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Studies on the Interaction of Human Phospholipid Scramblase 1 with C‐Terminal Domain of Topoisomerase IIα
Author(s) -
Sivagnanam Ulaganathan,
Gummadi Sathyanarayaidu
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb78
Subject(s) - phospholipid scramblase , topoisomerase , biology , fusion protein , microbiology and biotechnology , c terminus , plasma protein binding , phosphatidylserine , chemistry , dna , recombinant dna , biochemistry , phospholipid , amino acid , gene , membrane
Human phospholipid scramblase 1 (hPLSCR1) is a multifunctional protein that plays key roles in several cellular processes including apoptosis, tumorigenesis, anti‐viral defense, cell signalling and several protein‐protein interactions. It has been shown that hPLSCR1 interacts with the C‐terminal domain of topoisomerase IIα (topo IIα) and enhances its decatenation activity in vitro. The interacting region in topo IIα was identified but till date, no reports exist on the binding region in hPLSCR1. This study aims to identify the region of hPLSCR1 that interacts with topo IIα. To identify the topo IIα interacting sites in hPLSCR1, N‐terminal deletion constructs of hPLSCR1 viz Δ25‐hPLSCR1, Δ50‐hPLSCR1, Δ75‐hPLSCR1, Δ100‐hPLSCR1 and Δ160‐hPLSCR1 were generated by PCR, cloned, overexpressed and purified to homogeneity using Ni 2+ ‐NTA purification. The C‐terminal domain (CTD) of topo IIα was cloned in pGEX6P‐1 and was expressed as a GST fusion protein. GST pull down assays will be performed with the deletion constructs of hPLSCR1 and the GST‐CTD‐topo IIα. The binding region in hPLSCR1 will be confirmed by peptide competition assays. Our initial results show that the decatenation activity of topo IIα was enhanced when topo II was pretreated with hPLSCR1. Δ100‐hPLSCR1 did not show any enhancement of the decatenation activity compared to full length hPLSCR1. Hence, the binding region could be in the 1–100 region of hPLSCR1. Further deletions were done in the 1–100 region of hPLSCR1 as described earlier. GST‐pull down assays and decatenation assays will be performed for the deletion constructs to narrow down the region of hPLSCR1 that binds to topo IIα. We conclude that hPLSCR1 interacts with and enhances the activity of topo IIα and the 1–100 region of hPLSCR1 is critical for enhancement of decatenation activity. Further work is under progress to identify the exact topo IIα binding region of hPLSCR1 and the physiological relevance of this interaction in the cell. Support or Funding Information This work was funded by Council of Scientific and Industrial Research (CSIR), Government of India. Authors thank Ministry of Human Resources and Development Govt. of India and Indian Institute of Technology Madras for fellowship.