Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-01-16
2004-10-26
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S007100, C435S091100, C435S091500, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024500, C514S001000
Reexamination Certificate
active
06808873
ABSTRACT:
TECHNICAL FIELD
The invention relates generally to assays for screening for agents that affect mitochondrial activity. More specifically, the invention is directed to screening methods for use in identifying agents that alter mitochondrial regulation of intracellular calcium. An assay for the presence of extramitochondrial calcium and for factors that influence levels of intramitochondrial and/or extramitochondrial calcium, such as the calcium uniporter (CaUP), is provided herein.
BACKGROUND OF THE INVENTION
Mitochondria are organelles that are the main energy source in cells of higher organisms. These organelles provide direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes, including metabolic energy production, aerobic respiration and intracellular calcium regulation. For example, mitochondria are the site of 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. These processes require the maintenance of a mitochondrial membrane electrochemical potential, and defects in such membrane potential can result in a variety of disorders.
In addition to their role in energy production in growing cells, mitochondria (or at least mitochondrial components) participate in programmed cell death (PCD), also known as apoptosis (see Newmeyer et al.,
Cell
79:353-364, 1994; Liu et al.,
Cell
86:147-157, 1996). Apoptosis is apparently required for normal development of the nervous system and functioning of the immune system. Some disease states are associated with insufficient apoptosis (e.g., cancer and autoimmune diseases) or excessive levels of apoptosis (e.g., stroke and neurodegeneration). For general reviews of apoptosis, and the role of mitochondria therein, see Green and Reed,
Science
281:1309-1312, 1998; Green,
Cell
94:695-698, 1998; and Kromer,
Nature Medicine
3:614-620, 1997.
Mitochondria contain 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 review, see, e.g., Emster et al.,
J. Cell Biol.
91:227s, 1981). 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 (greater than about 10 kD) molecules.
Four of the five multisubunit protein complexes (Complexes I, III, IV and V) that mediate ETC activity are localized to the inner mitochondrial membrane. 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, protons are moved from the mitochondrial matrix, across the inner membrane, to the intermembrane space. This disequilibrium of charged species creates an electrochemical membrane potential of approximately 220 mV referred to as the “protonmotive force” (PMF). The PMF, which is often represented by the notation &Dgr;p, corresponds to the sum of the electric potential (&Dgr;&PSgr;m) and the pH differential (&Dgr;pH) across the inner membrane according to the equation
&Dgr;
p=&Dgr;&PSgr;m−Z&Dgr;pH
wherein Z stands for −2.303 RT/F. The value of Z is −59 at 25° C. when &Dgr;p and &Dgr;&PSgr;m are expressed in mV and &Dgr;pH is expressed in pH units (see, e.g., Emster et al.,
J. Cell Biol.
91:227s, 1981, and references cited therein).
&Dgr;&PSgr;m provides the energy for phosphorylation of adenosine diphosphate (ADP) 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 mitochondrial permeability transition (MPT) that accompanies certain diseases associated with altered mitochondrial function, protons are able to bypass the conduit of Complex V without generating ATP, thereby uncoupling respiration (i.e., ETC activity) from ATP production. During MPT, &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
+
and H
+
) and large (e.g., proteins). Loss of mitochondrial potential also appears to be a critical event in the progression of diseases associated with altered mitochondrial function, including degenerative diseases such as Alzheimer's Disease; diabetes mellitus; Parkinson's Disease; Huntington's disease; dystonia; Leber's hereditary optic neuropathy; schizophrenia; mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferative disorders; mitochondrial diabetes and deafness (MIDD) and myoclonic epilepsy ragged red fiber syndrome.
Normal alterations of intramitochondrial Ca
2+
are associated with normal metabolic regulation (Dykens, 1998 in
Mitochondria
&
Free Radicals in Neurodegenerative Diseases
, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 29-55; Radi et al., 1998 in
Mitochondria
&
Free Radicals in Neurodegenerative Diseases
, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 57-89; Gunter and Pfeiffer, 1991,
Am. J. Physiol.
27: C755; Gunter et al.,
Am. J. Physiol.
267:313, 1994). For example, fluctuating levels of mitochondrial free Ca
2+
may be responsible for regulating oxidative metabolism in response to increased ATP utilization, via allosteric regulation of enzymes (reviewed by Crompton and Andreeva,
Basic Res. Cardiol.
88:513-523, 1993); and the glycerophosphate shuttle (Gunter and Gunter,
J. Bioenerg. Biomembr.
26:471, 1994).
Normal mitochondrial function includes regulation of cytosolic free calcium levels by sequestration of excess Ca
2+
within the mitochondrial matrix. Depending on cell type, cytosolic Ca
2+
concentration is typically 50-100 nM. In normally functioning cells, when Ca
2+
levels reach 200-300 nM, mitochondria begin to accumulate Ca
2+
as a function of the equilibrium between influx via a Ca
2+
uniporter in the inner mitochondrial membrane and Ca
2+
efflux via both Na
+
dependent and Na
+
independent calcium carriers. The low affinity of this rapid uniporter mechanism suggests that the primary uniporter function may be to lower cytosolic Ca
2+
in response to pathological elevation of cytosolic free calcium levels, which may result from ATP depletion and/or abnormal calcium influx across the plasma membrane (Gunter and Gunter,
J. Bioenerg. Biomembr.
26:471, 1994; Gunter et al.,
Am. J. Physiol.
267:313, 1994). In certain instances, such perturbation of intracellular calcium homeostasis is a feature of diseases associated with altered mitochondrial function, regardless of whether the calcium regulatory dysfunction is causative of, or a consequence of, altered mitochondrial function including MPT.
In view of the significance of mitochondrial regulation of intracellular calcium and the relationship of this mitochondrial activity to several disease states, there is clearly a need for improved compositions and methods to control mitochondrial calcium homeostasis. To provide improved therapies for such diseases, agents that
Murphy Anne N.
Stout Amy K.
MitoKor, Inc.
Seed Intellectual Property Law Group PLLC
Siew Jeffrey
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