Tài liệu Báo cáo khoa học: Complex transcriptional and translational regulation of iPLA2c resulting

Complex transcriptional and translational regulation of iPLA2c

resulting in multiple gene products containing dual competing sites

for mitochondrial or peroxisomal localization

David J. Mancuso1,2, Christopher M. Jenkins1,2, Harold F. Sims1,2, Joshua M. Cohen1,2, Jingyue Yang1,2

and Richard W. Gross1,2,3,4

1

Division of Bioorganic Chemistry and Molecular Pharmacology, and Departments of 2

Medicine, 3

Chemistry and 4

Molecular Biology

and Pharmacology, Washington University School of Medicine, St. Louis, MO, USA

Membrane-associated calcium-independent phospholipase

A2c (iPLA2c) contains four potential in-frame methionine

start sites (Mancuso, D.J. Jenkins, C.M. & Gross, R.W.

(2000) J. Biol. Chem. 275, 9937–9945), but the mechanisms

regulating the types, amount and subcellular localization of

iPLA2c in cells are incompletely understood. We now:

(a) demonstrate the dramatic transcriptional repression of

mRNA synthesis encoding iPLA2c by a nucleotide sequence

nested in the coding sequence itself; (b) localize the site of

transcriptional repression to the most 5¢ sequence encoding

the iPLA2c holoprotein; (c) identify the presence of nuclear

protein constituents which bind to the repressor region by gel

shift analysis; (d) demonstrate the translational regulation of

distinct iPLA2c isoforms; (e) identify multiple novel exons,

promoters, and alternative splice variants of human iPLA2c;

(f) document the presence of dual-competing subcellular

localization signals in discrete isoforms of iPLA2c; and

(g) demonstrate the functional integrity of an N-terminal

mitochondrial localization signal by fluorescence imaging

and the presence of iPLA2c in the mitochondrial compart￾ment of rat myocardium. The intricacy of the regulatory

mechanisms of iPLA2c biosynthesis in rat myocardium is

underscored by the identification of seven distinct protein

products that utilize multiple mechanisms (transcription,

translation and proteolysis) to produce discrete iPLA2c

polypeptides containing either single or dual subcellular

localization signals. This unanticipated complex interplay

between peroxisomes and mitochondria mediated by com￾petition for uptake of the nascent iPLA2c polypeptide

identifies a new level of phospholipase-mediated metabolic

regulation. Because uncoupling protein function is regulated

by free fatty acids in mitochondria, these results suggest that

iPLA2c processing contributes to integrating respiration and

thermogenesis in mitochondria.

Keywords: phospholipase; mitochondria; peroxisomes; tran￾scription; translation.

Phospholipases A2 (PLA2s) play critical roles in cellular

growth, lipid homeostasis and lipid second messenger

generation by catalyzing the esterolytic cleavage of the

sn-2 fatty acid of glycerophospholipids [1–5]. The resultant

fatty acids and lysolipids are potent lipid mediators of signal

transduction and alter the biophysical properties of the

membrane bilayer, collectively contributing to the critical

roles that phospholipases play in cellular adaptation,

proliferation and signaling. PLA2s constitute a diverse

family of enzymes, which include the intracellular phos￾pholipase families, cytosolic PLA2s (cPLA2) and calcium￾independent PLA2s (iPLA2) as well as the secretory PLA2s

(sPLA2).

More than a decade ago, we identified multiple types of

kinetically distinguishable iPLA2 activities in the cytosolic,

microsomal and mitochondrial fractions from multiple

species of mammalian myocardium [6–10]. Utilizing the

synergistic power of HPLC in conjunction with MS of

intact phospholipids, initial insights into both the canine

and human mitochondrial lipidomes were made [8,11]. Both

human and canine cardiac mitochondria possess a high

plasmalogen content, and plasmalogens are readily hydo￾lyzed by heart mitochondrial phospholipases [7,8]. Both

cytosolic and membrane-associated iPLA2 activities are

inhibited by the nucleophilic serine-reactive mechanism￾based inhibitor (E)-6-(bromomethylene)-3-(1-naphthale￾nyl)-2H-tetrahydropyran-2-one (BEL) [12–14]. Recent

studies have shown that BEL has potent effects on

mitochondrial bioenergetics [15] and that fatty acids are a

Correspondence to R. W. Gross, Washington University School of

Medicine, Division of Bioorganic Chemistry and Molecular Phar￾macology, 660 South Euclid Avenue, Campus Box 8020, St. Louis,

MO 63110, USA. Fax: +1 314 362 1402; Tel: +1 314 362 2690;

E-mail: [email protected]

Abbreviations: BEL, (E)-6-(bromomethylene)-3-(1-naphthalenyl)-

2H-tetrahydropyran-2-one; cPLA2, cytosolic phospholipase A2; ECL,

enhanced chemoluminescence; EMSA, electrophoretic mobility shift

analyses; EST, expressed sequence tag; GAPDH, glyceraldehye￾3-phosphate dehydrogenase; iPLA2, calcium-independent phosphol￾ipase A2; iPLA2c, membrane associated calcium-independent phos￾pholipase A2 (AF263613); MOI, multiplicity of infection; PLA2,

phospholipase A2; Sf9, Spodoptera frugiperda cells; sPLA2, secretory

phospholipase A2; TAMRA, 6-carboxytetramethylrhodamine;

UCP, uncoupling protein.

(Received 25 August 2004, revised 10 October 2004,

accepted 13 October 2004)

Eur. J. Biochem. 271, 4709–4724 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04435.x