Porzio, Elena (2010) The relationship of the ADP-ribosylating enzyme from S. solfataricus with DING proteins and its intracellular localization. [Tesi di dottorato] (Unpublished)

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Item Type: Tesi di dottorato
Language: English
Title: The relationship of the ADP-ribosylating enzyme from S. solfataricus with DING proteins and its intracellular localization
Creators:
CreatorsEmail
Porzio, Elenae.porzio@alice.it
Date: 30 November 2010
Number of Pages: 74
Institution: Università degli Studi di Napoli Federico II
Department: Biologia strutturale e funzionale
Doctoral School: Scienze biologiche
PHD name: Biochimica e biologia cellulare e molecolare
PHD cycle: 23
PHD Coordinator:
nameemail
Arcari, Paoloarcari@unina.it
Tutor:
nameemail
Faraone Mennella, Maria Rosariafaraone@unina.it
Date: 30 November 2010
Number of Pages: 74
Uncontrolled Keywords: PARP; DING; Sulfolobus solfataricus
MIUR S.S.D.: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Date Deposited: 09 Dec 2010 16:50
Last Modified: 30 Apr 2014 19:46
URI: http://www.fedoa.unina.it/id/eprint/8426

Abstract

The PARPSso thermoprotein from Sulfolobus solfataricus has been identified as a PARP-like enzyme that cleaves -NAD+ to synthesize oligomers of ADP-ribose and cross-reacts with polyclonal anti-PARP-1 catalytic site antibodies. Despite the biochemical properties that allow to correlate it to PARP enzymes, the N-terminal and partial amino acid sequence suggest the sulfolobal enzyme belongs to a different class of enzymes, the DING proteins. Considering the high sequence identity with the human DING protein HPBP and the lack of a nucleotide coding sequence in both human and sulfolobal genomes, we hypothesized that PARPSso might share other features with the human DING. Further analysis of PARPSso amino acid sequence addressed the research towards studying other possible similarities between human and sulfolobal protein and then to explain how PARPSso correlates with canonic PARPs. For the latter question, the peculiar behaviour of the thermozyme, that is biochemically, but not structurally related to the classic PARPs, stimulated to investigate by computational analysis and databank, whether the protein might be phylogenetically related to any already known PARP amino acid sequence. Moreover, immunochemical and enzymatic crossed analyses were performed to establish whether purified HPBP and PARPSso have common immunoreactive and functional behaviour. The second part of the research was focused on the localization of PARPSso within the sulfolobal cell. Our interest to this item arose from the property of some DING proteins to be membrane bound, suggested to work as membrane transporters. On the other hand, from previous studies, it is known that PARPSso is only partially solubilized from the starting cell homogenate provided by ICMIB (CNR), and the soluble enzyme is strictly associated with DNA. In this thesis work, whole cells collected by centrifugation from culture medium were subjected to a different extraction procedure. This procedure included also experimental conditions used to differentiate between soluble (i.e. cytoplasmic) and insoluble (i.e. membrane-bound) protein fractions. PARPSso and DNA distributions were determined by enzyme assay, immunoblotting and agarose gel electrophoresis. Reciprocal interactions of thermozyme, nucleic acid and membrane lipids were investigated with different techniques and methodologies (nucleoid preparation, fluorescence binding assays, fluorescence microscopy analysis).

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