256
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.

          Related collections

          Most cited references54

          • Record: found
          • Abstract: found
          • Article: not found

          Poly(ADP-ribose): novel functions for an old molecule.

          The addition to proteins of the negatively charged polymer of ADP-ribose (PAR), which is synthesized by PAR polymerases (PARPs) from NAD(+), is a unique post-translational modification. It regulates not only cell survival and cell-death programmes, but also an increasing number of other biological functions with which novel members of the PARP family have been associated. These functions include transcriptional regulation, telomere cohesion and mitotic spindle formation during cell division, intracellular trafficking and energy metabolism.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Selection and identification of single domain antibody fragments from camel heavy-chain antibodies.

            Functional heavy-chain gamma-immunoglobulins lacking light chains occur naturally in Camelidae. We now show the feasibility of immunising a dromedary, cloning the repertoire of the variable domains of its heavy-chain antibodies and panning, leading to the successful identification of minimum sized antigen binders. The recombinant binders are expressed well in E. coli, extremely stable, highly soluble, and react specifically and with high affinity to the antigens. This approach can be viewed as a general route to obtain small binders with favourable characteristics and valuable perspectives as modular building blocks to manufacture multispecific or multifunctional chimaeric proteins.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Immunolabeling artifacts and the need for live-cell imaging.

              Fluorescent fusion proteins have revolutionized examination of proteins in living cells. Still, studies using these proteins are met with criticism because proteins are modified and ectopically expressed, in contrast to immunofluorescence studies. However, introducing immunoreagents inside cells can cause protein extraction or relocalization, not reflecting the in vivo situation. Here we discuss pitfalls of immunofluorescence labeling that often receive little attention and argue that immunostaining experiments in dead, permeabilized cells should be complemented with live-cell imaging when scrutinizing protein localization.
                Bookmark

                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                7 March 2016
                2016
                : 11
                : 3
                : e0151041
                Affiliations
                [1 ]ChromoTek GmbH, IZB, Planegg, Martinsried, Germany
                [2 ]Livestock Center of the Faculty of Veterinary Medicine, Ludwig Maximilians University, Munich, Germany
                [3 ]Department of Radiation Oncology, Ludwig Maximilians University, Munich, Germany
                [4 ]Institute for Applied Physics and Metrology, Bundeswehr University Munich, Neubiberg, Germany
                [5 ]Department of Radiation Oncology, Klinikum rechts der Isar, Technical University Munich, Munich, Germany
                [6 ]Clinical Cooperation Group ‘Personalized Radiotherapy of Head and Neck Cancer’, Helmholtz Center Munich, Neuherberg, Germany
                [7 ]Natural and Medical Institute at the University of Tuebingen, Reutlingen, Germany
                [8 ]Pharmaceutical Biotechnology, University of Tuebingen, Tuebingen, Germany
                University of Pecs Medical School, HUNGARY
                Author notes

                Competing Interests: UR and KZ are shareholders of ChromoTek GmbH. AB, LY, JB, BR and TR are employees at ChromoTek GmbH. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: AB LY TR AAF UR. Performed the experiments: AB LY JB BR KZ. Analyzed the data: AB LY KZ. Contributed reagents/materials/analysis tools: AB LY TR AAF SED GAD SG CG JR CS SN AMS. Wrote the paper: AB LY AAF UR.

                ‡ These authors also contributed equally to this work.

                Article
                PONE-D-15-28783
                10.1371/journal.pone.0151041
                4780744
                26950694
                ae0f4428-510e-4e92-9d61-16d1d90e162a
                © 2016 Buchfellner et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 8 July 2015
                : 23 February 2016
                Page count
                Figures: 8, Tables: 0, Pages: 23
                Funding
                This work was funded in part by the GoBio grant of the German Federal Ministry of Education and Research (BMBF) to ChromoTek. GoBio and ChromoTek GmbH provided support in form of salaries for authors (AB, LY, JB, BR, KZ and TR). The funder did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Microirradiation at SNAKE was partially supported by the DFG-Cluster of Excellence "Munich-Centre for Advanced Photonics".
                Categories
                Research Article
                Research and Analysis Methods
                Precipitation Techniques
                Immunoprecipitation
                Research and analysis methods
                Biological cultures
                Cell lines
                HeLa cells
                Research and analysis methods
                Biological cultures
                Cell cultures
                Cultured tumor cells
                HeLa cells
                Biology and life sciences
                Genetics
                DNA
                DNA damage
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA damage
                Biology and life sciences
                Genetics
                DNA
                DNA repair
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA repair
                Research and Analysis Methods
                Microscopy
                Light Microscopy
                Fluorescence Microscopy
                Research and Analysis Methods
                Imaging Techniques
                In Vivo Imaging
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Mammals
                Rodents
                Hamsters
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

                Uncategorized
                Uncategorized

                Comments

                Comment on this article