Human phospholipid scramblase 4 (hPLSCR4), an isoform of the scramblase family, is a type II single-pass transmembrane protein whose function remains unknown. To understand its role, recombinant hPLSCR4 was obtained by cloning the ORF into a pET28 a(+) vector and overexpressed in Escherichia coli. Functional assay showed that Ca2+, Mg2+, and Zn2+ activate hPLSCR4 and mediate scrambling activity independent of the phospholipid head group. Far-UV-CD and fl uorescence spectroscopy revealed that Ca2+ and Mg2+ binding induces conformation change in hPLSCR4, exposing hydrophobic patches of the protein, and Ca2+ has more affi nity than Mg2+ and Zn2+. Stains-all studies further confirm that hPLSCR4 is a Ca2+-binding protein. Point mutation (Asp 290→Ala) in hPLSCR4 decreased the Ca2+-binding affi nity as well as Tb3+ luminescence, suggesting residues of the predicted Ca2+-binding motif are involved in Ca2+ binding. Functional reconstitution with (Asp290→Ala) mutant led to ~50% and ~40% decrease in scramblase activity in the presence of Ca2+ and Mg2+, respectively. Copyright © 2011-2012 by Walter de Gruyter.

Sathyanarayana Naidu Gummadi

Department Of Biotechnology

Alanine

aspartic acid

CALCIUM BINDING PROTEIN

calcium ion

magnesium ion

Phospholipid

PHOSPHOLIPID SCRAMBLASE 4

PLASMID VECTOR

RECOMBINANT ENZYME

SCRAMBLASE

unclassified drug

zinc ion

article

binding affinity

Biochemistry

Circular dichroism

controlled study

enzyme activity

Escherichia coli

fluorescence spectroscopy

gene overexpression

hydrophobicity

molecular cloning

nonhuman

priority journal

protein function

Anilino Naphthalenesulfonates

calcium

Humans

Hydrophobic and Hydrophilic Interactions

Mutagenesis, Site-Directed

mutation

PHOSPHOLIPID TRANSFER PROTEINS

Spectrometry, Fluorescence

Fulltext

IIT Madras is a public technical and research university located in Chennai, Tamil Nadu. Founded in 1959, it is recognised as an Institute of National Importance.

IIT Madras has been ranked as the top engineering institute in India for four years in a row by the National Institutional Ranking Framework of the MHRD

It currently offers undergraduate, postgraduate and research degrees across 16 disciplines in Engineering, Sciences, Humanities and Management. About 596 faculty belonging to science and engineering departments and centres of the Institute are engaged in teaching, research and industrial consultancy.

IIT Madras

Biological Chemistry

Human phospholipid scramblases (hPLSCRs) are a family of four homologous single pass transmembrane proteins (hPLSCR1-4) initially identified as the proteins responsible for Ca2+ mediated bidirectional phospholipid translocation in plasma membrane. Though in-vitro assays had provided evidence, the role of hPLSCRs in phospholipid translocation is still debated. Recent reports revealed a new class of proteins, TMEM16 and Xkr8 to exhibit scramblase activity challenging the function of hPLSCRs. Apart from phospholipid scrambling, numerous reports have emphasized the multifunctional roles of hPLSCRs in key cellular processes including tumorigenesis, antiviral defense, protein and DNA interactions, transcriptional regulation and apoptosis. In this review, the role of hPLSCRs in mediating cell death through phosphatidylserine exposure, interaction with death receptors, cardiolipin exposure, heavy metal and radiation induced apoptosis and pathological apoptosis followed by their involvement in cancer cells are discussed. This review aims to connect the multifunctional characteristics of hPLSCRs to their decisive involvement in apoptotic pathways. © 2017

Biochimica et Biophysica Acta - Molecular Cell Research

The role of human phospholipid scramblases in apoptosis: An overview

Human phospholipid scramblases (hPLSCRs) are a group of transmembrane ATP independent lipid transporters mediating bi-directional transport of phospholipids. There are four homologues hPLSCR1-hPLSCR4 and hPLSCR1 is the extensively studied homologue among them. hPLSCR4 shares 48% homology with hPLSCR1 and mediates scrambling of PLs similar to hPLSCR1 in Ca2 + dependent manner. Transcriptional regulation helps in better understanding of the function and the expression of a protein. Till date there are no reports suggesting the transcriptional regulation of hPLSCR4. In this study, we identified Snail to be a potent regulator of hPLSCR4. ConSite tool predicted the presence of a putative Snail binding site with a consensus sequence of − 1521CAGGTG− 1516 on hPLSCR4 promoter. Luciferase assays depicted a dose dependent decrease in hPLSCR4 promoter activity with an increase in amount of Snail. Deletion analysis revealed that the region from − 1380 to − 2100 to be the regulatory region of hPLSCR4. Knock down studies further confirmed Snail mediated downregulation of hPLSCR4, as the mRNA and the protein levels of hPLSCR4 considerably increased under knock down conditions. The in vivo interaction of Snail with hPLSCR4 promoter was further confirmed by ChIP assay. This is the first report on the transcriptional regulation of hPLSCR4, where Snail was shown to downregulate the expression of hPLSCR4. © 2016 Elsevier B.V.

