DOCSLIB.ORG

Genetic Definition of a Protein-Splicing Domain: Functional Mini-Inteins Support Structure Predictions and a Model for Intein Evolution

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Genetic Definition of a Protein-Splicing Domain: Functional Mini-Inteins Support Structure Predictions and a Model for Intein Evolution Proc. Natl. Acad. Sci. USA Vol. 94, pp. 11466–11471, October 1997 Genetics Genetic definition of a protein-splicing domain: Functional mini-inteins support structure predictions and a model for intein evolution VICTORIA DERBYSHIRE*, DAVID W. WOOD*†,WEI WU*, JOHN T. DANSEREAU*, JACOB Z. DALGAARD‡, AND MARLENE BELFORT*§ *Molecular Genetics Program, Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, PO Box 22002, Albany, NY 12201-2002; †Howard P. Isermann Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180-3590; and ‡National Cancer Institute–Frederick Cancer Research and Development Center, Advanced BioScience Laboratories–Basic Research Program, PO Box B, Building 549, Room 154, Frederick, MD 21701-1201 Communicated by John Abelson, California Institute of Technology, Pasadena, CA, August 19, 1997 (received for review July 1, 1997) ABSTRACT Inteins are protein-splicing elements, most two activities may have evolved independently. First, the of which contain conserved sequence blocks that define a crystal structure of PI-SceI has recently been solved (12). This family of homing endonucleases. Like group I introns that 454-aa protein is folded into two distinct structural domains. encode such endonucleases, inteins are mobile genetic ele- Second, hidden Markov models have been used to define two ments. Recent crystallography and computer modeling stud- conserved functional domains of inteins, corresponding to ies suggest that inteins consist of two structural domains that independent endonuclease and splicing modules, separated by correspond to the endonuclease and the protein-splicing nonconserved spacer regions of variable lengths (ref. 13; elements. To determine whether the bipartite structure of J.Z.D., A. Klar, M. J. Moser, W. R. Holley, A. Chatterjee, and inteins is mirrored by the functional independence of the I. S. Mian, unpublished results; Fig. 1B). Third, three putative protein-splicing domain, the entire endonuclease component inteins have recently been reported that are in the 150-aa size was deleted from the Mycobacterium tuberculosis recA intein. range and lack endonuclease motifs, although it is not clear Guided by computer modeling studies, and taking advantage whether these smaller elements retain splicing function (4, 5, of genetic systems designed to monitor intein function, the 13). Finally, a newly identified Synechocystis intein does not 440-aa Mtu recA intein was reduced to a functional mini-intein contain a LAGLIDADG endonuclease but instead contains a of 137 aa. The accuracy of splicing of several mini-inteins was member of the H-N-H family of group I intron endonucleases verified. This work not only substantiates structure predic- (13, 28). tions for intein function but also supports the hypothesis that, Site-directed mutagenesis experiments have shown that like group I introns, mobile inteins arose by an endonuclease endonuclease activity is not required for protein-splicing func- gene invading a sequence encoding a small, functional splicing tion (14, 15), and deletion of a region encompassing the element. LAGLIDADG motifs of PI-SceI has confirmed this conclusion (16). However, despite the apparent structural autonomy of Inteins are protein-splicing elements that exist as in-frame the protein-splicing and endonuclease domains of PI-SceI, they fusions with flanking protein sequences called exteins. Inteins do appear to collaborate in interacting with the homing site are self-splicing at the protein level, with their excision being DNA (12). Therefore, it is important to determine whether the coupled to extein ligation (1–3). Most of the inteins that have bipartite structure of inteins is mirrored by the functional been described are in the 400- to 500-aa range with little independence of their two components. We tested the predic- absolute sequence conservation among the elements (4, 5). tion that the entire endonuclease domain and spacer sequences However, Cys or Ser residues are required at the amino termini between the domains can be deleted from a protein-splicing of both the intein and the second extein, and a His and Asn are element to generate a mini-intein that is splicing proficient. To present at the carboxy terminus of the intein (Fig. 1A Top). this end, we used the 440-aa intein from the Mycobacterium Most inteins contain eight conserved sequence blocks (A–H), tuberculosis recA gene expressed in Escherichia coli. The Mtu two of these being the LAGLIDADG motifs (blocks C and E) recA intein contains a conventional LAGLIDADG endonu- that define a family of intron-homing endonucleases (refs. 4 clease domain (4, 5), although endonuclease activity has not and 5; Fig. 1A). Consistent with the occurrence of these motifs, yet