N-terminal Proline-rich Domain Is Required for Scrambling Activity of Human Phospholipid Scramblases

Abstract

Background:The role of PRD in scrambling of phospholipids by hPLSCR1 is not known. Results:The addition of PRD of hPLSCR1 to hPLSCR2 restored its PL scrambling activity, which in turn leads to aggregation of the protein. Conclusion: PRD is crucial for PL scrambling activity and could probably mediate its function by metal ion-dependent oligomerization. Significance: The results provide an insight in understanding the mechanism of scramblases.

Identification and characterisation of a phospholipid scramblase in the malaria parasite Plasmodium falciparum

“…A recent study provided first evidence for Ca 2+ -induced oligomerisation of hPLSCR1 which in turn activates PL scrambling (24). Hence, all steps of inclusion body solubilisation, protein refolding and purification of His::PfPLSCR were carried out in the presence of 0.5 mM Ca 2+ and Fos-Choline-12.…”

“…The hPLSCR1 protein contains a single EF-hand like Ca 2+ binding loop, which has been shown to bind Ca 2+ and other divalent cations in a co-ordinated manner, thus inducing conformational changes of the protein (24,25,35). The unconventional Ca 2+ binding motif of hPLSCR1 appears to be conserved in the parasite ortholog, including the residues in positions 1 (Asp 240 ), 3 (Asp 242 ) and 9 (Phe 248 ) ( Fig.…”

“…The best studied member to date is hPLSCR1; initially identified in and purified from erythrocytes (15,16), the protein is characterised by several functional domains: an N-terminal proline-rich domain containing multiple PXXP and PPXY motifs suggested to mediate protein-protein interactions (14); a DNA binding motif spanning Met 86 to Glu 118 which has been implicated in binding the promoter region of the inositol 1,4,5-triphosphate receptor type I (17); a palmitoylation site 184 CCCPCC 189 involved in trafficking to the plasma membrane (18); a non-classical nuclear leader sequence (NLS) 257 GKISKHWTGI 266 shown to bind importin a (19) precedes an EF-hand like Ca 2+ binding motif 273 D[A/S]DNFGIQFPLD 284 which is directly followed by a C-terminal a-helical stretch suggested to insert hPLSCR1 into artificial bilayers (16,20,21). The integral membrane protein is expressed in blood cells and a wide range of tissues and cancer cell lines (15,16,22) and is capable of translocating PLs in reconstituted membrane systems (15,20,(23)(24)(25) While its actual cellular role in PL scrambling remains a matter of debate, hPLSCR1 has been implicated in a range of different activities related to apoptosis, transcriptional regulation, autoimmunity, antiviral defence and the development of cancer and lipid-related diseases [reviewed in (13)].…”

Roles and regulation of phospholipid scramblases

“…PLSCR3, which is localized in mitochondrial membranes, externalizes cardiolipin from the inner to the outer mitochondrial membrane [121]. By contrast, PLSCR2 lacks scramblase activity; this was attributed to the absence of the proline‐rich N‐terminal region that is present in the other PLSCR family members [122]. Indeed, when fused to the proline‐rich domain of PLSCR1, PLSCR2 is capable of calcium‐dependent scrambling activity [122].…”

“…By contrast, PLSCR2 lacks scramblase activity; this was attributed to the absence of the proline‐rich N‐terminal region that is present in the other PLSCR family members [122]. Indeed, when fused to the proline‐rich domain of PLSCR1, PLSCR2 is capable of calcium‐dependent scrambling activity [122]. Although the above studies implicate PLSCRs in the redistribution of phospholipids between membrane leaflets, there may be other factors contributing to PS externalization in response to apoptotic stimuli [123].…”