a dimeric exoribonuclease that efficiently degrades mRNA 3' poly(A) tails while also simultaneously interacting with the mRNA 5' cap. The cap binding amplifies the processivity of PARN action. We used surface plasmon resonance kinetic analysis, quantitative equilibrium fluorescence titrations and circular dichroism to study the cap binding properties of PARN. The molecular mechanism of 5' cap recognition by PARN has been demonstrated to differ from interactions seen for other known cap-binding proteins in that: i) the auxiliary biological function of 5' cap binding by the 3' degrading enzyme is accomplished by negative cooperativity of PARN dimer subunits; ii) non-coulombic interactions are major factors in the complex formation; and iii) PARN has versatile activity toward alternative forms of the cap. These characteristics contribute to stabilization of the PARN cap complex needed for the deadenylation processivity. Our studies provide a consistent biophysical basis for elucidation of the processive mechanism of PARN-mediated 3' mRNA deadenylation and provide a new framework to interpret the role of the 5' cap in mRNA degradation.

Keywords
PARN, mRNA 5 ' cap, Protein-ligand interactions, Fluorescence spectroscopy, Surface plasmon resonance, Circular dichroism
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-282786 (URN)10.1016/j.bbapap.2016.01.002 (DOI)000371551700002 ()26772900 (PubMedID)
Funder
Swedish Research Council
Available from: 2016-04-13 Created: 2016-04-07 Last updated: 2017-11-30Bibliographically approved
Dhanraj, S., Gunja, S. M., Deveau, A. P., Nissbeck, M., Boonyawat, B., Coombs, A. J., . . . Dror, Y. (2015). Bone Marrow Failure and Developmental Delay Caused By Mutations in Poly(A)-Specific Ribonuclease. Paper presented at 57th Annual Meeting of the American-Society-of-Hematology, DEC 05-08, 2015, Orlando, FL. Blood, 126(23)
Open this publication in new window or tab >>Bone Marrow Failure and Developmental Delay Caused By Mutations in Poly(A)-Specific Ribonuclease
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2015 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 126, no 23Article in journal, Meeting abstract (Other academic) Published
National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-284674 (URN)000368020101242 ()
Conference
57th Annual Meeting of the American-Society-of-Hematology, DEC 05-08, 2015, Orlando, FL
Available from: 2016-04-19 Created: 2016-04-19 Last updated: 2017-11-30Bibliographically approved
Dhanraj, S., Gunja, S. M., Deveau, A. P., Nissbeck, M., Boonyawat, B., Coombs, A. J., . . . Dror, Y. (2015). Bone marrow failure and developmental delay caused by mutations in poly(A)-specific ribonuclease (PARN). Journal of Medical Genetics, 52(11), 738-748
Open this publication in new window or tab >>Bone marrow failure and developmental delay caused by mutations in poly(A)-specific ribonuclease (PARN)
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2015 (English)In: Journal of Medical Genetics, ISSN 0022-2593, E-ISSN 1468-6244, Vol. 52, no 11, p. 738-748Article in journal (Refereed) Published
Abstract [en]

Background Deadenylation regulates RNA function and fate. Poly(A)-specific ribonuclease (PARN) is a deadenylase that processes mRNAs and non-coding RNA. Little is known about the biological significance of germline mutations in PARN. Methods We identified mutations in PARN in patients with haematological and neurological manifestations. Genomic, biochemical and knockdown experiments in human marrow cells and in zebrafish have been performed to clarify the role of PARN in the human disease. Results We identified large monoallelic deletions in PARN in four patients with developmental delay or mental illness. One patient in particular had a severe neurological phenotype, central hypomyelination and bone marrow failure. This patient had an additional missense mutation on the non-deleted allele and severely reduced PARN protein and deadenylation activity. Cells from this patient had impaired oligoadenylation of specific H/ACA box small nucleolar RNAs. Importantly, PARN-deficient patient cells manifested short telomeres and an aberrant ribosome profile similar to those described in some variants of dyskeratosis congenita. Knocking down PARN in human marrow cells and zebrafish impaired haematopoiesis, providing further evidence for a causal link with the human disease. Conclusions Large monoallelic mutations of PARN can cause developmental/mental illness. Biallelic PARN mutations cause severe bone marrow failure and central hypomyelination.

