6
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      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

          The covalent addition of ubiquitin to target proteins is a key post-translational modification that is linked to a myriad of biological processes. Here, we report a fast, single-molecule, and label-free method to probe the ubiquitination of proteins employing an engineered Cytolysin A (ClyA) nanopore. We show that ionic currents can be used to recognize mono- and polyubiquitinated forms of native proteins under physiological conditions. Using defined conjugates, we also show that isomeric monoubiquitinated proteins can be discriminated. The nanopore approach allows following the ubiquitination reaction in real time, which will accelerate the understanding of fundamental mechanisms linked to protein ubiquitination.

          Related collections

          Most cited references43

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

          Ubiquitination in disease pathogenesis and treatment.

          Ubiquitination is crucial for a plethora of physiological processes, including cell survival and differentiation and innate and adaptive immunity. In recent years, considerable progress has been made in the understanding of the molecular action of ubiquitin in signaling pathways and how alterations in the ubiquitin system lead to the development of distinct human diseases. Here we describe the role of ubiquitination in the onset and progression of cancer, metabolic syndromes, neurodegenerative diseases, autoimmunity, inflammatory disorders, infection and muscle dystrophies. Moreover, we indicate how current knowledge could be exploited for the development of new clinical therapies.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter.

            The detection of organic molecules is important in many areas, including medicine, environmental monitoring and defence. Stochastic sensing is an approach that relies on the observation of individual binding events between analyte molecules and a single receptor. Engineered transmembrane protein pores are promising sensor elements for stochastic detection, and in their simplest manifestation they produce a fluctuating binary ('on/off') response in the transmembrane electrical current. The frequency of occurrence of the fluctuations reveals the concentration of the analyte, and its identity can be deduced from the characteristic magnitude and/or duration of the fluctuations. Genetically engineered versions of the bacterial pore-forming protein alpha-haemolysin have been used to identify and quantify divalent metal ions in solution. But it is not immediately obvious how versatile binding sites for organic ligands might be obtained by engineering of the pore structure. Here we show that stochastic sensing of organic molecules can be procured from alpha-haemolysin by equipping the channel with an internal, non-covalently bound molecular 'adapter' which mediates channel blocking by the analyte. We use cyclodextrins as the adapters because these fit comfortably inside the pore and present a hydrophobic cavity suitable for binding a variety of organic analytes. Moreover, a single sensing element of this sort can be used to analyse a mixture of organic molecules with different binding characteristics. We envisage the use of other adapters, so that the pore could be 'programmed' for a range of sensing functions.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              The structure of a cytolytic alpha-helical toxin pore reveals its assembly mechanism.

              Pore-forming toxins (PFTs) are a class of potent virulence factors that convert from a soluble form to a membrane-integrated pore. They exhibit their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores. Among the group of bacterial PFTs are some of the most dangerous toxins, such as diphtheria and anthrax toxin. Examples of eukaryotic PFTs are perforin and the membrane-attack complex, proteins of the immune system. PFTs can be subdivided into two classes, alpha-PFTs and beta-PFTs, depending on the suspected mode of membrane integration, either by alpha-helical or beta-sheet elements. The only high-resolution structure of a transmembrane PFT pore is available for a beta-PFT--alpha-haemolysin from Staphylococcus aureus. Cytolysin A (ClyA, also known as HlyE), an alpha-PFT, is a cytolytic -helical toxin responsible for the haemolytic phenotype of several Escherichia coli and Salmonella enterica strains. ClyA is cytotoxic towards cultured mammalian cells, induces apoptosis of macrophages and promotes tissue pervasion. Electron microscopic reconstructions demonstrated that the soluble monomer of ClyA must undergo large conformational changes to form the transmembrane pore. Here we report the 3.3 A crystal structure of the 400 kDa dodecameric transmembrane pore formed by ClyA. The tertiary structure of ClyA protomers in the pore is substantially different from that in the soluble monomer. The conversion involves more than half of all residues. It results in large rearrangements, up to 140 A, of parts of the monomer, reorganization of the hydrophobic core, and transitions of -sheets and loop regions to -helices. The large extent of interdependent conformational changes indicates a sequential mechanism for membrane insertion and pore formation.
                Bookmark

                Author and article information

                Journal
                ACS Nano
                ACS Nano
                nn
                ancac3
                ACS Nano
                American Chemical Society
                1936-0851
                1936-086X
                29 March 2017
                23 May 2017
                : 11
                : 5
                : 4387-4394
                Affiliations
                []Chemical Biology I, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , 9747 AG Groningen, The Netherlands
                []Department of Chemistry, University of Leuven , 3001 Leuven, Belgium
                [§ ]Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , 9747 AG Groningen, The Netherlands
                Author notes
                Article
                10.1021/acsnano.6b07760
                5444049
                28353339
                064aac38-bfa7-4b22-be05-d1c363698986
                Copyright © 2017 American Chemical Society

                This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes.

                History
                : 17 November 2016
                : 17 March 2017
                Categories
                Article
                Custom metadata
                nn6b07760
                nn-2016-07760j

                Nanotechnology
                nanotechnology,nanopore,single-molecule kinetics,protein modifications,ubiquitin
                Nanotechnology
                nanotechnology, nanopore, single-molecule kinetics, protein modifications, ubiquitin

                Comments

                Comment on this article