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

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Tipologia del documento: Tesi di dottorato
Lingua: English
Titolo: The relationship of the ADP-ribosylating enzyme from S. solfataricus with DING proteins and its intracellular localization
Autori:
AutoreEmail
Porzio, Elenae.porzio@alice.it
Data: 30 Novembre 2010
Numero di pagine: 74
Istituzione: Università degli Studi di Napoli Federico II
Dipartimento: Biologia strutturale e funzionale
Scuola di dottorato: Scienze biologiche
Dottorato: Biochimica e biologia cellulare e molecolare
Ciclo di dottorato: 23
Coordinatore del Corso di dottorato:
nomeemail
Arcari, Paoloarcari@unina.it
Tutor:
nomeemail
Faraone Mennella, Maria Rosariafaraone@unina.it
Data: 30 Novembre 2010
Numero di pagine: 74
Parole chiave: PARP; DING; Sulfolobus solfataricus
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Depositato il: 09 Dic 2010 16:50
Ultima modifica: 30 Apr 2014 19:46
URI: http://www.fedoa.unina.it/id/eprint/8426
DOI: 10.6092/UNINA/FEDOA/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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