been demonstrated. Guided by junctions inferred from several inteins have been shown to have site-specific endonu- structure models (ref. 13; Fig. 1B), a series of mini-intein clease activity (6), and PI-SceI, the VMA1 intein of Saccha- derivatives was tested in two genetic systems developed to romyces cerevisiae, is capable of homing into a cognate intein- screen for splicing activity in vivo and in vitro. A number of less allele (7). The sporadic distribution of inteins in all three mini-inteins deleted for the entire endonuclease domain were biological kingdoms is consistent with their being mobile shown to be capable of protein splicing in both contexts, elements. consistent with structure predictions. These results support the Endonuclease genes have been assumed to be invasive model that homing inteins evolved through an endonuclease genetic elements that colonized group I introns, converting gene invading a DNA sequence encoding a functional mini- them into mobile genetic elements (8–11). Similarly, mobile intein. inteins appear to be derived from invasive endonuclease genes. Recent structural studies indeed suggest that the protein- splicing and endonuclease domains are separate and that their MATERIALS AND METHODS Construction of td:Mini-Intein Fusions. Plasmid pKKtd- The publication costs of this article were defrayed in part by page charge C238-I containing the Mtu recA intein is derived from pKK223 payment. This article must therefore be hereby marked ‘‘advertisement’’ in (Pharmacia). The plasmid contains an in-frame fusion of the accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9411466-6$2.00y0 Abbreviation: TS, thymidylate synthase. PNAS is available online at http:yywww.pnas.org. §e-mail: [email protected]. 11466 Downloaded by guest on October 3, 2021 Genetics: Derbyshire et al. Proc. Natl. Acad. Sci. USA 94 (1997) 11467 FIG. 1. Structural features of inteins. (A) Intein and endonuclease maps. The top map shows the generic relationship of an intein to its flanking exteins. Conserved amino acids are circled (see text). In the intein and endonuclease maps below, conserved sequence blocks are labeled A–H, with the LAGLIDADG endonuclease motifs, C and E, boxed (4, 5). (B) Intein structures. The top schematic depicts the two domains and spacer regions of the 440-aa Mtu recA intein according to modeling predictions (ref. 13; J.Z.D., et al., unpublished data) and the structure of PI-SceI (12), with corresponding linear maps below. Solid areas, protein-splicing domain; shaded, endonuclease domain; FIG. 2. Summary of phenotypes of mini-intein constructs. (A) The open, spacer regions. The smallest functional mini-inteins from this td-based genetic system. Schematic of the TS:intein in-frame fusion study (137 aa; Fig. 2B) are shown in both representations, with the system shows precursors and splice products (Left). TS phenotype of 2 linear form below a map of the Mtu recA intein with conserved td:intein in-frame fusion derivatives (Right). Patches of TS cells R sequence blocks. (D1210DthyA::Kan ) containing plasmids expressing td:intein fusions were tested for growth on minimal medium plates at different tem- intein with the intronless T4 td gene, which encodes thymidy- peratures (27). td, no intein; td Mtu, full-length Mtu recA intein; td late synthase (TS), such that Cys-238 of TS is the N-terminal MtuAA, full-length Mtu recA intein with C-terminal His and Asn residues mutated to Ala; remainder, td:mini-intein constructs. Num- residue of the second extein (circled in Fig. 2A; D.W.W., V.D., bering in brackets is as in B.(B) Summary of protein-splicing activity. W.W., Georges Belfort, and M.B., unpublished results). Full-length and mini-intein constructs are numbered 1–21. The con- pKKtdC238-I was used as a template for inverse PCRs to structs are defined by the deletion junctions, with Ala residues inserted generate a series of plasmids for expression of TS:mini-intein in the cloning listed in parentheses [e.g., 94D383 has all residues in-frame fusion proteins. The coding sequences for the fusion between 94 and 383 deleted, whereas 101D405(A7) has residues proteins were sequenced in their entirety. between 101 and 405 deleted and replaced with 7 Ala residues]. The primers used in the PCRs carried a terminal BssHII Constructs 4, 9, 12, and 18 have Ala–Arg inserted between the junction residues as indicated by a prime. TS phenotype: 15, growth at 23°C, restriction site, such that digestion of the products with this 30°C, and 37°C; 14, growth at 23°C, 30°C, and weak growth at 37°C; enzyme and religation generated in-frame td:mini-intein fu- 13, growth at 23°C and at 30°C; 12, growth at 23°C; 11, weak growth sions with central deletions marked by a BssHII site and, at 23°C; —, no growth on minimal media. MIC splicing is quantitated hence, an Ala–Arg dipeptide. Where possible, these residues as the percentage of precursor that was converted to ligated exteins replaced the same or similar amino acids in the final fusion after3hofinduction: 15, 80–100%; 14, 60–80%; 13, 40–60%; 12, proteins.
