1.B.8 The Mitochondrial and Plastid Porin (MPP) Family
Porins of the MPP family are found in eukaryotic organelles. The organelles include mitochondria of many eukaryotes as well as chloroplasts and plastids of plants. The best characterized members of the MPP family are the voltage-dependent anion-selective channel (VDAC) porins in the mitochondrial outer membrane. These porins have an estimated channel diameter of 2.5-3 nm. Topological models have been proposed in which VDAC consists of an N-terminal, globular α-helix and (1) 12 or 13 β-strands, (2) 16 β-strands or (3) 19 β-strands (now favored; see below) (Casadio et al., 2002). VDAC also appears to be present in plasma membranes (De Pinto et al., 2010). Prostacyclin receptor-mediated ATP release from ethrocytes requires VDAC (Sridharan et al., 2011). Phylogenetic analyses of eukaryotic VDAC proteins from diverse organisms have been reported (Wojtkowska et al. 2012). Over-oxidation of cysteines and succinylation of cysteines in VDACs has been noticed (Saletti et al. 2018).
A murine VDAC, VDAC-1, exhibits more than one topological type due to the use of alternative first exons. Thus, two different porins, differing only with respect to their N-termini, have been identified. One porin isoform (plasmalemmal VDAC-1) has a hydrophobic leader peptide that targets the protein through the golgi apparatus to the plasma membrane; the other isoform (mitochondrial VDAC-1) is translocated to the outer mitochondrial membrane because it lacks the N-terminal hydrophobic leader. The former is believed to account for the plasma membrane Maxi (large conductance) Cl- channel (Bahamonde et al., 2003).
VDACs play a role in forming the mitochondrial permeability transition pore (PTP) which is important for Ca2+ homeostasis and programmed cell death. PTP is triggered by Ca2+ influx into mitochondria, and VDAC is permeable to Ca2+. It is also regulated by various compounds such as glutamate, NADH and nucleotides. VDAC has two nucleotide binding sites (Yehezkel et al., 2006). In VDAC1 the two cysteine residues seem not to be required for apoptosis or VDAC1 oligomerization (Aram et al., 2010). Ions interact intimately with the inner walls of the channel and are selected by their 3-dimensional structure, not merely by their size and charge (Colombini 2016). The N-terminus acts not as a gate on a stable barrel, but rather stabilizes the barrel, preventing its shift into a partially collapsed, low-conductance, closed state (Shuvo et al. 2016).
Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons. Misfolded mutant SOD1 binds directly to VDAC1. Direct binding of mutant SOD1 to VDAC1 inhibits conductance of channels when reconstituted in a lipid bilayer (Israelson et al., 2010).
VDAC-protein interactions for each mammalian isoform (VDAC1, 2 and 3) showed that VDAC1 is mainly involved in the maintenance of cellular homeostasis and in pro-apoptotic processes, whereas VDAC2 displays an anti-apoptotic role, while VDAC3 may contribute to mitochondrial protein quality control and act as a marker of oxidative status (Caterino et al. 2017). In pathological conditions, namely neurodegenerative and cardiovascular diseases, both VDAC1 and VDAC2 establish abnormal interactions aimed to counteract the mitochondrial dysfunction which contributes to end-organ damage.
Persistent opening of permeability transition pore,PTP, creates a bioenergetic crisis with collapse of the membrane potential, ATP depletion, Ca2+ deregulation, and release of proteins such as cytochrome c into the cytoplasm. These events promote cell death. The PTP traverses the inner and outer membranes and involves the ATP/ADP exchanger (ANT) in the inner membrane and VDAC in the other membrane (Cesura et al., 2003). A calcium-triggered conformational change of the mitochondrial phosphate carrier (PiC), facilitated by cyclophilin-D (CyP-D), may induce pore opening. This is enhanced by an association of the PiC with the 'c' conformation of the ANT. Agents that modulate pore opening may act on either or both the PiC and the ANT (Leung and Halestrap, 2008). Chitosan quaternary ammonium salts induce mitochondrial PTP opening (Xia et al. 2020).
The selective anti-tumour agent erastin causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin. Using purified mitochondria expressing a single VDAC isoform, erastin alters the permeability of the outer mitochondrial membrane by binding directly to VDAC2. Thus, ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway (Yagoda et al., 2007).
VDACs form 19-stranded beta barrels with the first and last strand parallel. The hydrophobic outside perimeter of the barrel is covered by detergent molecules in a beltlike fashion (Hiller et al., 2010). In the presence of cholesterol, recombinant VDAC-1 can form voltage-gated channels in phospholipid bilayers similar to those of the native protein. The NMR measurements revealed the binding sites of VDAC-1 for the Bcl-2 protein, Bcl-x(L), for reduced beta-nicotinamide adenine dinucleotide, and for cholesterol. Bcl-x(L) interacts with the VDAC barrel laterally at strands 17 and 18 (Hiller et al., 2008). The position of the voltage-sensing N-terminal segment is oriented against the interior wall, causing a partial narrowing at the center of the pore. This segment is ideally positioned to regulate the conductance of ions and metabolites passing through the VDAC pore (Ujwal et al., 2008).