Keywords
Genetics, Haematology (incl Blood transfusion), Copy-number, Molecular genetics, Neurology
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-269251 (URN)10.1136/jmedgenet-2015-103292 (DOI)000364633100003 ()26342108 (PubMedID)
Funder
Swedish Research Council
Available from: 2015-12-17 Created: 2015-12-15 Last updated: 2018-01-10Bibliographically approved
Ren, Y.-G., Henriksson, N. & Virtanen, A. (2014). Identification of Divalent Metal Ion Binding Sites in RNA/DNA-Metabolizing Enzymes by Fe(II)-Mediated Hydroxyl Radical Cleavage (2ed.). In: Roland K. Hartmann, Albrecht Bindereif, Astrid Schön and Eric Westhof (Ed.), Handbook of RNA Biochemistry: Second, Completely Revised and Enlarged Edition (pp. 397-406). Wiley-Blackwell, 1-2
Open this publication in new window or tab >>Identification of Divalent Metal Ion Binding Sites in RNA/DNA-Metabolizing Enzymes by Fe(II)-Mediated Hydroxyl Radical Cleavage
2014 (English)In: Handbook of RNA Biochemistry: Second, Completely Revised and Enlarged Edition / [ed] Roland K. Hartmann, Albrecht Bindereif, Astrid Schön and Eric Westhof, Wiley-Blackwell, 2014, 2, Vol. 1-2, p. 397-406Chapter in book (Refereed)
Place, publisher, year, edition, pages
Wiley-Blackwell, 2014 Edition: 2
Keywords
3′ exonuclease, 3′ exonucleolytic site of Escherichia coli DNA polymerase I, Bovine heart protein kinase A catalytic subunit, Displacement of metal ions by aminoglycosides, Displacement of Mg(II) by Fe(II) ions, Fe(II)-mediated hydroxyl radical cleavage of proteins, Fenton reaction, Identification of divalent metal ion binding sites in proteins, Klenow (Pol) fragment, Labeling of polypeptide with 32P, Metalloenzymes, Poly(A)-specific ribonuclease (PARN), Protein kinase recognition motif, RNA/DNA-metabolizing enzymes
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-307112 (URN)10.1002/9783527647064.ch19 (DOI)2-s2.0-84977654207 (Scopus ID)9783527647064 (ISBN)9783527327645 (ISBN)
Available from: 2016-11-21 Created: 2016-11-08 Last updated: 2017-01-04Bibliographically approved
Virtanen, A., Henriksson, N., Nilsson, P. & Nissbeck, M. (2013). Poly(A)-specific ribonuclease (PARN): An allosterically regulated, processive and mRNA cap-interacting deadenylase. Critical reviews in biochemistry and molecular biology, 48(2), 192-209
Open this publication in new window or tab >>Poly(A)-specific ribonuclease (PARN): An allosterically regulated, processive and mRNA cap-interacting deadenylase
2013 (English)In: Critical reviews in biochemistry and molecular biology, ISSN 1040-9238, E-ISSN 1549-7798, Vol. 48, no 2, p. 192-209Article, review/survey (Refereed) Published
Abstract [en]

Deadenylation of eukaryotic mRNA is a mechanism critical for mRNA function by influencing mRNA turnover and efficiency of protein synthesis. Here, we review poly(A)-specific ribonuclease (PARN), which is one of the biochemically best characterized deadenylases. PARN is unique among the currently known eukaryotic poly(A) degrading nucleases, being the only deadenylase that has the capacity to directly interact during poly(A) hydrolysis with both the m 7 G-cap structure and the poly(A) tail of the mRNA. In short, PARN is a divalent metal-ion dependent poly(A)-specific, processive and cap-interacting 3'-5' exoribonuclease that efficiently degrades poly(A) tails of eukaryotic mRNAs. We discuss in detail the mechanisms of its substrate recognition, catalysis, allostery and processive mode of action. On the basis of biochemical and structural evidence, we present and discuss a working model for PARN action. Models of regulation of PARN activity by trans-acting factors are discussed as well as the physiological relevance of PARN.