Load more

Recommended publications
  • Control of Protein Function
    3 Control of Protein Function In the cell, precise regulation of protein function is essential to avoid chaos. This chapter describes the most important molecular mechanisms by which protein function is regulated in cells. These range from control of a protein’s location and lifetime within the cell to the binding of regulatory molecules and covalent modifications such as phosphorylation that rapidly switch protein activity on or off. Also covered here are the nucleotide-driven switches in conformation that underlie the action of motor proteins and that regulate many signal transduction pathways. 3-0 Overview: Mechanisms of Regulation 3-1 Protein Interaction Domains 3-2 Regulation by Location 3-3 Control by pH and Redox Environment 3-4 Effector Ligands: Competitive Binding and Cooperativity 3-5 Effector Ligands: Conformational Change and Allostery 3-6 Protein Switches Based on Nucleotide Hydrolysis 3-7 GTPase Switches: Small Signaling G Proteins 3-8 GTPase Switches: Signal Relay by Heterotrimeric GTPases 3-9 GTPase Switches: Protein Synthesis 3-10 Motor Protein Switches 3-11 Regulation by Degradation 3-12 Control of Protein Function by Phosphorylation 3-13 Regulation of Signaling Protein Kinases: Activation Mechanism 3-14 Regulation of Signaling Protein Kinases: Cdk Activation 3-15 Two-Component Signaling Systems in Bacteria 3-16 Control by Proteolysis: Activation of Precursors 3-17 Protein Splicing: Autoproteolysis by Inteins 3-18 Glycosylation 3-19 Protein Targeting by Lipid Modifications 3-20 Methylation, N-acetylation, Sumoylation and Nitrosylation 3-0 Overview: Mechanisms of Regulation Protein function in living cells is precisely regulated A typical bacterial cell contains a total of about 250,000 protein molecules (comprising different amounts of each of several thousand different gene products), which are packed into a volume so small that it has been estimated that, on average, they are separated from one another by a distance that would contain only a few molecules of water.
    [Show full text]
  • Enzyme-Catalyzed Expressed Protein Ligation
    ENZYME-CATALYZED EXPRESSED PROTEIN LIGATION by Samuel Henager A dissertation submitted to The Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland August, 2017 Abstract Expressed protein ligation involves the chemoselective reaction of recombinant protein thioesters produced via inteins with N-Cys containing synthetic peptides and has proved to be a valuable method for protein semisynthesis. Expressed protein ligation requires a cysteine residue at the ligation junction which can limit its use. Here we employ subtiligase, a re-engineered form of the protease subtilisin, to ligate a range of synthetic peptides, without the requirement of an N-terminal cysteine, to a variety of recombinant protein thioesters in rapid fashion. We have further broadened the scope of subtiligase-mediated protein ligations by employing a second-generation form (E156Q/G166K subtiligase) and a newly developed form (Y217K subtiligase) for ligation junctions with acidic residues. We have applied subtiligase-mediated expressed protein ligation to the generation of tetraphosphorylated, monophosphorylated, and non-phosphorylated forms of the tumor suppressor lipid phosphatase PTEN. In this way, we have demonstrated that the natural sequence around the ligation junction produced by subtiligase rather than cysteine-mediated ligation is necessary to confer the dramatic impact of tail phosphorylation on driving PTEN's closed conformation and reduced activity. We thus propose that subtiligase-mediated expressed protein ligation is an attractive traceless technology for precision analysis of protein post- translational modifications. Thesis Advisor: Dr. Philip Cole Second Reader: Dr. Jungsan Sohn ii To my family and friends, without whom none of this would have been possible.
