Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Structural and functional studies of a novel Botulinum neurotoxin and of MTH1
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-0010-5091
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

X-ray crystallography visualizes the three dimensional molecular structures of proteins at atomic resolution. Seeing the molecular structure of a biomedically interesting protein enables a higher understanding of its function. The process of producing pure protein from genetic material to generate crystals and determine the molecular structure can be a long and challenging process. My thesis involves structural and functional studies of two different proteins, which are both biomedically interesting and important to learn about. X-ray crystallography is the method which has been used to determine the majority of the protein structures that we know of today and is also the method used in the results presented in my thesis. 

Today there are no cancer therapies defeating all types of cancers and they do not come without side effects. Battling cancer diseases often include long and painful treatments. Finding an anti-cancer drug targeting phenotypes characteristic of cancer cells is a compelling thought. MutT homolog-1 (MTH1) is an enzyme present in all proliferating cells. The enzyme seems to be crucial for cancer cell survival but not for the viability of normal cells. MTH1 cleans out oxidized and thereby damaged nucleotides from the free nucleotide pool and stops them from being used in DNA synthesis. This process is very important in fast proliferating cancer cells. The hypothesis is to inhibit MTH1 and thereby allow a limitless amount of DNA damage in the cancer cells. This action will eventually kill cancer cells while not affecting normal cells. The molecular structure of MTH1 with (PDB ID: 3ZR0) and without a product bound (PDB ID: 3ZR1) was determined and is presented in my thesis. These two structures aided in the synthesis of inhibitors. 

Botulinum neurotoxins (BoNTs) are the most potent toxins known. As little as one gram of pure toxin could potentially kill one million people. Due to its potency BoNT is a potential  bioterrorism threat. The toxin is also a very potent drug used clinically to relieve the symptoms of an array of neuromuscular disorders. Most people know this neurotoxin by one of its commercial names: Botox™. Additionally BoNTs are the cause of botulism. BoNTs are neuro-specific enzymes that target neuromuscular signaling, inducing flaccid paralysis and potentially death. It is of importance to learn more about these toxins to enable the development of new countermeasures, vaccines or more efficient neuroparalytic drugs. BoNTs consist of three domains with different functions, all crucial for intoxication. The toxins are fragile and can easily be destroyed by harsh surroundings if not protected by non-toxic non-hemagglutinin (NTNH) proteins. The complex of some BoNT serotypes and their protective NTNH have proven to be pH-dependent. Parts of the intoxication process are not yet clear and their mechanisms are still puzzling researchers. Until recently seven BoNT serotypes were identified. We have now identified and characterized a novel serotype called BoNT/X. The molecular structure of the active domain is presented here (PDB ID: 6F47). The pH-dependent mechanism forming a complex as seen in other serotypes, is confirmed to be present in BoNT/X as well.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2018. , p. 50
Keywords [en]
Botulinum neurotoxin, botulism, cancer, gene cluster, molecular structures, MTH1, NTNH, oxidised nucleotides, progenitor complex, protein expression, protein purification, X-ray crystallography
National Category
Other Chemistry Topics
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-159295ISBN: 978-91-7797-384-3 (print)ISBN: 978-91-7797-385-0 (electronic)OAI: oai:DiVA.org:su-159295DiVA, id: diva2:1241924
Public defence
2018-10-10, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2018-09-17 Created: 2018-08-26 Last updated: 2018-09-17Bibliographically approved
List of papers
1. Crystal structure of human MTH1 and the 8-oxo-dGMP product complex
Open this publication in new window or tab >>Crystal structure of human MTH1 and the 8-oxo-dGMP product complex
Show others...
2011 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 585, no 16, p. 2617-2621Article in journal (Refereed) Published
Abstract [en]

MTH1 hydrolyzes oxidized nucleotide triphosphates, thereby preventing them from being incorporated into DNA. We here present the structures of human MTH1 (1.9 angstrom) and its complex with the product 8-oxo-dGMP (1.8 angstrom). Unexpectedly MTH1 binds the nucleotide in the anti conformation with no direct interaction between the 8-oxo group and the protein. We suggest that the specificity depends on the stabilization of an enol tautomer of the 8-oxo form of dGTP. The binding of the product induces no major structural changes. The structures reveal the mode of nucleotide binding in MTH1 and provide the structural basis for inhibitor design.

