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Characterization of the endopeptidase activity of tripeptidyl-peptidase II
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Interfaculty institute of Biochemistry, University of Tübingen.
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2012 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 424, no 3, 503-507 p.Article in journal (Refereed) Published
Abstract [en]

Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity.

Place, publisher, year, edition, pages
2012. Vol. 424, no 3, 503-507 p.
Keyword [en]
TPP II, endopeptidase, alternate activity
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-134620DOI: 10.1016/j.bbrc.2012.06.144ISI: 000307618800024OAI: oai:DiVA.org:uu-134620DiVA: diva2:373151
Available from: 2010-11-30 Created: 2010-11-30 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Interpreting a Giant: Studies of Structure and Function of Tripeptidyl-peptidase II
Open this publication in new window or tab >>Interpreting a Giant: Studies of Structure and Function of Tripeptidyl-peptidase II
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine peptidase that forms a gigantic homooligomeric complex, and is involved in the degradation of peptides in the cytosol. In addition, TPP II has been implicated in specific cellular processes, such as apoptosis control and adipogenesis, but if this is dependent on its endo- or exopeptidase activity remains to be determined. This work is devoted to the structure and function of TPP II, and to finding connections between the two.

Evolutionarily conserved regions of TPP II have been identified, and sequence signatures have been constructed as an aid in identification of TPP II homologues. The conserved regions highlight amino acid residues of potential importance to structure, function or both. In addition, the first TPP II homologue in a prokaryote has been documented, which was likely the result of a horizontal gene transfer.

Substrate binding for the exopeptidase activity of TPP II has been studied through mutagenesis of Glu-331, which revealed a molecular ruler mechanism that positions substrates for cleavage at the third peptide bond from the N-terminus. Thus, the well-known tripeptidyl-releasing property of TPP II could be explained. The exopeptidase activity was also probed by pH dependence studies, which revealed that a substrate with a smaller residue in the P1 position could bind non-productively to the active site. Furthermore, a difference in the pH dependence of KM between TPP II from Drosophila and homologues from mammals indicated a difference in the configuration of the binding pockets between these species.

The endopeptidase activity of TPP II has also been investigated, and was found to differ from the exopeptidase activity. The endopeptidase activity appeared to be promiscuous and the preference for basic amino acid residues in the P1 position reported earlier could not be substantiated.

In conclusion, many structural and mechanistic features have been observed in this work. This might be of value to future drug discovery efforts towards TPP II, and in elucidating the physiological role of this gigantic enzyme.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 45 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 791
Keyword
Tripeptidyl-peptidas II, molecular ruler, sequence signatures, pH-dependence, endopeptidase
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-134633 (URN)978-91-554-7966-4 (ISBN)
Public defence
2011-01-21, B7:101a, BMC, Husargatan 3, Uppsala, 09:15 (English)
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Note
Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 721Available from: 2010-12-22 Created: 2010-11-30 Last updated: 2011-03-21Bibliographically approved

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Dogan, JakobJemth, PerTomkinson, Birgitta

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