Single-Molecule Force Spectroscopy on the N2A Element of Titin : Effects of Phosphorylation and CARP

Lade...
Vorschaubild
Dateien
Lanzicher_2-lbfx9948my7r5.pdf
Lanzicher_2-lbfx9948my7r5.pdfGröße: 3.51 MBDownloads: 215
Datum
2020
Autor:innen
Lanzicher, Thomas
Saripalli, Chandra
Keschrumrus, Vic
Smith, John E.
Sbaizero, Orfeo
Granzier, Henk
Herausgeber:innen
Kontakt
ISSN der Zeitschrift
Electronic ISSN
ISBN
Bibliografische Daten
Verlag
Schriftenreihe
Auflagebezeichnung
ArXiv-ID
Internationale Patentnummer
Link zur Lizenz
Angaben zur Forschungsförderung
Projekt
Open Access-Veröffentlichung
Open Access Gold
Sammlungen
Core Facility der Universität Konstanz
Gesperrt bis
Titel in einer weiteren Sprache
Forschungsvorhaben
Organisationseinheiten
Zeitschriftenheft
Publikationstyp
Zeitschriftenartikel
Publikationsstatus
Published
Erschienen in
Frontiers in Physiology. Frontiers Research Foundation. 2020, 11, 173. eISSN 1664-042X. Available under: doi: 10.3389/fphys.2020.00173
Zusammenfassung

Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104-residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). The N2A element is of interest because it assembles a signalosome with CARP (Cardiac Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2A-Us and I81 and is highly upregulated in response to mechanical stress. The mechanical properties of the N2A element were studied using single-molecule force spectroscopy, including how these properties are affected by CARP and phosphorylation. Three protein constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80 and I81 domains and with specific handles at their ends for study by atomic force microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence length (Lp) of ∼0.35 nm and contour length (Lc) of ∼39 nm. CARP increased the Lp of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest that CARP might function as a molecular chaperone that protects I81 from unfolding when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite (Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81. AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor the Ig domain unfolding force (Funfold). Simulating the force-sarcomere length relation of a single titin molecule containing all spring elements showed that the compliance of the N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element has a mechanical function per se. Instead, it is likely that this compliance has local effects on binding of signaling molecules and that it contributes thereby to strain- and phosphorylation- dependent mechano-signaling.

Zusammenfassung in einer weiteren Sprache
Fachgebiet (DDC)
570 Biowissenschaften, Biologie
Schlagwörter
Konferenz
Rezension
undefined / . - undefined, undefined
Zitieren
ISO 690LANZICHER, Thomas, Tiankun ZHOU, Chandra SARIPALLI, Vic KESCHRUMRUS, John E. SMITH, Olga MAYANS, Orfeo SBAIZERO, Henk GRANZIER, 2020. Single-Molecule Force Spectroscopy on the N2A Element of Titin : Effects of Phosphorylation and CARP. In: Frontiers in Physiology. Frontiers Research Foundation. 2020, 11, 173. eISSN 1664-042X. Available under: doi: 10.3389/fphys.2020.00173
BibTex
@article{Lanzicher2020Singl-49714,
  year={2020},
  doi={10.3389/fphys.2020.00173},
  title={Single-Molecule Force Spectroscopy on the N2A Element of Titin : Effects of Phosphorylation and CARP},
  volume={11},
  journal={Frontiers in Physiology},
  author={Lanzicher, Thomas and Zhou, Tiankun and Saripalli, Chandra and Keschrumrus, Vic and Smith, John E. and Mayans, Olga and Sbaizero, Orfeo and Granzier, Henk},
  note={Article Number: 173}
}
RDF
<rdf:RDF
    xmlns:dcterms="http://purl.org/dc/terms/"
    xmlns:dc="http://purl.org/dc/elements/1.1/"
    xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#"
    xmlns:bibo="http://purl.org/ontology/bibo/"
    xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#"
    xmlns:foaf="http://xmlns.com/foaf/0.1/"
    xmlns:void="http://rdfs.org/ns/void#"
    xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > 
  <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/49714">
    <dc:creator>Smith, John E.</dc:creator>
    <dcterms:title>Single-Molecule Force Spectroscopy on the N2A Element of Titin : Effects of Phosphorylation and CARP</dcterms:title>
    <dc:creator>Lanzicher, Thomas</dc:creator>
    <dc:contributor>Smith, John E.</dc:contributor>
    <foaf:homepage rdf:resource="http://localhost:8080/"/>
    <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/49714/1/Lanzicher_2-lbfx9948my7r5.pdf"/>
    <dc:contributor>Sbaizero, Orfeo</dc:contributor>
    <dcterms:issued>2020</dcterms:issued>
    <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/49714/1/Lanzicher_2-lbfx9948my7r5.pdf"/>
    <dc:contributor>Zhou, Tiankun</dc:contributor>
    <dc:contributor>Lanzicher, Thomas</dc:contributor>
    <dc:creator>Mayans, Olga</dc:creator>
    <dcterms:rights rdf:resource="http://creativecommons.org/licenses/by/4.0/"/>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2020-05-28T08:56:17Z</dc:date>
    <dc:creator>Zhou, Tiankun</dc:creator>
    <dcterms:abstract xml:lang="eng">Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104-residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). The N2A element is of interest because it assembles a signalosome with CARP (Cardiac Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2A-Us and I81 and is highly upregulated in response to mechanical stress. The mechanical properties of the N2A element were studied using single-molecule force spectroscopy, including how these properties are affected by CARP and phosphorylation. Three protein constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80 and I81 domains and with specific handles at their ends for study by atomic force microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence length (Lp) of ∼0.35 nm and contour length (Lc) of ∼39 nm. CARP increased the Lp of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest that CARP might function as a molecular chaperone that protects I81 from unfolding when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite (Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81. AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor the Ig domain unfolding force (F&lt;sub&gt;unfold&lt;/sub&gt;). Simulating the force-sarcomere length relation of a single titin molecule containing all spring elements showed that the compliance of the N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element has a mechanical function per se. Instead, it is likely that this compliance has local effects on binding of signaling molecules and that it contributes thereby to strain- and phosphorylation- dependent mechano-signaling.</dcterms:abstract>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dc:creator>Saripalli, Chandra</dc:creator>
    <dc:contributor>Mayans, Olga</dc:contributor>
    <dc:contributor>Saripalli, Chandra</dc:contributor>
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2020-05-28T08:56:17Z</dcterms:available>
    <dc:contributor>Keschrumrus, Vic</dc:contributor>
    <dc:creator>Granzier, Henk</dc:creator>
    <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/49714"/>
    <dc:creator>Keschrumrus, Vic</dc:creator>
    <dc:creator>Sbaizero, Orfeo</dc:creator>
    <dc:contributor>Granzier, Henk</dc:contributor>
    <dc:language>eng</dc:language>
    <dc:rights>Attribution 4.0 International</dc:rights>
  </rdf:Description>
</rdf:RDF>
Interner Vermerk
xmlui.Submission.submit.DescribeStep.inputForms.label.kops_note_fromSubmitter
Kontakt
URL der Originalveröffentl.
Prüfdatum der URL
Prüfungsdatum der Dissertation
Finanzierungsart
Kommentar zur Publikation
Allianzlizenz
Corresponding Authors der Uni Konstanz vorhanden
Internationale Co-Autor:innen
Universitätsbibliographie
Ja
Begutachtet
Ja
Diese Publikation teilen