7970, a novel ATPase-like molecule and uses thereof

Details 7970, a novel ATPase-like molecule and uses thereof 7970, a novel ATPase-like molecule and uses thereof

2001-02-21

2002-11-12

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S006120, C435S325000, C536S023200

Reexamination Certificate

active

06479268

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to novel ATPase-like nucleic acid sequences and proteins. Also provided are vectors, host cells, and recombinant methods for making and using the novel molecules.
BACKGROUND OF THE INVENTION
Enzymes that bind to and hydrolyze ATP play a pivotal role in translating chemically stored energy into biological activity. ATPases can function in a variety of cellular processes including, selective ion transport events, actin-based motility, membrane traffic and numerous biosynthetic pathways. Multiple ATPase families exist, including ion pumps, DEAD box-helicases, ABC transporters, and AAA (ATPases Associated to a variety of cellular Activities).
AAA proteins play essential roles in cellular housekeeping, cell division and differentiation and have been identified in prokaryotes and eukaryotes. All members of the AAA family are Mg
2+
dependent ATPases and comprise a conserved region that binds ATP. Cytosolic, transmembrane, as well as, membrane-associated AAA family members have been identified in various cellular locations and multimeric states.
The biological role of the AAA family members in the cell is diverse. Currently, members of this ATPase family are known to be involved in organelle biogenesis, cell-cycle regulation, vesicle-mediated transport, assembly of proteins through membranes, peroxisome biogenesis, gene expression in yeast and in human, and 26S proteasome function. For are view, see, Confalonieri et al. (1995)
BioEssays
17:639-650.
The SEC18 gene product from
S. cerevisiae
is an AAA family member that influences the transport of proteins between the endoplasmic reticulum and the golgi complex. It has been shown that SEC18 is an essential component of a multisubunit 20S “fusion machine” that promotes membrane bilayer fusion coupled to ATP hydrolysis. The 20S fusion machine has been proposed to be involved in the assembly, fusion or division of a variety of other membrane-bound subcellular compartments such as vacuoles, nuclei, mitochondria, orperoxisomes (Wilson et al. (1992)
J. Cell. Bio
. 117:531-538). Other AAA family members are involved in mitochondrial function. YME1 is a putative ATP and zinc-dependent protease. Its inactivation leads to several morphological and functional defects, such as the escape of DNA from mitochondria (Thorsness et al. (1993)
Mol Cell Biol
13: 5418-5426).
MSP1 is another AAA ATPase protein family member from yeast that influences mitochondrial function. MSP1 is an intrinsic mitochondrial outer membrane protein with an apparent molecular mass of 40 KDa. MSP1 is known to influence intramitochondrial protein sorting. Nakai et al. have demonstrated that the 61 mCl fusion protein, normally localized to the outer mitochondrial membrane, is mislocalized to the inner membrane of the mitochondria upon overexpression of MSP1 in yeast cell (Nakai et al. (1993)
J. Biol. Chem
. 268:24262-9).
Several members of the AAA family are involved in the biogenesis of peroxisomes. These organelles contain enzymes responsible for fatty acid oxidation and the elimination of peroxides. AAA family members, such as the PAS genes of
S. cerevisiae
, appear to be required for peroxisome growth, and proliferation (Subramani et al. (1993)
Annu. Rev. Cell Biol
. 9:445-478). Furthermore, mutations in the AAA proteins Pex1p or Pex6p accumulate abnormal peroxisomal vesicles, suggesting a defect in vesicle fusion during peroxisome assembly (Song et al. (1993)
J. Cell Biol
. 123:535-548 and Heyman et al. (1994)
J. Cell Biol
. 127:1269-1273).
AAA family members are also known to regulate transcription. Nelbock et al. described the TBP1 protein that binds human HIV TAT transactivator, thus impairing its activity in cotransfection experiments (Nelbock et al. (1990)
Science
248: 1650-1653). TBP1 has since been identified as an AAA family member which acts as a transcriptional activator for various promoters (Ohana et al. (1993)
Proc. Natl. Acad. Scie
. 90:138-142).
Various ATP-dependent protease, such as the regulatory components Lon and Clp, are also members of the AAA ATPase family. Evidence suggests the Lon and Clp proteases are involved in DNA replication, recombination and restriction. For instance, human Lon binds specifically to single-stranded DNA in a region of the mitochondrial genome involved in regulation of DNA replication and transcription. It has been suggested that Lon may target and remodel specific DNA binding proteins either for selective degredation or for assembly (Fu et al. (1998)
Biochemistry
37:1905-1909).
Dubiel et al. discovered that subunit 4 of the human proteasome was in fact a member of the AAA family (Dubiel et al. (1992)
J. Biol. Chem
. 267:22699-22702). Subsequently, at least 5 of the 26S-proteasome subunits already described as transcription factors or cell cycle proteins have now been identified as representatives of the AAA family. Therefore, members of the family are likely to play an essential role in ATP-dependent and ubiquitin-dependent degradation of abnormal proteins and short-lived regulatory proteins and in antigen processing.
Macromolecular machines (protein complexes) carry out nearly every major process in a cell with highly coordinated moving parts driven by energy dependent conformational changes. Examples of such structures include the proteasomes, spliceosomes, ribosomes, peroxisomes and chromosomal replicases. The intricacy of these machines require additional devices to assist in their assembly. The AAA family of ATPase is thought of as a class of molecular chaperones that assist in the noncovalent assembly of other proteins or protein complexes. Thus, the AAA family members play critical regulatory roles in the assembly or regulation of various molecular machines associated with diverse cellular activities. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize novel ATPases. The present invention advances the state of the art by providing a novel human ATPase-like nucleic acid and polypeptide.
SUMMARY OF THE INVENTION
Isolated nucleic acid molecules corresponding to ATPase-like nucleic acid sequences are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO:2. Further provided are ATPase-like polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein.
The present invention also provides vectors and host cells for recombinant expression of the nucleic acid molecules described herein, as well as methods of making such vectors and host cells and for using them for production of the polypeptides or peptides of the invention by recombinant techniques.
The ATPase molecules of the present invention are useful for modulating agents in a variety of cellular processes including organelle biogenesis, cell-cycle regulation, vesicle-mediated transport, assembly of proteins through membranes, peroxisome biogenesis, protein sorting, gene expression, and 26S proteasome function. The molecules are also useful for the diagnosis and treatment of a variety of clinical conditions.
Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding ATPase-like proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of ATPase-like-encoding nucleic acids.
Another aspect of this invention features isolated or recombinant ATPase-like proteins and polypeptides. Preferred ATPase-like proteins and polypeptides possess at least one biological activity possessed by naturally occurring ATPase proteins.
Variant nucleic acid molecules and polypeptides substantially homologous to the nucleotide and amino acid sequences set forth in the sequence listings are encompassed by the present invention. Additionally, fragments and substantially homologous fragments of the nucleotide and amino acid se

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