    [Show full text]
  • NMR Evidence for an Unusual Peptide Bond at the N-Extein–Intein Junction
    Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction Alessandra Romanelli*†, Alexander Shekhtman†‡, David Cowburn‡, and Tom W. Muir*§ *Laboratory of Synthetic Protein Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021; and ‡New York Structure Biology Center, New York, NY 10027 Edited by Rowena G. Matthews, University of Michigan, Ann Arbor, MI, and approved March 9, 2004 (received for review October 13, 2003) Protein splicing is a posttranslational autocatalytic process in which (or O 3 N) acyl shift. This is known to be a spontaneous chemical an intervening sequence, termed an intein, is removed from a host rearrangement (10) and presumably does not require the intein. protein, the extein. Although we have a reasonable picture of the Although we have a reasonable overview of the various steps in basic chemical steps in protein splicing, our knowledge of how protein splicing, the mechanistic details of autocatalysis remain these are catalyzed and regulated is less well developed. In the poorly understood. There have been several high-resolution crystal current study, a combination of NMR spectroscopy and segmental structures of inteins (11–16) and related proteins (5, 7, 17). These isotopic labeling has been used to study the structure of an active structures have shed some light on the mechanism of the first step protein splicing precursor, corresponding to an N-extein fusion of in protein splicing, the N 3 S (or N 3 O) acyl shift. As illustrated ؊ 1 the Mxe GyrA intein. The JNC؅ coupling constant for the ( 1) in Fig.
    [Show full text]
  • The Splicing Factor XAB2 Interacts with ERCC1-XPF and XPG for RNA-Loop Processing During Mammalian Development
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.20.211441; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The Splicing Factor XAB2 interacts with ERCC1-XPF and XPG for RNA-loop processing during mammalian development Evi Goulielmaki1*, Maria Tsekrekou1,2*, Nikos Batsiotos1,2, Mariana Ascensão-Ferreira3, Eleftheria Ledaki1, Kalliopi Stratigi1, Georgia Chatzinikolaou1, Pantelis Topalis1, Theodore Kosteas1, Janine Altmüller4, Jeroen A. Demmers5, Nuno L. Barbosa-Morais3, George A. Garinis1,2* 1. Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology- Hellas, GR70013, Heraklion, Crete, Greece, 2. Department of Biology, University of Crete, Heraklion, Crete, Greece, 3. Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal, 4. Cologne Center for Genomics (CCG), Institute for Genetics, University of Cologne, 50931, Cologne, Germany, 5. Proteomics Center, Netherlands Proteomics Center, and Department of Biochemistry, Erasmus University Medical Center, the Netherlands. Corresponding author: George A. Garinis ([email protected]) *: equally contributing authors bioRxiv preprint doi: https://doi.org/10.1101/2020.07.20.211441; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers.
    [Show full text]
  • Site Selection of Pyrococcus Horikoshi Rada Intein
    Murray State's Digital Commons Honors College Theses Honors College Spring 5-8-2020 Site Selection of Pyrococcus horikoshi RadA Intein Madison Paige White Murray State University Follow this and additional works at: https://digitalcommons.murraystate.edu/honorstheses Part of the Biology Commons, and the Microbiology Commons Recommended Citation White, Madison Paige, "Site Selection of Pyrococcus horikoshi RadA Intein" (2020). Honors College Theses. 44. https://digitalcommons.murraystate.edu/honorstheses/44 This Thesis is brought to you for free and open access by the Honors College at Murray State's Digital Commons. It has been accepted for inclusion in Honors College Theses by an authorized administrator of Murray State's Digital Commons. For more information, please contact [email protected]. Murray State University Honors College HONORS THESIS Certificate of Approval Site Selection of Pyrococcus horikoshi RadA Intein Madison White May/2021 Approved to fulfill the _________________________________________ requirements of HON 437 Dr. Christopher Lennon, Assistant Professor Biology Approved to fulfill the _________________________________________ Honors Thesis requirement Dr. Warren Edminster, Executive Director of the Murray State Honors Honors College Diploma Examination Approval Page Author: Madison White Project Title: Site Selection of Pyrococcus horikoshi RadA Intein Department: Biology Date of Defence: April 24, 2020 Approval by Examining Committee: ____________________________________________ __________________
    [Show full text]
  • Expanding the Uses of Split-Intein Through Protein Engineering
