Thioredoxin family active site molecules and uses therefor

Details Thioredoxin family active site molecules and uses therefor Thioredoxin family active site molecules and uses therefor

1999-03-08

2001-01-16

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S189000, C435S252300, C435S320100, C435S810000, C536S023200, C536S023400, C536S023500

Reexamination Certificate

active

06174682

ABSTRACT:

BACKGROUND OF THE INVENTION
Thioredoxin family active site proteins are a superfamily of proteins that participate in redox reactions and are distributed among a wide range of living organisms (Holmgren, A. (1985)
Ann. Rev. Biochem.
54:237-271; Eklund, H. et al. (1991)
Proteins
11:13-28; Freedman, R. B. et al. (1994)
Trends in Biochem. Sci.
19:331-336). The thioredoxin family active site is characterized by a CXXC motif (C represents cysteine and X represents any of the 20 amino acids incorporated into proteins). The neighboring cysteine residues cycle between a reduced sulfhydryl and an oxidized disulfide form.
The reduced form of thioredoxin is known to activate some enzymes by reducing disulfide bridges that control their activity. In addition, thioredoxin is an electron donor in the reaction sequence that reduces ribonucleotides to deoxyribonucleotides catalyzed by ribonucleotide reductase (Stryer, L. (1995)
Biochemistry
4th Edition, W.H. Freeman and Company, pages 677, and 750-751.). It has been reported that in humans, thioredoxin and the cellular redox state modified by thioredoxin play a crucial role in arterial neointima formation in atherosclerosis (Takagi, Y. et al. (1998)
Laboratory Investigation
78:957-66). Thioredoxin is believed to be involved in cellular defense mechanisms against oxidative damage (see, for example, Tanaka, T. et al. (1997)
Laboratory Investigation
77:145-55). Thioredoxin has also been implicated in regulating glucocorticoid responsiveness to cellular oxidative stress response pathways. In particular, thioredoxin is believed to be capable of sensing, and transmitting, the redox state of the cell, to the glucocorticoid receptor by targeting both the ligand- and DNA-binding domains of the receptor (Makino, Y. et al. (1996)
Journal of Clinical Investigation
98:2469-77). Human thioredoxin has been suggested to act as a free radical scavenger and has been shown to limit the extent of ischaemia reperfusion injury (Fukuse, T. et al. (1995)
Thorax
50:387-91).
Protein disulfide isomerases are an important class of thioredoxin family active site-containing proteins that catalyze the oxidation of thiols, reduction of disulfide bonds, and isomerization of disulfides, depending on the reaction conditions (Freedman, R. B. et al. (1994)
Trends in Biochem. Sci.
19:331-336).
Protein disulfide isomerases catalyze the formation of correct disulfide pairings in nascent proteins. Protein disulfide isomerases preferentially interact with peptides that contain cysteine residues but are otherwise undiscriminating. The broad substrate specificity of protein disulfide isomerases enables them to speed the folding of diverse disulfide-containing proteins. By shuffling disulfide bonds, protein disulfide isomerases enable proteins to quickly find the most thermodynamically stable pairings amongst those that are accessible. Consequently, protein disulfide isomerases are involved in protein processing, protein folding, and protein secretion. Certain protein disulfide isomerases are also involved in collagen and collagen-like protein biosynthesis because a protein disulfide isomerase is a subunit of prolyl 4-hydroxylase, a collagen and collagen-like protein biosynthetic enzyme (Kivirikko, K. I. et al. (1998)
Matrix Biol.
16:357-368; Kivirikko, K. I. et al. (1998)
Adv. Enzymol. Relat. Areas Mol. Biol.
72:325-398). Because mutations in prolyl 4-hydroxylase cause Ehlers-Danlos Syndrome, protein disulfide isomerases have been implicated in Ehlers-Danlos Syndrome.
Given the important biological roles and properties of thioredoxin family active site-containing proteins, there exists a need for the identification of novel genes encoding such proteins as well as for the discovery of modulators of such molecules for use in regulating a variety of normal and/or pathological cellular processes.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of novel members of the thioredoxin family active site molecules, referred to herein as MP-4 nucleic acid and protein molecules. The MP-4 nucleic acid and protein molecules of the present invention are useful as targets for developing modulating agents that regulate a variety of cellular processes, e.g., cellular redox reactions. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding MP-4 polypeptides or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of MP-4-encoding nucleic acids.
In one embodiment, a MP-4 nucleic acid molecule of the invention is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1, SEQ ID NO:3, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-73 of SEQ ID NO:1. In yet another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 917-1207 of SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:1 or 3. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 395 nucleotides of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or a complement thereof.
In another embodiment, a MP-4 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, a MP-4 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to the amino acid sequence of SEQ ID NO:2.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human MP-4. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2. In yet another preferred embodiment, the nucleic acid molecule is at least 842 nucleotides in length and encodes a protein having a MP-4 activity (as described herein).
Another embodiment of the invention features nucleic acid molecules, preferably MP-4 nucleic acid molecules, which specifically detect MP-4 nucleic acid molecules relative to nucleic acid molecules encoding non-MP-4 proteins. For example, in one embodiment, such a nucleic acid molecule is at least 395, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, or a complement thereof, or a complement thereof In preferred embodiments, the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 1-78 and 973-976 of SEQ ID NO:1. In other preferred embodiments, the nucleic acid molecules comprise nucleotides 1-78 and 973-976 of SEQ ID NO:1.
In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 or SEQ ID NO:3 under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a MP-4 nucleic acid molecule, e.g., the coding strand of a MP-4 nucleic acid molecule.
Another aspect of the invention provides a vector comprising a MP-4 nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. The invention also provides

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