Binding of Endostatin to Phosphatidylserine-Containing Membranes and Formation of Amyloid-like Fibers

Abstract

Endostatin, the 20-kDa C-terminal NC1 domain of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis and tumor growth. A major problem in reconciling the many reported in vitro effects of endostatin is the lack of a high-affinity receptor, and a search for the latter continues. In accordance with the above, the molecular mechanisms of action of endostatin remain elusive. We show here that endostatin binds to membranes containing acidic phospholipids, phosphatidylserine (PS) or phosphatidylglycerol (PG). More specifically, a red shift in the fluorescence emission of Trp of endostatin in the presence of liposomes containing these anionic lipids was evident, revealing the average environment of Trps to become less hydrophobic. This shift was not observed for phosphatidylcholine (PC) liposomes, demonstrating the acidic lipid to be required. Quenching by endostatin of the fluorescence of a pyrene-labeled phospholipid analogue in PS containing membranes was seen, while there was no effect for PC liposomes. Resonance energy transfer from the Trp residues of endostatin to a dansyl-labeled phospholipid further confirmed the association of endostatin with PS-containing membranes, whereas there was no binding to PC liposomes. Intriguingly, the association of endostatin with PS-containing liposomes triggered the formation of fibers, with Congo red staining producing green birefringence characteristic for amyloid. Lipid was incorporated into these fibers, as shown by staining when a trace amount (X = 0.02) of fluorescent phospholipid analogues was present in the liposomes. No fiber formation was seen when endostatin was added to liposomes composed of PC only. Because PS has been reported to be exposed in the outer surface of the plasma membrane of cancer cells and vascular endothelial cells, our results suggest that this lipid could represent a target for endostatin in the cancer cell surface and tumors, thus suggesting a novel mechanism of its action. More specifically, analogous to a number of other cytotoxic proteins interacting with negatively charged lipids, PS-triggered fiber formation by endostatin on the surface of cancer cells would impair the permeability barrier function of the plasma membrane, resulting in cell death.

The Yeast Prion Ure2p Native-like Assemblies Are Toxic to Mammalian Cells Regardless of Their Aggregation State

“…For example, it is known that an ␣-lactalbumin folding variant with molten globule features is able to kill specifically tumoral cells (35); similarly, apoptin multimers, exposing extensive hydrophobic surfaces (36), kill specifically tumoral cells (37). Endostatin also displays antitumoral specificity upon aggregation triggered by its interaction with membrane phosphatidylserine (38), whereas the 32-kDa ectodomain fragment of CD44 displays selective toxicity to differing cell lines (39). Most pore-forming toxins of bacterial or eukaryotic origin kill target cells by oligomerizing into their plasma membranes, where they produce nonspecific pores (40 -42).…”

“…most often hydrophobic) surfaces as well as of a suitable membrane lipid composition in exposed cells. Indeed, it has been repeatedly reported that anionic phospholipids, such as phosphatidylserine, which becomes exposed in the outer membrane leaflet in apoptotic and tumoral cells, are putative docking sites for amyloid aggregates or can favor aggregate formation, thus initiating membrane impairment (38,50,51). Membrane cholesterol modulates membrane fluidity and may affect the incorporation of toxic aggregates into exposed cells (24,52) as well as membrane-associated Abeta fibrillogenesis and toxicity in neuronal cells (53,54).…”

The role of lipid–protein interactions in amyloid-type protein fibril formation

“…In vivo abnormal partial unfolding or folding may result from destabilization of the protein structure upon mutation, oxidation, heat stress, or changes in pH and the dielectric properties of the protein's local environment (Zerovnik, 2002;Trzesniewska et al, 2004;Gamblin et al, 2000). Evidently, protein-membrane interactions may play an important role in modulating conformational transitions of both membrane and soluble proteins, including amyloidogenic ones (Zhao et al, 2004(Zhao et al, , 2005). An increasing number of studies provides support to the idea that lipid bilayers can lower the activation energy barrier for protein unfolding.…”

“…Cell membrane is further thought to be the direct target mediating amyloidinduced cell death. Amyloid formation has been reported to induce membrane permeabilization resulting from alterations in bilayer structure and/or uptake of lipids into the forming fiber (Sparr et al, 2004;Zhao et al, 2004Zhao et al, , 2005Lin et al, 2001;Michikawa et al, 2001). It has also been hypothesized that extraction of membrane lipids by the forming amyloid (Sparr et al, 2004;Zhao et al, 2004) may be the direct cause for membrane permeabilization and cell death (Zhao et al, 2005).…”

Islet Amyloid Polypeptide Forms Rigid Lipid–Protein Amyloid Fibrils on Supported Phospholipid Bilayers

“…16,18 As a result, amyloid fibrillogenesis is dramatically accelerated by negatively charged lipids, as has been shown, for example, for Aβ, 19,20 prion, 21 α-synuclein, 22 and IAPP. 18 Formation of amyloid-like fibrils was also induced by negatively charged phospholipids for several other cytotoxic and apoptotic proteins, 17,23 including antimicrobial peptides. 24,25 Amyloid-type fibrils obtained in vitro in the presence of lipid membranes were found to incorporate phospholipids.…”