    Expanding The Uses of Split-Intein Through Protein Engineering by Stanley Siu Cheung Wong A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Biomaterials and Biomedical Engineering University of Toronto © Copyright by Stanley Siu Cheung Wong 2013 Expanding the Uses of Split-Intein Through Protein Engineering Stanley Siu Cheung Wong Doctor of Philosophy Institute of Biomaterials and Biomedical Engineering University of Toronto 2013 Abstract Split-protein systems are invaluable tools used for the discovery and investigations of the complexities of protein functions and interactions. Split-protein systems rely on the non-covalent interactions of two fragments of a split protein to restore protein function. Because of this, they have the ability to restore protein functions post-translationally, thus allowing for quick and efficient responses to a milieu of cellular mechanisms. Despite this, split-protein systems ha ve been largely limited as a reporting tool for protein-protein interactions. The recent discovery of inteins has the potential of broadening the scope of split-protein systems. Inteins are protein elements that possess the unique ability of post-translationally ligating protein fragments together with a native peptide bond, a process termed protein splicing. This allows split-proteins to reassemble in a more natural state. Exploiting this property and utilizing protein engineering techniques and methodologies, several approaches are described here for restoring and controlling split-protein functions using inteins. ii First, the protein splicing behaviour was demonstrated with the development of a simple in vitro visual fluorescence assay that relies on examining the subcellular localization of different fluorescent proteins.
    [Show full text]
  • Split Inteins As Versatile Tools in Applications of Downstream Purification And
    Split Inteins As Versatile Tools in Applications of Downstream Purification and Bioconjugation Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yamin Fan Graduate Program in Chemical Engineering The Ohio State University 2019 Dissertation Committee David W. Wood, Advisor Shang-Tian Yang Andre Palmer Copyrighted by Yamin Fan 2019 Abstract Over the past two decades, inteins have been extensively used in a wide variety of applications in biotechnology. Split inteins are a subset of inteins, which are identified more recently and expressed in two separate segments naturally. They catalyze the splicing reaction in trans upon association of the two halves. Due to their unique features, split inteins offer improved controllability and flexibility in trans-splicing and trans- cleaving over the previous tools based on contiguous inteins. The engineered split inteins would allow the development of efficient self-cleaving affinity tags for purification applications and new methods for protein conjugation. In this work, an engineered split intein derived from Nostoc punciforme (Npu) was applied in a column-free purification strategy in combination with the aggregating tag, elastin-like polypeptide (ELP), as an initial capture step for recombinant proteins expressed in E. coli. Meanwhile, on-column purification strategy using the same engineered split intein was employed for the production of value-added biosimilar target, Granulocyte-colony stimulating factor (G-CSF). To adapt the split intein-based purification platform for the production of protein therapeutics expressed in mammalian cells, multiple leader sequences were designed and screened for optimal expression and secretion of intein-tagged precursor proteins.
    [Show full text]
  • Npgrj Nmeth 729 31..37
    ARTICLES Using protein-DNA chimeras to detect and count small numbers of molecules methods Ian Burbulis1, Kumiko Yamaguchi1, Andrew Gordon1, Robert Carlson2 & Roger Brent1 We describe general methods to detect and quantify small antibodies4. These experiments demonstrated the application of .com/nature e numbers of specific molecules. We redirected self-splicing the extraordinary sensitivity of PCR detection to non-nucleic acid protein inteins to create ‘tadpoles’, chimeric molecules molecules. Since these initial developments, other investigators .natur comprised of a protein head covalently coupled to an have described detection of DNA–linked antibodies bound to w oligonucleotide tail. We made different classes of tadpoles that targets by PCR5–8, T7 runoff transcription9 and hybridization10. bind specific targets, including Bacillus anthracis protective For a number of reasons, including in many cases the molecular antigen and the enzyme cofactor biotin. We measured the heterogeneity of the antibodies and the lack of precise chemical http://ww amount of bound target by quantifying DNA tails by T7 RNA control of the site(s) of DNA attachment, assays based on DNA– polymerase runoff transcription and real-time polymerase chain linked antibodies have not gained wide use. We envisioned that the oup r G reaction (PCR) evaluated by rigorous statistical methods. These ability to effect well-controlled synthesis of defined protein-DNA assays had a dynamic range of detection of more than 11 orders conjugates in large quantities might enable wider development of of magnitude and distinguished numbers of molecules that target-binding and amplification measurement methods. lishing differed by as little as 10%. At their low limit, these assays were Here, we devised the means to covalently link chemically b used to detect as few as 6,400 protective antigen molecules, homogeneous affinity proteins and DNA molecules by controlled Pu 600 biotin molecules and 150 biotinylated protein molecules.
