Polyhydroxybutyrate Targets Mammalian Mitochondria and Increases Permeability of Plasmalemmal and Mitochondrial Membranes

Abstract

Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle.

Physiological roles of the mitochondrial permeability transition pore

“…Recent reports have also confirmed increased propensity toward PT caused by the presence of polyphosphates, chains of 10s to 100 s of repeating phosphates linked by ATP-like high energy bonds (Abramov et al, 2007; Seidlmayer et al, 2012; Holmstrom et al, 2013; Stotz et al, 2014). The actions of Ca 2+ may also require polyhydroxybutyrate (PHB), which enters mitochondria and enhances the ability of Ca 2+ to induce PT (Elustondo et al, 2013). Although the above conditions occur under pathological circumstances, evidence for physiological opening of the mPTP has been described including flickering short of permanent opening (Hausenloy et al, 2004; Crompton, 1999; Huser & Blatter, 1999; Korge et al, 2011; Petronilli et al, 1999; Wang et al, 2008; Ichas & Mazat, 1998; Jouaville et al, 1998).…”

“…When viewed from the inter-membrane space, the denuded c-subunit oligomer appears to have a central pore-like structure that is normally obscured by the F 1 stalk components, suggesting that the mouth of the pore may be unmasked by removal of F 1 (Pogoryelov et al, 2007). Although it has been suggested that phospholipids occupy the central cavity of the c-subunit ring in F 1 F O ATP synthases from different species (Oberfeld et al, 2006; Meier et al, 2001; Matthies et al, 2009) other evidence provides for formation of a proteolipid or proteophospholipid channel structure within the central lipid region upon activation of PT (Abramov et al, 2007; Elustondo et al, 2013; Azarashvili et al, 2014; McGeoch & McGeoch, 2008; Pavlov et al, 2005). Polyhydroxybutyrate (PHB) is a polymer distinct from the lipids that may form an ion channel within the lipid milieu if mammalian c-subunit is indeed filled with PHB (Elustondo et al, 2013; Pavlov et al, 2005).…”

“…Although it has been suggested that phospholipids occupy the central cavity of the c-subunit ring in F 1 F O ATP synthases from different species (Oberfeld et al, 2006; Meier et al, 2001; Matthies et al, 2009) other evidence provides for formation of a proteolipid or proteophospholipid channel structure within the central lipid region upon activation of PT (Abramov et al, 2007; Elustondo et al, 2013; Azarashvili et al, 2014; McGeoch & McGeoch, 2008; Pavlov et al, 2005). Polyhydroxybutyrate (PHB) is a polymer distinct from the lipids that may form an ion channel within the lipid milieu if mammalian c-subunit is indeed filled with PHB (Elustondo et al, 2013; Pavlov et al, 2005). Data suggest a working model whereby the c-subunit pore forms within the proteolipid milieu (for example by elevated matrix Ca 2+ ) whereupon the ring expands and F 1 shifts; the pore is closed by a decrease in diameter of the ring and inactivated by binding of the F 1 components to the ring (Fig.…”

Cell death disguised: The mitochondrial permeability transition pore as the c-subunit of the F1FO ATP synthase

“…Recent reports have also confirmed increased activity of PT by polyphosphates, chains of 10s to 100s of repeating phosphates linked by ATP-like high energy bonds [58–61]. The actions of Ca 2+ may also require polyhydroxybutyrate (PHB), which enters mitochondria and enhances the ability of Ca 2+ to induce PT [62]. …”

“…When viewed from the inter-membrane space, the denuded c-subunit oligomer appears as a ring with a central pore-like structure that is normally obscured by the F 1 stalk components gamma, delta and epsilon, suggesting that the pore may form within the center of the ring given the proper hydrophilic conformation [129]. Although it has been suggested that phosphlipids occupy the central cavity of the c-subunit ring in F 1 F O ATP synthases from different species [156–158] other evidence provides for formation of a proteolipid or proteophospholipid channel structure within the central lipid region [58,62,118,131,159]. Data suggest a working model whereby the c-subunit pore forms within the proteolipid milieu upon activation of mPTP (for example by elevated matrix Ca 2+ ) whereupon the ring expands and F 1 shifts; the pore is closed by a decrease in diameter of the ring and inactivated by binding of the F 1 components to the ring (Figs.…”