Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-09
2001-04-03
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023200, C435S320100
Reexamination Certificate
active
06210890
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of a human peroxisomal thioesterase and to the use of these sequences in the diagnosis, prevention, and treatment of cancer, inflammation, and disorders associated with fatty acid metabolism.
BACKGROUND OF THE INVENTION
Two soluble thioesterases involved in fatty acid biosynthesis have been isolated from mammalian tissues, one which is active only toward long-chain fatty-acyl thioesters and one which is active toward thioesters with a wide range of fatty-acyl chain-lengths. These thioesterases catalyze the chain-terminating step in the de novo biosynthesis of fatty acids. Chain termination involves the hydrolysis of the thioester bond which links the fatty acyl chain to the 4′-phosphopantetheine prosthetic group of the acyl carrier protein (ACP) subunit of the fatty acid synthase (Smith, S. (1981a) Methods Enzymol. 71:181-188; Smith, S. (1981b) Methods Enzymol. 71:188-200).
E. coli
contains two soluble thioesterases, thioesterase I which is active only toward long-chain acyl thioesters, and thioesterase II (TEII) which has a broad chain-length specificity (Naggert, J. et al. (1991) J. Biol. Chem. 266:11044-11050).
E. coli
TEII does not exhibit sequence similarity with either of the two types of mammalian thioesterases which function as chain-terminating enzymes in de novo fatty acid biosynthesis. Unlike the mammalian thioesterases,
E. coli
TEII lacks the characteristic serine active site gly-X-ser-X-gly sequence motif and is not inactivated by the serine modifying agent diisopropyl fluorophosphate. However, modification of histidine 58 by iodoacetamide and diethylpyrocarbonate abolished TEII activity. Overexpression of TEII did not alter fatty acid content in
E. coli
, which suggests that it does not function as a chain-terminating enzyme in fatty acid biosynthesis (Naggert et al., supra). For that reason, Naggert et al. (supra) proposed that the physiological substrates for
E. coli
TEII may be coenzyme A (CoA)-fatty acid esters instead of ACP-phosphopanthetheine-fatty acid esters.
CoA plays an important role in the synthesis and metabolism of fatty acids. Esterification of the fatty acid carboxylic acid group with CoA creates a thioester bond which activates the fatty acid molecule for nucleophilic attack and subsequent metabolic conversions. Likewise, hydrolysis of the fatty acyl-CoA thioester bond renders the fatty acid carboxylate group unreactive toward nucleophilic attack.
Peroxisomes are single, membrane-bound, spheroid organelles present in virtually all eukaryotic cells. The peroxisome matrix contains more than forty enzymes which are involved in a variety of metabolic processes including peroxide-based respiration, synthesis of plasmalogen and bile acids, beta-oxidation of fatty acids, and glyoxylate transamination. Peroxisomal matrix enzymes are synthesized on free cytoplasmic polysomes and are imported into peroxisomes without subsequent proteolytic processing. Most peroxisomal enzymes contain a C-terminal SKL (ser-lys-leu) matrix targeting sequence.
More than half of the enzymes present in mammalian peroxisomes are associated with lipid metabolism (Baumgart, E. et al. (1996) Proc. Nat. Acad. Sci. 93:13748-13753). Beta-oxidation of very long straight-chain fatty acids, branched-chain fatty acids, dicarboxylic fatty acids, and eicosanoids occurs within peroxisomes. Beta-oxidation of the side chain of the bile acid intermediates di- and trihydroxycoprostanic acids, which results in the formation of the primary bile acids (chenodeoxycholic and cholic acid, respectively), also takes place in peroxisomes. The different fatty acid substrates are likely to be degraded in distinct beta-oxidation pathways (Baumgart, et al., supra).
Disorders associated with defective peroxisomal fatty acid metabolism include adrenoleukodystrophy, adrenomyeloneuropathy, cerebrohepatorenal syndrome (Zellweger syndrome), Refsum's disease, and peroxisomal thiolase deficiency. Patients with defective peroxisomal fatty acid metabolism exhibit neuronal demyelination, disordered neuronal migration, hypotonia, mental retardation, tapetoretinal degeneration, sensorineural hearing loss, cystic changes in the kidneys, skeletal changes, and death. The clinical distinction between patients with a disorder of peroxisome assembly and those with a defect in a peroxisomal fatty acid metabolic enzyme can be difficult (Watkins, P. A. et al. (1995) Ann. Neurol. 38:472-477).
The discovery of a new human peroxisomal thioesterase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of cancer, inflammation, and disorders associated with fatty acid metabolism.
SUMMARY OF THE INVENTION
The invention features a substantially purified polypeptide, human peroxisomal thioesterase (PxTE), having the amino acid sequence shown in SEQ ID NO:1, or fragments thereof.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of said polynucteotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO.2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2.
In another aspect the invention provides a composition comprising an isolated and purified polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence 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 thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding PxTE 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 PxTE having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist which decreases the activity of a polypeptide of SEQ ID NO:1. In one aspect the invention provides a purified antibody which binds to a polypeptide comprising at least a fragment of the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist which modulates the activity of the polypeptide of SEQ ID NO:1.
The invention also provides a method for treating or preventing a disorder associated with fatty acid metabolism comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising purified PxTE.
The invention also provides a method for treating or preventing cancer comprising administering to a subject in need of such treatment an effective amount of an antagonist of PxTE.
The invention also provides a method for treating or preventing inflammation comprising administering to a subject in need of such treatment an effective amoun
Corley Neil C.
Hillman Jennifer L.
Shah Purvi
Bugaisky Gabriele E.
Incyte Genomics Inc.
Incyte Genomics, Inc.
Low Christopher S. F.
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