DTA

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Tesi etd-05052018-111337

Type of thesis
Perfezionamento
Author
PERUZZI, SARA
URN
etd-05052018-111337
Title
Structural insight into PNKD function
Scientific disciplinary sector
BIO/15
Course
SCIENZE MEDICHE - Translational Medicine
Committee
relatore Prof. RECCHIA, FABIO ANASTASIO
Relatore Prof.ssa MARTINO, SABATA
Membro Dott. REGGIANI, ANGELO
Membro MINUTOLO, FILIPPO
Keywords
  • Paroxysmal dyskinesia
  • PNKD
  • protein characterization
  • x-ray crystallography
  • single particle cryo electron microscopy
Exam session start date
;
Availability
parziale
Abstract
PNKD is a membrane protein expressed only in the synaptic terminals of the central nervous system, whose dysfunction is responsible for the neurological movement disorder paroxysmal non-kinesigenic dyskinesia. This rare disease causes involuntary hyperkinetic attacks with dystonia and choreoathetosis. The dyskinesia attacks start mainly during childhood with variable frequency and duration, and the symptoms are aggravated by stress, fatigue, alcohol and coffee consumption. The paroxysmal non-kinesigenic dyskinesia is not efficiently responsive to the treatment with antiepileptic or anticonvulsant drugs, and there are no effective therapies for its cure. Despite its dramatic consequences, the physiological function of PNKD protein and its implication in the development of the paroxysmal non-kinesigenic dyskinesia have not yet been clarified. <br>The main aim of this PhD project was the structural characterization of human PNKD, in order to obtain accurate information about its specific function and structural targets for the design of small-molecule PNKD modulators, with potential therapeutic applications for the disorder.<br>To investigate the membrane target we carried on several biophysical characterizations, including protein X-ray crystallography and single particle cryo-electron microscopy. Different constructs, expression systems and purification strategies were obtained to over-produce useful and stable samples for protein crystallization and structure determination. We succeed to obtain small PNKD crystals, but their size was not sufficient to have good diffraction for structure determination. In parallel, several PNKD fusion proteins were designed and successfully produced for structural characterization by single particle cryo electron microscopy. From the analysis of a PNKD fusion protein containing a maltose binding protein and a green fluorescent protein, we obtained a protein structural model at ~1 nm resolution. To improve this result we produced a PNKD fusion protein using glutamine synthetase as fusion partner. Preliminary microscopy images demonstrated that the use of human glutamine synthase with a decametric assembly as fusion protein is a promising and innovative strategy for structural determination at higher resolution. Data processing and analysis are currently under way. The structural characterization of PNKD at atomic level will provide essential clues for its biochemical role and interactions, its involvement in the movement disorder, and the identification of specific ligands that can act as PNKD chemical modulators.<br>
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