Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-11-03
2003-05-13
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S029000, C435S325000, C435S404000, C435S026000, C435S014000, C435S015000, C536S022100
Reexamination Certificate
active
06562563
ABSTRACT:
TECHNICAL FIELD
The invention relates generally to methods for identifying agents that affect mitochondrial membrane permeability transition. More specifically, the invention relates to compositions and screening methods for use in identifying agents that alter the interaction between the mitochondrial adenine nucleotide translocator and cyclophilin D.
BACKGROUND OF THE INVENTION
Mitochondria are the main energy source in cells of higher organisms, and provide direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes. Such processes include electron transport chain (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP), and which also underlies a central mitochondrial role in intracellular calcium homeostasis.
Mitochondrial ultrastructural characterization reveals the presence of an outer mitochondrial membrane that serves as an interface between the organelle and the cytosol, a highly folded inner mitochondrial membrane that appears to form attachments to the outer membrane at multiple sites, and an intermembrane space between the two mitochondrial membranes. The subcompartment within the inner mitochondrial membrane is commonly referred to as the mitochondrial matrix. (For a review, see, e.g., Emster et al., 1981
J. Cell Biol
. 91:227s.) The cristae, originally postulated to occur as infoldings of the inner mitochondrial membrane, have recently been characterized using three-dimensional electron tomography as also including tube-like conduits that may form networks, and that can be connected to the inner membrane by open, circular (30 nm diameter) junctions (Perkins et al., 1997
, Journal of Structural Biology
119:260). While the outer membrane is freely permeable to ionic and non-ionic solutes having molecular weights less than about ten kilodaltons, the inner mitochondrial membrane exhibits selective and regulated permeability for many small molecules, including certain cations, and is impermeable to large (>~10 kDa) molecules.
Altered or defective mitochondrial activity, including but not limited to failure at any step of the ETC, may result in catastrophic mitochondrial collapse that has been termed “permeability transition” (PT) or “mitochondrial permeability transition” (MPT). According to generally accepted theories of mitochondrial function, proper ETC respiratory activity requires maintenance of an electrochemical potential (&Dgr;&PSgr;m) in the inner mitochondrial membrane by a coupled chemiosmotic mechanism. Altered or defective mitochondrial activity may dissipate this membrane potential, thereby preventing ATP biosynthesis and halting the production of a vital biochemical energy source. In addition, mitochondrial proteins such as cytochrome c may leak out of the mitochondria after permeability transition and may induce the genetically programmed cell suicide sequence known as apoptosis or programmed cell death (PCD).
Four of the five multi-subunit protein complexes (Complexes I, III, IV and V) that mediate ETC activity are localized to the inner mitochondrial membrane, which is the most protein rich of biological membranes in cells (75% by weight); the remaining ETC complex (Complex II) is situated in the matrix. In at least three distinct chemical reactions known to take place within the ETC, positively-charged protons are moved from the mitochondrial matrix, across the inner membrane, to the intermembrane space. This disequilibrium of charged species creates an electrochemical potential of approximately 220 mV referred to as the “proton motive force” (PMF), which is often represented by the notation &Dgr;&psgr; or &Dgr;&psgr;m and represents the sum of the electric potential and the pH differential across the inner mitochondrial membrane (see, e.g., Emster et al., 1981
J. Cell Biol
. 91:227s and references cited therein).
This membrane potential provides the energy contributed to the phosphate bond created when adenosine diphosphate (ADP) is phosphorylated to yield ATP by ETC Complex V, a process that is “coupled” stoichiometrically with transport of a proton into the matrix; &Dgr;&psgr;m is also the driving force for the influx of cytosolic Ca
2+
into the mitochondrion. Under normal metabolic conditions, the inner membrane is largely impermeable to proton movement from the intermembrane space into the matrix, leaving ETC Complex V as the primary means whereby protons can return to the matrix. When, however, the integrity of the inner mitochondrial membrane is compromised, as occurs during MPT that may accompany a disease associated with altered mitochondrial function, protons are able to bypass the conduit of Complex V without generating ATP, thereby “uncoupling” respiration because electron transfer and associated proton pumping yields no ATP. Thus, mitochondrial permeability transition involves the opening of a mitochondrial membrane “pore”, a process by which, inter alia, the ETC and &Dgr;&psgr;m are uncoupled, &Dgr;&psgr;m collapses and mitochondrial membranes lose the ability to selectively regulate permeability to solutes both small (e.g., ionic Ca
2+
, Na
+
, K
+
, H
+
) and large (e.g., proteins).
The mitochondrial permeability transition “pore” may not be a discrete assembly or multi-subunit complex, and the term thus refers instead to any mitochondrial molecular component (including, e.g., a mitochondrial membrane per se) that regulates the inner membrane selective permeability where such regulated function is impaired during MPT. A mitochondrial molecular component may be a protein, polypeptide, peptide, amino acid or derivative thereof; a lipid, fatty acid or the like, or derivative thereof; a carbohydrate, saccharide or the like or derivative thereof; a nucleic acid, nucleotide, nucleoside, purine, pyrimidine or related molecule, or derivative thereof, or the like; or any other biological molecule that is a constituent of a mitochondrion. A mitochondrial permeability transition pore component, also referred to as a mitochondrial pore component, may be any mitochondrial molecular component that regulates the selective permeability characteristic of mitochondrial membranes as described above, including those responsible for establishing &Dgr;&PSgr;m and those that are functionally modified during MPT. Mitochondrial pore components may also include factors that interact with mitochondria, for example through transient or stable association with other mitochondrial pore components, in a manner that regulates MPT. Examples of such factors include cyclophilins (described in greater detail below), calcium modulating cyclophilin ligand (CAML, see, e.g., Table 1, infra, and references cited therein) and members of the Bcl-2 family including Bcl-2 (e.g., Green et al., 1998
Science
281:1309), Bax (Marzo et al., 1998
Science
281:2027) and Bak (Narita et al., 1998
Proc. Nat. Acad. Sci. USA
95:14681).
Without wishing to be bound by theory, it is unresolved whether this pore is a physically discrete conduit that is formed in mitochondrial membranes, for example by assembly or aggregation of particular mitochondrial and/or cytosolic proteins and possibly other molecular species, or whether the opening of the “pore” may simply represent a general increase in the porosity of the mitochondrial membrane. In any event, certain mitochondrial molecular components may contribute to the MPT mechanism, including ETC components or other mitochondrial components described herein. For example, some non-limiting examples of mitochondrial permeability transition pore components that appear to contribute to the MPT mechanism include members of the following families of gene products (see, e.g., Table 1, infra, and references cited therein): adenine nucleotide translocator (ANT); peripheral benzodiazepine receptor (PBzR; McEnery et al., 1992
Proc. Nat. Acad. USA
89:3170); PBzR-associated protein (PRAX); voltage dependent anion channel (VDAC, also known as porin); cyclophilin (Cyp); calcium modulating cyclophi
Andreyev Alexander Y.
Clevenger William
Davis Robert E.
Frigeri Luciano G.
Murphy Anne N.
Chakrabarti Arun Kr.
Jones W. Gary
Mitokor
Seed Intellectual Property Law Group PLLC
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