Human phospholipid scramblase 1 (hPLSCR1) is a proapoptotic protein whose expression is deregulated in a variety of cancers cells. However till date the transcription regulation of hPLSCR1 is unknown. Transcriptional regulation of hPLSCR1 was studied by cloning the 5′-flanking region of hPLSCR1. Luciferase assays revealed that -1525 to -1244 region of hPLSCR1 was found to regulate its promoter activity. A putative Snail transcription factor (TF) binding site was found within the regulatory region of the promoter. Snail binding was found to down regulate the expression of hPLSCR1 both at the transcriptional and translational levels. Snail knock down using Snail-shRNA confirmed that down regulation of hPLSCR1 by Snail was specific. Point mutation studies confirm that the predicted Snail TF binds to -1123 to -1117 site. ChIP assay further confirms the physical interaction of Snail with hPLSCR1 promoter. This is the first report showing the transcriptional regulation of hPLSCR1 expression by Snail TF and its possible implications in cancer progression. © 2014 Elsevier Inc. All rights reserved.

Sathyanarayana Naidu Gummadi

Department Of Biotechnology

PHOSPHOLIPID SCRAMBLASE 1

SCRAMBLASE

short hairpin RNA

TRANSCRIPTION FACTOR SNAIL

unclassified drug

article

binding site

CHROMATIN IMMUNOPRECIPITATION

down regulation

LUCIFERASE ASSAY

molecular cloning

point mutation

priority journal

promoter region

protein expression

protein protein interaction

transcription regulation

Gastropoda

Chip

HPLSCR1

PROMOTER

SNAIL

TRANSCRIPTIONAL REGULATION

Base Sequence

Cell Line, Tumor

Down-Regulation

Humans

Molecular Sequence Data

neuroblastoma

PHOSPHOLIPID TRANSFER PROTEINS

Promoter Regions, Genetic

protein binding

Transcription Factors

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

Biochemical and Biophysical Research Communications

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

Fulltext

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

Background: Human phospholipid scramblase 1 (hPLSCR1) was initially identified as a Ca2+ dependent phospholipid translocator involved in disrupting membrane asymmetry. Recent reports revealed that hPLSCR1 acts as a multifunctional signaling molecule rather than functioning as scramblase. hPLSCR1 is overexpressed in a variety of tumor cells and is known to interact with a number of protein molecules implying diverse functions. Results: In this study, the nuclease activity of recombinant hPLSCR1 and its biochemical properties have been determined. Point mutations were generated to identify the critical region responsible for the nuclease activity. Recombinant hPLSCR1 exhibits Mg2+ dependent nuclease activity with an optimum pH and temperature of 8.5 and 37 °C respectively. Experiments with amino acid modifying reagents revealed that histidine, cysteine and arginine residues were crucial for its function. hPLSCR1 has five histidine residues and point mutations of histidine residues to alanine in hPLSCR1 resulted in 60 % loss in nuclease activity. Thus histidine residues could play a critical role in the nuclease activity of hPLSCR1. Conclusions: This is the first report on the novel nuclease activity of the multi-functional hPLSCR1. hPLSCR1 shows a metal dependent nuclease activity which could play a role in key cellular processes that needs to be further investigated. © 2016 Sivagnanam et al.

BMC Biochemistry

Identification and characterization of the novel nuclease activity of human phospholipid scramblase 1

Human phospholipid scramblase 1 (hPLSCR1) is a type II endofacial membrane protein which mediates bi-directional transport of phospholipids across the plasma membrane. hPLSCR1, a multifunctional protein with variety of roles in apoptosis, tumor progression, cell signaling and anti-viral defense. The expression of such a multifunctional protein should be under tight regulation. Apart from a single report showing snail mediated down regulation of hPLSCR1, the molecular mechanisms regulating the expression of scramblases are not well elucidated. In this study we identified c-Myc as a transcriptional regulator of hPLSCR1. Transcription factor prediction tool ConSite predicted three binding sites for c-Myc. Reporter gene assays and western blot analysis revealed c-Myc mediated up regulation of hPLSCR1 expression. Deletion construct -790 lacking one c-Myc binding site showed a 27% decrease in promoter activity while deletion construct -469 lacking two c-Myc binding sites showed a 62% decrease in promoter activity. Site directed mutagenesis revealed the importance of c-Myc binding sites from -751 to -756 and -548 to -553 on the promoter of hPLSCR1in transcriptionally regulating the expression of hPLSCR1. The results were further confirmed by shRNA mediated knock down of endogenous c-Myc and in vivo interactions by ChIP assay. © 2015 Elsevier Inc. All rights reserved.

Two c-Myc binding sites are crucial in upregulating the expression of human phospholipid scramblase 1 gene

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

BMC Cancer

Snail transcription factor negatively regulates maspin tumor suppressor in human prostate cancer cells

FEBS Letters

Phospholipid scramblase 1 mediates hepatitis C virus entry into host cells

Gastroenterology

SNAIL Regulates Interleukin-8 Expression, Stem Cell–Like Activity, and Tumorigenicity of Human Colorectal Carcinoma Cells

Molecular Medicine

Identification of Phospholipid Scramblase 1 as a Biomarker and Determination of Its Prognostic Value for Colorectal Cancer

Snail interacts with hPLSCR1 promoter and down regulates its expression in IMR-32

Journal	Biochemical and Biophysical Research Communications
Publisher	Academic Press Inc.
ISSN	0006291X
Open Access	No