Keywords
Deadenylase, mRNA degradation, mRNA poly(A) tail degradation, mRNA turnover, ribonuclease
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-199609 (URN)10.3109/10409238.2013.771132 (DOI)000316955000006 ()
Available from: 2013-05-08 Created: 2013-05-08 Last updated: 2017-12-06Bibliographically approved
Virtanen, A., Henriksson, N., Nilsson, P. & Lindell, M. (2012). Mechanism of processive and cap-stimulated mRNA poly(A) tail degradation. Paper presented at Experimental Biology Meeting, APR 21-25, 2012, San Diego, CA, USA. The FASEB Journal, 26, 950.3
Open this publication in new window or tab >>Mechanism of processive and cap-stimulated mRNA poly(A) tail degradation
2012 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, p. 950.3-Article in journal, Meeting abstract (Other academic) Published
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-190158 (URN)000310711306919 ()
Conference
Experimental Biology Meeting, APR 21-25, 2012, San Diego, CA, USA
Available from: 2013-01-07 Created: 2013-01-07 Last updated: 2017-12-06Bibliographically approved
Thuresson, A.-C., Kirsebom, L. A. & Virtanen, A. (2007). Inhibition of poly(A) polymerase by aminoglycosides. Biochimie, 89(10), 1221-1227
Open this publication in new window or tab >>Inhibition of poly(A) polymerase by aminoglycosides
2007 (English)In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 89, no 10, p. 1221-1227Article in journal (Refereed) Published
Abstract [en]

Aminoglycosides are potent inhibitors of bacterial growth and are used clinically as antibiotics. However, their usage has declined in recent years due to the emergence of resistance and severe toxic side effects. Here we show that human poly(A) polymerase gamma (PAPgamma) is inhibited by aminoglycosides. The inhibition was pH dependent and could be released by Mg(II) ions in a competitive manner suggesting that electrostatic interactions are important for inhibition and that the binding sites for aminoglycosides overlap with Mg(II) ion binding sites. Kinetic analysis revealed that aminoglycosides of the neomycin and kanamycin families behaved as mixed non-competitive inhibitors for the PAPgamma substrates oligoA15 and ATP. Interestingly, sisomicin and 5-epi-sisomycin showed a competitive mechanism of inhibition for the oligoA15 whereas they inhibited the ATP substrate mixed non-competitive. This implies that different aminoglycosides bind in different ways to a common binding pocket and suggests that the binding sites for related aminoglycosides are not overlapping even if they may share molecular determinants. Our study emphasizes the possibility that aminoglycoside toxicity could be due to interference with housekeeping enzymes involved in breaking and forming phosphodiester bonds.

Keywords
Polyadenylation, aminoglycosides, inhibition, divalent metal ions
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-106236 (URN)10.1016/j.biochi.2007.06.001 (DOI)000250613600008 ()17629605 (PubMedID)
Available from: 2009-06-17 Created: 2009-06-17 Last updated: 2017-12-13Bibliographically approved
Kirsebom, L. A., Virtanen, A. & Mikkelsen, N.-E. (2006). Aminoglycoside interactions with RNAs and nucleases. In: RNA towards medicine (pp. 23). : Springer Verlag
Open this publication in new window or tab >>Aminoglycoside interactions with RNAs and nucleases
2006 (English)In: RNA towards medicine, Springer Verlag , 2006, p. 23-Chapter in book (Other scientific)
Place, publisher, year, edition, pages
Springer Verlag, 2006
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-77394 (URN)0171-2004 (ISBN)
Available from: 2006-12-11 Created: 2006-12-11
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