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Interpreting a Giant: Studies of Structure and Function of Tripeptidyl-peptidase II
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry. (Birgitta Tomkinson)
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. , p. 45
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 791
Keywords [en]
Tripeptidyl-peptidas II, molecular ruler, sequence signatures, pH-dependence, endopeptidase
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-134633ISBN: 978-91-554-7966-4 (print)OAI: oai:DiVA.org:uu-134633DiVA, id: diva2:373175
Public defence
2011-01-21, B7:101a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
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
List of papers
1. Development, evaluation and application of tripeptidyl-peptidase II sequence signatures
Open this publication in new window or tab >>Development, evaluation and application of tripeptidyl-peptidase II sequence signatures
2009 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 484, no 1, p. 39-45Article in journal (Refereed) Published
Abstract [en]

Tripeptidyl-peptidase II (TPP II) is a cytosolic peptidase that has been implicated in fat formation and cancer, apparently independent of the enzymatic activity. In search for alternative functional regions, conserved motifs were identified and eleven signatures were constructed. Seven of the signatures covered previously investigated residues, whereas the functional importance of the other motifs is unknown. This provides directions for future investigations of alternative activities of TPP II. The obtained signatures provide an efficient bioinformatic tool for the identification of TPP II homologues. Hence, a TPP II sequence homologue from fission yeast, Schizosaccharomyces pombe, was identified and demonstrated to encode the TPP II-like protein previously reported as multicorn. Furthermore, an homologous protein was found in the prokaryote Blastopirellula marina, albeit the TPP II function was apparently not conserved. This gene is probably the result of a rare gene transfer from eukaryote to prokaryote.

Keywords
Serine protease, subtilase, sequence motif, cytosolic protein degradation, tripeptidyl-peptidase II
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-119529 (URN)10.1016/j.abb.2009.01.007 (DOI)000264927700006 ()19467630 (PubMedID)
Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2017-12-12
2. Investigation of a role for Glu-331 and Glu-305 in substrate binding of tripeptidyl-peptidase II
Open this publication in new window or tab >>Investigation of a role for Glu-331 and Glu-305 in substrate binding of tripeptidyl-peptidase II
2008 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1784, no 12, p. 1899-1907Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to investigate the mechanism by which tripeptidyl-peptidase II (TPP II) can specifically release tripeptides from the free N-terminus of an oligopeptide. The subtilisin-like N-terminal part of TPP II was modelled using subtilisin as template. Two glutamate residues (Glu-305 and Glu-331) appeared to be positioned so as to interact with the positively charged N-terminus of the substrate. In order to test this potential interaction, both residues were replaced by glutamine and lysine. The catalytic efficiency was reduced 400-fold for the E331Q variant and 20000-fold for the E331K variant, compared with the wild-type (wt). A substantial part of this reduction was due to decreased substrate affinity, since the K(M) for both mutants was at least two orders of magnitude greater than for the wt. This decrease was linked specifically to interaction with the free N-terminal amino group, based on inhibition studies. Glu-305 appears to be essential for enzymatic activity, but the extremely low activity of the E305Q variant prevented an investigation of the involvement of Glu-305 in substrate binding. The present work is, to our knowledge, the first report to investigate a mechanism for a tripeptidyl-peptidase activity through site-directed mutagenesis.

Keywords
Tripeptidyl-peptidase II, Exopeptidase, Substrate specificity, Intracellular protein degradation, Oligomerization, Molecular ruler
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-100055 (URN)10.1016/j.bbapap.2008.08.017 (DOI)000261673300002 ()18822395 (PubMedID)
Available from: 2009-03-24 Created: 2009-03-24 Last updated: 2017-12-13Bibliographically approved
3. Inter-species variation in the pH dependence of tripeptidyl-peptidase II
Open this publication in new window or tab >>Inter-species variation in the pH dependence of tripeptidyl-peptidase II
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Tripeptidyl-peptidase II (TPP II) is a large enzyme complex (>4 MDa) participating in the general protein turn-over in the cell downstream of the proteasome. In addition, there have been reports of involvement of TPP II in different physiological situations. To facilitate further investigations of the physiological role of TPP II and its enzymatic properties, a characterization at protein level is necessary. Therefore, an expression system for murine TPP II using Escherichia coli has been developed. The pH-optimum for cleavage of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was investigated for mTPP II, and compared with human TPP II and TPP II from Drosophila melanogaster. It was shown that the mouse enzyme had similar pH dependence as the human enzyme, while dTPP II had a slightly lower optimum. Surprisingly, the investigation also demonstrated that TPP II from all sources showed a different pH-profile for hydrolysis of AAA-pNA compared to AAF-pNA. To investigate this observation further, steady-state kinetic parameters were determined at various pH. Since both the KM and Vmax are lower for cleavage of AAA-pNA, a potential explanation could be that the substrate AAA-pNA is non-productively bound to the active site of the enzyme.

Keywords
tripeptidyl-peptidase II, TPP II, AAF-pNA, AAA-pNA, steady-state kinetics, pH-dependence
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:nbn:se:uu:diva-134621 (URN)
Available from: 2010-11-30 Created: 2010-11-30 Last updated: 2011-06-28
4. Characterization of the endopeptidase activity of tripeptidyl-peptidase II
Open this publication in new window or tab >>Characterization of the endopeptidase activity of tripeptidyl-peptidase II
Show others...
2012 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 424, no 3, p. 503-507Article 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.

Keywords
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:nbn:se:uu:diva-134620 (URN)10.1016/j.bbrc.2012.06.144 (DOI)000307618800024 ()
Available from: 2010-11-30 Created: 2010-11-30 Last updated: 2017-12-12Bibliographically approved

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