Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-09
2001-08-21
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S023200, C536S023500, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06277572
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of new human NADH dehydrogenase protein subunits and to the use of these sequences in the diagnosis, prevention, and treatment of cancer, smooth muscle disorders, and neurological disorders.
BACKGROUND OF THE INVENTION
NADH dehydrogenase (NADH:ubiquinone oxidoreductase, NADH-D) is the first multienzyme complex (Complex I) in a chain of three complexes that make up the mitochondrial electron transport chain. The mitochondrial electron transport chain is responsible for the transport of electrons from NADH to oxygen and the coupling of this oxidation to the synthesis of ATP (oxidative phosphorylation) which provides the energy source for driving a cell's many energy-requiring reactions. NADH-D accomplishes the first step in this process by accepting electrons from NADH and passing them through a flavin molecule to ubiquinone, which transfers the electrons to the second enzyme complex in the chain.
NADH-D and the other members of the electron transport chain are located in the mitochondrial membrane. NADH-D is the largest of the three complexes with an estimated mass of 800 kDa comprising some 40 polypeptide subunits of widely varying size and composition. The polypeptide composition of NADH-D in a variety of mammalian species including rat, rabbit, cow, and man is very similar (Cleeter, M. W. J. and Ragan, C. I. (1985) Biochem. J. 230: 739-46). The best characterized NADH-D is from bovine heart mitochondria and is composed of 41 polypeptides (Walker, J. E. et al. (1992) J. Mol. Biol. 226: 1051-72). Seven of these polypeptides are encoded by mitochondrial DNA while the remaining 34 are nuclear gene products that are imported into the mitochondria. Many of these imported polypeptides are characterized by various N-terminal peptide sequences or modified N-terminal amino acids (myristoylation or acetylation) that target them to the mitochondria and are then cleaved from the mature protein. However several of these polypeptides have neither N-terminal targeting sequences nor modified-N terminal amino acids. Their import signals appear to lie within the mature protein (Walker et al., supra).
The functions of many of the individual subunits in NADH-D are largely unknown. The 24-, 51-, and 75-kDa subunits have been identified as being catalytically important in electron transport, with the 51-kDa subunit forming part of the NADH binding site and containing the flavin moiety that is the initial electron acceptor (Ali, S. T. et al. (1993) Genomics 18:435-39). The location of other functionally important groups, such as the electron-carrying iron sulfate centers, remains to be determined. Many of the smaller subunits (<30 k Da) contain hydrophobic sequences that may be folded into membrane spanning &agr;-helices. These subunits presumably are anchored into the inner membrane of the mitochondria and interact via more hydrophilic parts of their sequence with globular proteins in the large extrinsic domain of NADH-D.
Defects and altered expression of NADH-D are associated with a variety of disease conditions in man, including neurodegenerative diseases, myopathies, and cancer. In addition, NADH-D reduction of the quinone moiety in chemotherapeutic agents such as doxorubicin is believed to contribute to the antitumor activity and/or mutagenicity of these drugs.
The discovery of new NADH dehydrogenase subunits and the polynucleotides encoding them provides a means to investigate mitochondrial respiratory mechanisms under normal and disease conditions and satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment or prevention of cancer and immune disorders.
SUMMARY OF THE INVENTION
The present invention features two new human NADH-D protein subunits hereinafter designated as NDS-5 and NDS-6 and collectively as NDS, and characterized as having similarity to other NADH-D protein subunits.
Accordingly, the invention features substantially purified NDS-5 and NDS-6 having the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3, respectively.
One aspect of the invention features isolated and substantially purified polynucleotides that encode NDS-5 and NDS-6. In a particular aspect, the polynucleotides are the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:4, respectively.
The invention also relates to polynucleotide sequences comprising the complement of SEQ ID NO:2 or SEQ ID NO:4 or variants thereof. In addition, the invention features polynucleotide sequences which hybridize under stringent conditions to SEQ ID NO:2 or SEQ ID NO:4.
The invention additionally features expression vectors and host cells comprising polynucleotides that encode NDS, and a method for producing NDS using the vectors and host cells. The present invention also features antibodies which bind specifically to NDS, and pharmaceutical compositions comprising substantially purified NDS. The invention also features agonists and antagonists of NDS. The invention also provides methods for treating disorders associated with expression of NDS by administration of NDS and methods for detection of polynucleotides encoding a regulator of gene transcription in a biological sample.
REFERENCES:
Cleeter, M.W.J., et al., “The polypeptide composition of the mitochondrial NADH: ubiquinone reductase complex from several mammalian species,”Biochem. J., 230:739-746 (1985).
Walker, J.E., et al., “Sequences of 20 Subunits of NADH: Ubiquinone Oxidoreductase from Bovine Heart Mitochondria,”J. Mol. Biol., 226:1051-1072 (1992) (GI 250; GI 254).
Ali, S., et al., “Chromosomal Localization of the Human Gene Encoding the 51-kDa Subunit of Mitochondrial Complex I (NDUFV1) to 11q13,”Genomics, 18:435-439 (1993).
The WashU-Merck EST Project, Accession No. AA009413 (1995).
The WashU-Merck EST Project, Accession No. W24219 (1995).
The WashU-Merck EST Project, Accession No. AA203643 (1995).
The WashU-Merck EST Project, Accession No. W93566 (1995).
The WashU-Merck EST Project, Accession No. AA111999 (1995).
The WashU-Merck EST Project, Accession No. N47307 (1995).
The WashU-Merck EST Project, Accession No. W72958 (1995).
Hillman Jennifer L.
Lal Preeti
Shah Purvi
Incyte Pharmaceuticals Inc.
Incyte Pharmaceuticals, Inc.
LeGuyader John L.
Sather Susan K.
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