Mitochondria import 90-99% of their proteins from the cytosol. Three protein families including Sam50, VDAC and Tom40 together with Mdm10 compose the set of integral beta-barrel proteins embedded in the mitochondrial outer membrane in S. cerevisiae (MOM) (Zeth 2010). The 16-stranded Sam50 protein forms part of the sorting and assembly machinery (SAM) and shows a clear evolutionary relationship to members of the bacterial Omp85 family (1.B.33). VDAC and Tom40 both adopt the same fold with 19 probable TMSs. Tom40 is in the TOM complex (3.A.8). Models of Tom40 and Sam50 have been developed using X-ray structures of related proteins. These models have been analyzed with respect to properties such as conservation and charge distribution yielding features related to their individual functions (Zeth 2010).
The gene for VDAC1 in humans is over-expressed in many cancer types, and silencing of VDAC1 expression inhibits tumor development. Along with regulating cellular energy production and metabolism, VDAC1 is involved in the process of apoptosis by mediating the release of apoptotic proteins and interacting with anti-apoptotic proteins. The engagement of VDAC1 in the release of apoptotic proteins located in the inter-membranal space involves VDAC1 oligomerization that mediates the release of cytochrome c and AIF to the cytosol, subsequently leading to apoptotic cell death (Shoshan-Barmatz et al. 2015).
The mitochondrial permeability transition pore complex (PTPC) is involved in the control of the mitochondrial membrane permeabilization during apoptosis, necrosis and autophagy. Indeed, the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC), two major components of the PTPC, are the targets of a variety of proapoptotic inducers. Verrier et al. 2004 identified some of the interacting partners of ANT. During chemotherapy-induced apoptosis, some of these interactions were constant (e.g. ANT-VDAC), whereas others changed. Glutathione-S-transferase (GST) also interacts. This interaction is lost during apoptosis induction, suggesting that GST behaves as an endogenous repressor of PTPC and ANT pore opening. Thus, ANT is connected to mitochondrial proteins as well as to proteins from other organelles such as the endoplasmic reticulum, forming a dynamic polyprotein complex. Changes within this ANT interactome coordinate the lethal response of cells to apoptosis induction (Verrier et al. 2004).
Under cellular stress, human VDACs hetero-oligomerize and coaggregate with proteins that can form amyloidogenic and neurodegenerative deposits, implicating a role for VDACs in proteotoxicity. Gupta and Mahalakshmi 2019 mapped aggregation-prone regions of human VDACs, using isoform 3 as the model VDAC, and showed that the region comprising strands beta7-beta9 is aggregation prone. An alpha1-beta7-beta9 interaction (involving the hVDAC3 N-terminal alpha1 helix) can lower protein aggregation, whereas perturbations of this interaction promote VDAC aggregation. hVDAC3 aggregation proceeds via a partially unfolded structure.
VDACs 1 - 3, also called porins 1 - 3 or Por1-3) regulate the formation of the mitochondrial protein import gate in the OM, the translocase of the outer membrane (TOM) complex, and its dynamic exchange between the major form of a trimer and the minor form of a dimer (Endo and Sakaue 2019). The TOM complex dimer lacks the core subunit, Tom22, and mediates the import of a subset of mitochondrial proteins while the TOM complex trimer facilitates the import of most other mitochondrial proteins. Porins interact with both a translocating inner membrane (IM) protein like a carrier protein accumulated at the small TIM chaperones in the intermembrane space and the TIM22 complex, a downstream translocator in the IM for carrier protein import. Porins (VDACs) thereby facilitate the efficient transfer of carrier proteins to the IM during their import. Finally, porins facilitate the transfer of lipids between the OM and IM and promote a back-up pathway for cardiolipin synthesis in mitochondria. Thus, porins have roles in addition to metabolite transport in mitochondria (Endo and Sakaue 2019). Δpor1 cells lacking VDAC1 show enhanced phospholipid biosynthesis, accumulate lipid droplets, increase vacuoles and cell size, and overproduce and excrete inositol (Magrì et al. 2019).
Using human VDAC as a template scaffold, Srivastava and Mahalakshmi 2020 designed and engineered odd- and even-stranded structures of smaller (V2(16), V2(17), V2(18)) and larger (V2(20), V2(21)) barrel diameters. Determination of the structures, dynamics, and energetics of these engineered structures in bilayer membranes revealed that the 19-stranded barrel holds modest to low stability, but possesses superior voltage-gated channel regulation, efficient mitochondrial targeting and in vivo cell survival, with lipid-modulated stability, all of which supersede the occurrence of a metastable 19-stranded scaffold.
The generalized transport reaction catalyzed by VDACs is:
(Anionic) metabolites (out) ↔ anionic metabolites (intermembrane space)