Factors Regulating the Substrate Specificity of A-type Phospholipases - A Mass-Spectrometric study
Year: 2016
Type: dissertation
Abstract: The aim of this thesis was to study the substrate specificity of A-type phospholipases (PLAs) that belong to different sub-families in order to understand the key factors that regulate their activity. The experimental part of this thesis consists of three studies each one focusing on PLAs that belong to a specific subgroup. In the first study, we developed a mass-spectrometric (MS) assay and implemented it to study in detail the effect of acyl chain length and unsaturation of glycerophospholipids (GPLs) on their hydrolysis by three different secretory PLA2s (sPLA2s) from various sources. The key finding of this study was that efflux of the GPL substrate from the bilayer largely determines the rate of hydrolysis by these PLAs. In micelles, accommodation of the GPL acyl chains in the catalytic active site seems to be more important for substrate specificity. In the next study we used the MS assay developed in study I, to investigate whether substrate efflux propensity regulates the activity of the Ca2+ -independent PLA-Beta (iPLAβ). Our results strongly suggest that the activity of iPLAβ is also determined by the efflux of the GPL substrate from the membrane bilayer. Our last study was on the cytoplasmic PLA-alpha (cPLA2α) that has been implicated in the initiation of the inflammatory lipid-mediator cascade generating eicosanoids and platelet-activating factor. The study was carried out to understand to what extent accommodation in the catalytic site determines specificity for arachidonic acid (AA) and if efflux propensity plays a role in the substrate specificity of cPLA2α. Our results indicate that while accommodation of the substrate in the active site greatly contributes to the preferential hydrolysis of AA-containing GPLs by cPLA2α, efflux of the substrate from a membrane bilayer also plays a significant role. In summary, these studies not only provide information on the factors regulating the substrate specificity of various PLAs but also indicate that lateral arrangement of GPLs could be a key regulator of homeostatic PLAs like iPLAβ.
Source: Työväentutkimus Vuosikirja
Author: Krishna Chaithanya Batchu
Language: English
Subfield: Biochemistry
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2196922446