Delta 1-pyrroline-5-carboxylate reductase homolog

Details Delta 1-pyrroline-5-carboxylate reductase homolog Delta 1-pyrroline-5-carboxylate reductase homolog

2000-05-05

2001-07-31

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S007100, C435S025000, C424S185100, C530S325000

Reexamination Certificate

active

06268192

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of a delta 1-pyrroline-5-carboxylate reductase homolog and to the use of these sequences in the diagnosis, treatment, and prevention of neuronal disorders, connective tissue disorders, and disorders of cell proliferation.
BACKGROUND OF THE INVENTION
Many pathways of biogenesis and biodegradation require oxidoreductase (dehydrogenase or reductase) activity, coupled to reduction or oxidation of a cofactor, such as NAD(P)
+
/NAD(P)H. (Newsholme, E. A. and Leech, A. R. (1983)
Biochemistry for the Medical Sciences
, John Wiley and Sons, Chichester, U.K. pp. 779-793.) Reductase activity catalyzes the transfer of electrons with the oxidation of NADH and NADPH. The reverse dehydrogenase reaction reduces NAD
+
and NADP
+
.
Delta 1-pyrroline-5-carboxylate reductase (P5CR) catalyzes the NAD(P) dependent oxidation of 1-pyrroline-5-carboxylate to proline, the terminal step in the biosynthesis of proline from glutamate. Proline is a critical substrate for the synthesis of collagen in tissues such as bone and interstitial fluid. In chondrocytes, P5CR is active during bone formation, and has a peak in activity which correlates with maximal collagen synthesis. P5CR may be involved in the regulation of nucleotide metabolism mediated by interaction with enzymes in the production of ribose phosphate. (Yeh et al. (1984) J. Biol. Chem. 259:5454-5458.) The enzyme activity is regulated by non-dietary controls. (Samuels et al. (1989) J. Nutr. 119:1900-1906.)
Proline is involved in a variety of disorders, including fibrosis. Cardiac fibrosis occurs, after myocardial infarction, in non-infarcted ventricular tissue and is associated with abnormal cardiac function. In hypertensive heart disease, reactive myocardial fibrosis is observed as an excessive accumulation of fibrillar collagen within the normal connective tissue of the myocardium. This accumulation increases in the presence of aldosterone, deoxycortisone, or angiotensin II. Collagen synthesis of cardiac fibroblasts decreases in the presence of prostaglandin E2. (Funck et al. (1997) Adv. Exp. Med. Biol. 432:35-44.) Proline levels are also increased in liver undergoing fibrosis due to chronic liver damage. Abnormalities of liver regeneration can contribute to chronic hepatitis, cirrhosis, and/or liver cancer. (Leevy, C. B. (1998) 16:88-98.) Lung hydroxyproline is an index of fibrosis in interstitial lung fibrosis. Leiomyomas are characterized by increased cell proliferation and tissue fibrosis. (Lee (1998) J. Clin. Endocrinol. Metab. 83:219-223.) Fibrosis in heart, liver, and lung, may be modified by inhibiting metabolites that regulate collagen deposition.
Some cancerous growth may involve increased or decreased P5CR activity. Treatments for chronic liver disease that inhibit the collagen-producing stellate cells have been reported to inhibit the development of hepatocellular carcinoma. (Sakaida et al. (1998) J. Hepatol. 28:298-306.) Deficiency of P5CR in a human leukemic lymphoblastoid cell line relative to normal human lymphocytes has been observed. (Lorans et al. (1978) Cancer Res. 38:3950-3953.) Raised P5CR activity distinguishes neoplastic from non-neoplastic colon cells. (Herzfeld et al. (1980) Cancer 46:2047-2054.)
Proline appears to modulate synaptic transmission in the mammalian brain. A subset of glutamate pathways involve high affinity proline transport and may be implicated in the seizures and mental retardation associated with genetic disorders of proline metabolism. Disorders of proline metabolism resulting in hyperprolinemia have been observed, at least one of which is associated with neuropsychiatric disorders that may be due to proline potentiation of transmission in cells of the hippocampus. (Cohen et al. (1997) Brain Res. 769:333-339; Cohen et al. (1997) Brain Res. 761:271-282.) Deficiency of another key enzyme in proline metabolism, prolidase, is associated with skin lesions, recurrent infections, mental retardation, and splenomegaly. P5CR deficiency has been observed in retinal degeneration. (Matsuzawa et al. (1982)Adv. Exp. Med. Biol. 153:361-370.)
The sequences of P5CR from eubacteria, archaeobacteria and eukaryotes show only a moderate level of overall similarity. P5CR is characterized by a conserved motif. (Delauney et al. (1990) Mol. Gen. Genet. 221:299-305).
The discovery of a new delta 1-pyrroline-5-carboxylate reductase homolog and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of neuronal disorders, connective tissue disorders and disorders of cell proliferation.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a new human delta 1-pyrroline-5-carboxylate reductase homolog (P5CRH), the polynucleotides encoding P5CRH, and the use of these compositions for the diagnosis, treatment, or prevention of neuronal disorders, connective tissue disorders, and disorders of cell proliferation.
The invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector comprising at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell comprising an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a neurona

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