    [Show full text]
  • The RNA Splicing Response to DNA Damage
    Biomolecules 2015, 5, 2935-2977; doi:10.3390/biom5042935 OPEN ACCESS biomolecules ISSN 2218-273X www.mdpi.com/journal/biomolecules/ Review The RNA Splicing Response to DNA Damage Lulzim Shkreta and Benoit Chabot * Département de Microbiologie et d’Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-819-821-8000 (ext. 75321); Fax: +1-819-820-6831. Academic Editors: Wolf-Dietrich Heyer, Thomas Helleday and Fumio Hanaoka Received: 12 August 2015 / Accepted: 16 October 2015 / Published: 29 October 2015 Abstract: The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.
    [Show full text]
  • O-Glcnacylation of Small Heat Shock Proteins Enhances Their Anti-Amyloid Chaperone Activity
    bioRxiv preprint doi: https://doi.org/10.1101/869909; this version posted December 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. O-GlcNAcylation of small heat shock proteins enhances their anti-amyloid chaperone activity Aaron T. Balana1, Paul M. Levine1, Somnath Mukherjee2, Nichole J. Pedowitz,1 Stuart P. Moon1, Terry T. Takahashi,1 Christian F. W. Becker2, and Matthew R. Pratt1,3,* 1Departments of Chemistry and 3Biological Sciences, University of Southern California, Los Angeles, California, 90089, United States 2Institute of Biological Chemistry, Faculty of Chemistry, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria *Corresponding Author: Matthew R. Pratt Email: [email protected] Abstract A major role for the intracellular posttranslational modification O-GlcNAc appears to be the inhibition of protein aggregation. Most of the previous studies in this area have focused on O-GlcNAcylation of the amyloid-forming proteins themselves. Here, we use synthetic protein chemistry to discover that O-GlcNAc also activates the anti-amyloid activity of certain small heat shock proteins (sHSPs), a potentially more important modification event that can act broadly and substoichiometrically. More specifically, we find that O-GlcNAcylation increases the ability of sHSPs to block the amyloid formation of both α-synuclein and Aβ. Mechanistically, we show that O-GlcNAc near the sHSP IXI-domain prevents its ability to intramolecularly compete with substrate binding. Our results have important implications for neurodegenerative diseases associated with amyloid formation and potentially other areas of sHSP biology.
    [Show full text]
  • Proteolytic Cleavage—Mechanisms, Function
    Review Cite This: Chem. Rev. 2018, 118, 1137−1168 pubs.acs.org/CR Proteolytic CleavageMechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational Modification Theo Klein,†,⊥ Ulrich Eckhard,†,§ Antoine Dufour,†,¶ Nestor Solis,† and Christopher M. Overall*,†,‡ † ‡ Life Sciences Institute, Department of Oral Biological and Medical Sciences, and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada ABSTRACT: Proteases enzymatically hydrolyze peptide bonds in substrate proteins, resulting in a widespread, irreversible posttranslational modification of the protein’s structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissuefrom 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C- termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the structural biology aspects and mechanisms
    [Show full text]
  • Homology Modeling and Mutational Analysis of Ho Endonuclease of Yeast
    Copyright 2004 by the Genetics Society of America Homology Modeling and Mutational Analysis of Ho Endonuclease of Yeast Anya Bakhrat,* Melissa S. Jurica,†,1 Barry L. Stoddard† and Dina Raveh*,2 *Department of Life Sciences, Ben Gurion University of the Negev, Beersheva, 84105 Israel and †Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 Manuscript received August 7, 2003 Accepted for publication October 31, 2003 ABSTRACT Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.
    [Show full text]