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Characterization of the promoter of the gene encoding human tripeptidyl-peptidase II and identification of upstream silencer elements
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.
2007 (English)In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 393, no 1-2, 62-69 p.Article in journal (Refereed) Published
Abstract [en]

Tripeptidyl-peptidase II (TPP II) is one of the many proteases involved in the important process of intracellular proteolysis. The widespread distribution and broad substrate specificity suggest that TPP II is encoded by a "house-keeping gene". However, both TPP II protein and mRNA levels vary in different cells. To investigate whether these variations are due to regulation on a genetic level, the promoter of the TPP2 gene has previously been identified. The promoter contains two inverted CCAAT-boxes and an E-box. By means of reporter assays and electrophoretic mobility shift assays the promoter has now been further characterized. It could be concluded that USF-1 (upstream stimulatory factor-1) binds to the E-box in the promoter. The transcription factors NF-Y and USF-1 are present in protein-DNA complexes of different sizes. Mutation of the E-box had no effect, indicating that only binding of NF-Y to the two CCAAT-boxes was important for activation of transcription. However, this does not exclude the possibility that USF-1 can play an important role in transcription in other types of cells. Furthermore, the region upstream of the promoter was investigated due to its ability to inhibit transcription. Several silencer elements were identified and we also showed that Oct-1 binds to one of these elements. Thus, this investigation reveals that TPP II expression could be regulated through both positive and negative regulatory elements. Further studies are required to establish the involvement of different genetic elements, and how the interplay between different transcription factors will affect the transcriptional rate in vivo.

Place, publisher, year, edition, pages
2007. Vol. 393, no 1-2, 62-69 p.
Keyword [en]
transcriptional regulation, NF-Y, USF-1, Oct-1, EMSA, pGL3
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-95190DOI: 10.1016/j.gene.2007.01.015ISI: 000246407700007PubMedID: 17343995OAI: oai:DiVA.org:uu-95190DiVA: diva2:169309
Available from: 2006-11-24 Created: 2006-11-24 Last updated: 2011-02-01Bibliographically approved
In thesis
1. Tripeptidyl-Peptidase II: Structure, Function and Gene Regulation
Open this publication in new window or tab >>Tripeptidyl-Peptidase II: Structure, Function and Gene Regulation
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using Pichia pastoris was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the KMapp is more than 102 higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 52 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 243
Biochemistry, gene regulation, protein expression, enzyme kinetics, tripeptidyl-peptidase II, proteasome, intracellular protein degradation, exopeptidase, Biokemi
urn:nbn:se:uu:diva-7345 (URN)91-554-6733-4 (ISBN)
Public defence
2006-12-15, B7:113a, BMC, Husargatan 3, Uppsala, 09:15
Available from: 2006-11-24 Created: 2006-11-24Bibliographically approved

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