Keywords
MTH1, MutT, Oxidative damage, 8-oxo-dGTPase, NUDT1, 8-oxo-dGTP, Tautomer
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-68308 (URN)10.1016/j.febslet.2011.07.017 (DOI)000293826300012 ()
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research The Wenner-Gren FoundationSwedish Cancer Society
Note

authorCount :7

Available from: 2012-01-13 Created: 2012-01-03 Last updated: 2018-08-28Bibliographically approved
2. Identification and characterization of a novel botulinum neurotoxin
Open this publication in new window or tab >>Identification and characterization of a novel botulinum neurotoxin
Show others...
2017 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 14130Article in journal (Refereed) Published
Abstract [en]

Botulinum neurotoxins are known to have seven serotypes (BoNT/A-G). Here we report a new BoNT serotype, tentatively named BoNT/X, which has the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. Similar to BoNT/B/D/F/G, BoNT/X cleaves vesicle-associated membrane proteins (VAMP) 1, 2 and 3, but at a novel site (Arg66-Ala67 in VAMP2). Remarkably, BoNT/X is the only toxin that also cleaves non-canonical substrates VAMP4, VAMP5 and Ykt6. To validate its activity, a small amount of full-length BoNT/X was assembled by linking two non-toxic fragments using a transpeptidase (sortase). Assembled BoNT/X cleaves VAMP2 and VAMP4 in cultured neurons and causes flaccid paralysis in mice. Thus, BoNT/X is a novel BoNT with a unique substrate profile. Its discovery posts a challenge to develop effective countermeasures, provides a novel tool for studying intracellular membrane trafficking, and presents a new potential therapeutic toxin for modulating secretions in cells.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-145842 (URN)10.1038/ncomms14130 (DOI)000406846300001 ()
Available from: 2017-08-24 Created: 2017-08-24 Last updated: 2018-08-27Bibliographically approved
3. Structural characterisation of the catalytic domain of botulinum neurotoxin X - high activity and unique substrate specificity
Open this publication in new window or tab >>Structural characterisation of the catalytic domain of botulinum neurotoxin X - high activity and unique substrate specificity
Show others...
2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 4518Article in journal (Refereed) Published
Abstract [en]

Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used to treat an increasing number of medical disorders. There are seven well-established serotypes (BoNT/A-G), which all act as zinc-dependent endopeptidases targeting specific members of the SNARE proteins required for synaptic vesicle exocytosis in neurons. A new toxin serotype, BoNT/X, was recently identified. It cleaves not only the canonical targets, vesicle associated membrane proteins (VAMP) 1/2/3 at a unique site, but also has the unique ability to cleave VAMP4/5 and Ykt6. Here we report the 1.35 angstrom X-ray crystal structure of the light chain of BoNT/X (LC/X). LC/X shares the core fold common to all other BoNTs, demonstrating that LC/X is a bona fide member of BoNT-LCs. We found that access to the catalytic pocket of LC/X is more restricted, and the regions lining the catalytic pocket are not conserved compared to other BoNTs. Kinetic studies revealed that LC/X cleaves VAMP1 with a ten times higher efficiency than BoNT/B and the tetanus neurotoxin. The structural information provides a molecular basis to understand the convergence/divergence between BoNT/X and other BoNTs, to develop effective LC inhibitors, and to engineer new scientific tools and therapeutic toxins targeting distinct SNARE proteins in cells.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-156106 (URN)10.1038/s41598-018-22842-4 (DOI)000427366200048 ()29540745 (PubMedID)
Available from: 2018-05-11 Created: 2018-05-11 Last updated: 2018-08-27Bibliographically approved
4. pH dependence of the minimal progenitor complex of Botulinum neurotoxin X
Open this publication in new window or tab >>pH dependence of the minimal progenitor complex of Botulinum neurotoxin X
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-157951 (URN)
Available from: 2018-06-29 Created: 2018-06-29 Last updated: 2018-09-17Bibliographically approved

Open Access in DiVA

Structural and functional studies of a novel Botulinum neurotoxin and of MTH1(6785 kB)85 downloads
File information
File name FULLTEXT01.pdfFile size 6785 kBChecksum SHA-512
040dc4b6fbff2e8c99e2ba79be17c5524818caba4e4c2f3732e055c26573d8a05152fd78360cd77943dda20c1d297462fda00bd952e8ed67ec8ed54dd5e296a2
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Henriksson, Linda
By organisation
Department of Biochemistry and Biophysics
Other Chemistry Topics

Search outside of DiVA

GoogleGoogle Scholar
Total: 85 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1545 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf