Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor

Abstract

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.

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“…These PLs are involved in many functions that could alter cell growth or death. For example, PE serves as chaperone for proteins moving from cytoplasm to membrane [16], and PC and PI are sources of diacylglycerol for signaling function [17,18]. Changes in the n-3 fatty acid composition of PC have been demonstrated to reduce tumor cell growth, and alternations in PE composition lead to mitochondrial-mediated apoptosis [16,18,19].…”

“…For example, PE serves as chaperone for proteins moving from cytoplasm to membrane [16], and PC and PI are sources of diacylglycerol for signaling function [17,18]. Changes in the n-3 fatty acid composition of PC have been demonstrated to reduce tumor cell growth, and alternations in PE composition lead to mitochondrial-mediated apoptosis [16,18,19]. PI is a major source of arachidonic acid (AA; 20:4n-6), important for eicosanoid synthesis [17], compounds which promote tumor progression and metastasis [20].…”

Structural insights into the multiple functions of protein C inhibitor

“…The model placed the head group of PE into the helix A gap and the acyl chains in the hydrophobic D' and H' channels. This binding site appears to fit the conical shape of PE perfectly, and was verified by mutagenesis [43] ( Fig. 7B).…”

“…Review Article lipoprotein (LDL), and that the transferred PE supported improved prothrombinase activity. This factor was later identified by the same group as PCI [43]. PCI could transfer PE from LDL and small unilamellar vesicles to platelets and other cells, and specificity for PE was absolute.…”

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“…PE is found mainly in the inner leaflets of mammalian plasma membranes, and it accounts for 20% to 50% of total phospholipids. PE works as an anticoagulant, enhancing activated protein C (APC) activity in blood coagulation reactions, downregulating procoagulant function 9 , and inhibiting factor Xa-prothrombin (PT) 10 . Cell surfaceexposed PE plays a major role in translocating protein C inhibitor proteins across the plasma membrane 10 .…”