Gene

Snail represses the expression of human phospholipid scramblase 4 gene

Human phospholipid scramblase 1 (hPLSCR1) belongs to the ATP-independent class of phospholipid translocators which possess a single EF-hand-like Ca 2+-binding motif and also a C-terminal helix (CTH). The CTH domain of hPLSCR1 was believed to be a putative single transmembrane helix at the C-terminus. Recent homology modeling studies by Bateman et al. predicted that the hydrophobic nature of this helix is due to its packing in the core of the protein domain and proposed that this is not a true transmembrane helix [Bateman A, Finn RD, Sims PJ, Wiedmer T, Biegert A &amp; Johannes S. Bioinformatics 2008, 25, 159]. To determine the exact function of the CTH of hPLSCR1, we deleted the CTH domain and determined: (a) whether CTH plays any role beyond membrane anchorage, (b) the functional consequences of CTH deletion, and (c) any conformational changes associated with CTH in a lipid environment. In vitro reconstitution studies confirm that the predicted CTH is required for membrane insertion and scrambling activity. CTH deletion caused a 50% decrease in binding affinity of Ca2+ for CTH-hPLSCR1 (Ka = 115 μm) compared with hPLSCR1 (Ka = 249 μm). Far UV-CD studies revealed that the CTH peptide adopts α-helicity only in the presence of SDS micelles and negatively charged vesicles, indicating that electrostatic interactions are required for insertion of the peptide. CTH peptide-quenching studies confirm that the predicted CTH inserts into the membrane and its ability to interact with the membrane depends on the presence of charge interactions. TOXCAT assay revealed that CTH of hPLSCR1 does not oligomerize in the membrane. We conclude that CTH is required for membrane insertion and Ca2+ coordination and also plays an important role in the functional conformation of hPLSCR1. Phospholipid scramblases constitute a family of homologous proteins named as hPLSCR1-hPLSCR4. The C-terminal helix (CTH) is presently close to the EF-hand motif in the scramblase homologues indicating that it may play an important role in Ca2+ binding. This study shows that CTH is required for membrane insertion, Ca2+ co-ordination and also plays an important role in the functional conformation of hPLSCR1. © 2013 FEBS.

FEBS Journal

The single C-terminal helix of human phospholipid scramblase 1 is required for membrane insertion and scrambling activity

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6±1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents. © 2011 Rajasekharan, Gummadi.

PLoS ONE

Flip-flop of phospholipids in proteoliposomes reconstituted from detergent extract of chloroplast membranes: Kinetics and phospholipid specificity

Background: Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca2+ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca2+/Mg2+ binding to human scramblases and conformational changes taking place in them remains unknown. Methods: In the present study, we analyzed the Ca2+ and Mg2+ binding to the calcium binding motifs of hPLSCR1-4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry. Results: The results in this study show that (i) affinities of the peptides are in the order hPLSCR1 &gt; hPLSCR3 &gt; hPLSCR2 &gt; hPLSCR4 for Ca2+ and in the order hPLSCR1 &gt; hPLSCR2 &gt; hPLSCR3 &gt; hPLSCR4 for Mg2+, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca2+ and Mg2+ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families. Conclusions: Based on the above results, we hypothesize that the Ca2+ binding motif of hPLSCR1 is a novel type of Ca2+ binding motif. General significance: Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function. © 2009 Elsevier B.V. All rights reserved.

Biochimica et Biophysica Acta - General Subjects

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Human phospholipid scramblase 1 (hPLSCR1) scrambles plasma membrane phospholipids during cell activation, blood coagulation and apoptosis. It was over-expressed in E. coli with a histidine tag and purified from the inclusion bodies (∼30 mg/l culture broth) under denaturing conditions using 8 M urea. The denatured hPLSCR1 refolded into its native configuration when urea was removed as shown by a 10-fold increase in its intrinsic fluorescence. Active hPLSCR1 showed scrambling activity in vitro after reconstituting in proteoliposomes. hPLSCR1 showed higher rates of scrambling activity for phosphatidylethanolamine than phosphatidylcholine. Binding studies with the calcium analogue "Stains-all" dye showed a characteristic peak, termed as the J band, at 650 nm. This is the first report on high level expression of hPLSCR1 with histidine tag in E. coli. © 2008 Springer Science+Business Media B.V.

Biotechnology Letters

Over-expression of recombinant human phospholipid scramblase 1 in E. coli and its purification from inclusion bodies

Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic organisms. They are believed to be involved in destroying plasma membrane phospholipid asymmetry at critical cellular events like cell activation, injury and apoptosis. However, a detailed mechanism of phospholipid scrambling still awaits a proper understanding. The most studied member of this family, phospholipid scramblase 1 (PLSCR1) (a 37 kDa protein), is involved in rapid Ca2+ dependent transbilayer redistribution of plasma membrane phospholipids. Recently the function of PLSCR1 as a phospholipids translocator has been challenged and evidences suggest that PLSCR1 acts as signaling molecule. It has been shown to be involved in protein phosphorylation and as a potential activator of genes in response to interferon and other cytokines. Interferon induced rapid biosynthesis of PLSCR1 targets some of the protein into the nucleus, where it binds to the promoter region of inositol 1,4,5-triphosphate (IP3) receptor type 1 (IP3R1) gene and induces its expression. Palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus. In the present review, we discuss the current understanding of PLSCR1 in relation to its trafficking, localization and signaling functions. © 2007 Elsevier Inc. All rights reserved.

Archives of Biochemistry and Biophysics

Phospholipid scramblases: An overview

Journal of Fluorescence

Analysis of Lanthanide-Induced Conformational Change of the C-Terminal Domain on Centrin

Biochemical and functional characterization of human phospholipid scramblase 4 (hPLSCR4)

Journal	Biological Chemistry
ISSN	14316730
Open Access	No