Molecular and Biochemical Characterization of Phospholipase C zeta domains

Doctoral Dissertation uoadl:1309211 170 Read counter

Τομέας ΙΙ [Οργανική Χημεία – Οργανική Χημική Τεχνολογία – Χημεία Τροφίμων]
Library of the School of Science
Deposit date:
Θεοδωρίδου Μαρία
Dissertation committee:
Ντία Γαλανοπούλου Αναπλ. Καθηγήτρια ΕΚΠΑ (επιβλέπουσα), Κωνσταντίνος Βοργιάς Καθηγητής ΕΚΠΑ, Γεώργιος Νούνεσης Ερευνητής Α΄ ΕΚΕΦΕ "Δημόκριτος"
Original Title:
Μοριακός και Βιοχημικός Χαρακτηρισμός Δομικών Περιοχών της Φωσφολιπάσης C ζήτα (PLCζ)
Translated title:
Molecular and Biochemical Characterization of Phospholipase C zeta domains
Sperm-specific phospholipase C (PLC) activates early embryo development by
triggering intracellular calcium (Ca2+) oscillations in mammalian oocytes
indistinguishable from those seen at fertilization. PLC is the smallest with
the most elementary domain organization among all mammalian PLC isoforms.
Sperm-delivered PLC catalyzes the hydrolysis of its membrane-bound
phospholipid substrate PtdInsP2, generating cytosolic Ca2+ oscillations via the
InsP3 signaling pathway. The aim of this study was to investigate how discrete
PLC domains contribute to the novel biochemical and enzymatic properties of
this protein resulting in Ca2+ release in the fertilizing oocyte. A number of
techniques were employed including a chimeric analysis approach, site directed
mutagenesis, cRNA microinjection in mouse oocytes, recombinant protein
expression and purification, in vitro enzymatic assays, protein-lipid overlay
and liposome binding assays. These data suggest that the EF hand domains are
not solely responsible for the high Ca2+ sensitivity of PLC and that the
membrane association and binding of PLC to PIP2 is mediated by the C2 domain
and the XY-linker region. Unlike somatic PLCs, the sperm PLC XY-linker does
not mediate auto-inhibition of its enzymatic activity since the
positively-charged residues within the XY-linker play an important role in the
PLC interaction with PIP2 required for generating the Ca2+ oscillations
essential for mammalian fertilization. It is also demonstrated species-specific
differences in the in vivo Ca2+ oscillation-inducing activity of human and
mouse PLC. Chimeric analysis suggests a novel role of the EF hands and
XY-linker region in the species-specific differences in PLC activity.
Cell signalling, Phospholipase C zeta, Ca2+ oscillations, Fertilization, Sperm
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