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WO2000023589A2 - Proteines associees a la proliferation et a l'apoptose - Google Patents

Proteines associees a la proliferation et a l'apoptose Download PDF

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Publication number
WO2000023589A2
WO2000023589A2 PCT/US1999/024511 US9924511W WO0023589A2 WO 2000023589 A2 WO2000023589 A2 WO 2000023589A2 US 9924511 W US9924511 W US 9924511W WO 0023589 A2 WO0023589 A2 WO 0023589A2
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WIPO (PCT)
Prior art keywords
seq
proap
polynucleotide
sequence
sequences
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PCT/US1999/024511
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English (en)
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WO2000023589A3 (fr
Inventor
Y. Tom Tang
Henry Yue
Jennifer L. Hillman
Karl J. Guegler
Neil C. Corley
Preeti Lal
Yalda Azimzai
Mariah R. Baughn
Junming Yang
Leo L. Shih
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Incyte Pharmaceuticals, Inc.
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Priority to CA002347836A priority Critical patent/CA2347836A1/fr
Priority to JP2000577298A priority patent/JP2003524382A/ja
Priority to EP99955070A priority patent/EP1123394A2/fr
Priority to AU11260/00A priority patent/AU1126000A/en
Publication of WO2000023589A2 publication Critical patent/WO2000023589A2/fr
Publication of WO2000023589A3 publication Critical patent/WO2000023589A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins

Definitions

  • This invention relates to nucleic acid and amino acid sequences of proliferation and apoptosis related proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, immunological, and reproductive disorders.
  • Tissue growth involves complex and ordered patterns of cell proliferation, cell differentiation, and regulated cell death (apoptosis).
  • Cell proliferation and apoptosis are regulated to maintain both the number and the spatial organization of cells. This regulation depends on appropriate expression of proteins which control cell cycle progression in response to extracellular signals, such as growth factors and other mitogens, and intracellular cues, such as DNA damage or nutrient starvation.
  • Molecules which directly or indirectly modulate cell cycle progression fall into several categories, including growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, tumor-suppressor proteins, and mitosis-promoting factors. Cancers are characterized by continuous or uncontrolled cell proliferation. Some cancers are associated with suppression of nonnal apoptotic cell death. Growth Factors and Signal Transduction Machinery
  • Growth factors are typically large, secreted polypeptides that act on cells in their local environment to promote cell proliferation. Growth factors bind to and activate specific cell surface receptors that initiate intracellular signal transduction cascades. Many growth factor receptors are classified as receptor tyrosine kinases that undergo autophosphorylation upon ligand binding. Autophosphorylation enables the receptor to interact with signal transduction proteins such as SH2 or SH3 (Src homology regions 2 or 3) domain-containing proteins. Other proteins that act downstream of growth factor receptors contain unique signaling domains such as the SPRY (Spla and ryanodine receptor) domain. (See, for example, Schultz, J. et al. (1998) Proc. Natl. Acad. Sci.
  • G-proteins such as Ras, Rab, and Rho
  • GAPs GTPase activating proteins
  • GNRPs guanine nucleotide releasing proteins
  • Small G proteins act as molecular switches that turn on mitogen-activated protein kinase (MAP kinase) cascades.
  • MAP kinase activates transcription of the early-response genes discussed below.
  • EGF epidermal growth factor
  • Ras/Raf/MAP kinase pathway a transmembrane protein tyrosine kinase pathway
  • Other pathways potentially activated by EGF include the phosphatidylinositol pathway and the JAK/STAT signaling pathway (Tarnawski, A.S. et al. (1998) J. Clin. Gastroenterol. 27:S 12-S20).
  • GPCR G-protein coupled receptor
  • PKC Protein kinase C plays a central role in the control of proliferation and differentiation of various cell types by mediating the signal transduction response to hormones and growth factors.
  • the PKC family of serine/threonine kinases includes twelve different isoforms which have similar catalytic domains at their C-termini, but differ in their N-terminal regulatory domains. Since most cells express multiple PKC isoforms, the specificity of each enzyme for its substrate is achieved by targeting individual isoenzymes to a select location in the cell, either constitutively or upon cell stimulation.
  • PKC -binding proteins and lipids have been identified that may function to compartmentalize PKC isoenzymes, including RACKl , serum deprivation response (sdr) protein, and SRBC (sdr-related gene product that binds C-kinase).
  • RACKl serum deprivation response
  • SRBC sdr-related gene product that binds C-kinase.
  • both sdr and SRBC appear to provide localization of activated PKC to caveolae, but each has specificity for a different isoenzyme; sdr interacts specifically with PKC ⁇ and SRBC interacts with PKC ⁇ .
  • Both sdr and SRBC are induced during stages of growth arrest, and were originally isolated from serum-deprived cultured cells.
  • Oncogenes i.e. "cancer-causing genes” are involved in the reception and transduction of growth factor signals and in the modulation of gene expression in response to these signals. For example, stimulation of a cell by growth factor activates two sets of genes, the early-response genes and the delayed-response genes.
  • Early-response gene products include myc, fos, and jun, all of which encode gene regulatory proteins These regulatory proteins activate the transcription of the delayed-response genes which encode proteins directly involved in cell cycle progression, such as the cyclms and cychn dependent kinase discussed below Additional oncogene products which directly regulate gene expression include the Rel transcription factor, the Ret zinc finger protein, and the Tre oncoprotem (See, for example, Cao, T et al (1998) J Cell Sci 1 1 1 1319-1329, and Nakamura, T et al (1992) Oncogene 7 733-741 ) Some conserved regions of oncogenes have been identified, such as the C3HC4 RING finger motif Mutations m the C3HC4 RING finger domain of the Bm ⁇ -1 oncoprotem, for example, block lymphoma induction in mice (Hemenway, C S (1998) Oncogene 16 2541-2547) Apoptosis inhibition motifs have also been identified, such as the B
  • Tumor supressor genes often act as "gatekeepers" (Kmzler K W and Vogelstem, B ( 1996) Cell 87 159-170) Normally, the gatekeeper is responsible tor maintaining a balance ot cell division growth arrest, and death External signals may activate or inactivate the gatekeeper, or alter its location withm the cell In some cases, inactivation of the gatekeeper is necessary for cell proliferation, and activation is necessary for cell growth arrest and differentiation In other cases, the situation is reversed Proteins which interact with the gatekeeper modify its activity or intracellular location to provide the approp ⁇ ate response to external signals at any stage in the cell's development An example of a gatekeeper protein is the adenomatous polyposis co ( APC) protein Though APC is expressed ubiquitously, it appears to function as a gatekeeper in colorectal cells
  • the molecular machinery which drives the cell cycle in response to mitogens and growth factors has been extensively studied in model systems such as budding yeast, fission yeast, and the African clawed frog, Xenopus.
  • the cell cycle is comprised of four successive phases: Gl, S (DNA synthesis), G2, and M (mitosis).
  • Gl DNA synthesis
  • G2 mitochondrial
  • M mitochondrial
  • Cells which exit the cell cycle enter a quiescent phase called GO.
  • Studies in yeast have shown that exit from S and M phases is driven by the anaphase- promoting complex, an assembly of proteins that degrades cyclins via the ubiquitin-mediated protein degradation pathway. (See, for example, Kominami, K. et al. ( 1998) EMBO J.
  • dmplp novel, 142 amino acid protein from the yeast S. pombe, termed dmplp, that is required for proper spindle formation and entry into mitosis, but does not interact with cyclin-type proteins (Berry L.D. and Gould K.L. (1997) J. Cell Biol. 137: 1337-1354). Dimlp appears to be evolutionarily conserved, since a human homolog has recently been desc ⁇ bed (La ⁇ n D , et al (1997) Gl 2565275) Apoptosis Machinery
  • Apoptosis is the genetically controlled process by which unneeded or defective cells undergo programmed cell death Selective elimination of cells is as important for morphogenesis and tissue remodeling as is cell proliferation and differentiation Lack of apoptosis may result m hype ⁇ lasia and other disorders associated with increased cell proliferation Apoptosis is also a cntical component of the immune response Immune cells such as cytotoxic T-cells and natural killer cells prevent the spread of disease by inducing apoptosis in tumor cells and virus-mfected cells In addition, immune cells that fail to distinguish self molecules from foreign molecules must be eliminated by apoptosis to avoid an autoimmune response
  • apoptotic cells undergo distinct mo ⁇ hological changes Hallmarks of apoptosis include cell shnnkage, nuclear and cytoplasmic condensation, and alterations in plasma membrane topology Biochemically, apoptotic cells are characterized by increased intracellular calcium concentration, fragmentation of chromosomal DNA, and expression of novel cell surface components
  • the molecular mechanisms of apoptosis are highly conserved, and many of the key protein regulators and effectors of apoptosis have been identified
  • Apoptosis generally proceeds in response to a signal which is transduced intracellularly and results in altered patterns of gene expression and protein activity
  • Signaling molecules such as hormones and cytokines are known both to stimulate and to inhibit apoptosis through interactions with cell surface receptors Transcnption factors also play an important role in the onset of apoptosis
  • a number of downstream effector molecules, particularly proteases such as the cysteme proteases called caspases have been implicated in the degradation of cellular
  • DFF DNA fragmentation factor
  • DFF40/CAD a 40-kDa, caspase-activated nuclease termed DFF40/CAD
  • DFF45/ICAD Two mouse homologs of DFF45/ICAD, termed CIDE-A and CIDE-B, have recently been described (Inohara, N et al (1998) EMBO J. 17:2526-2533).
  • CIDE-A and CIDE-B expression in mammalian cells activated apoptosis, while expression of CIDE-A alone induced DNA fragmentation.
  • FAS-mediated apoptosis was enhanced by CIDE-A and CIDE-B, further implicating these proteins as effectors that mediate apoptosis.
  • Cancers are characterized by inappropriate cell proliferation, which may be due to uncontrolled cell growth or inadequate apoptosis.
  • Strategies for treatment may involve either reestablishing control over cell cycle progression, or selectively stimulating apoptosis in cancerous cells (Nigg, E.A. (1995) BioEssays 17:471-480).
  • the invention features substantially purified polypeptides, proliferation and apoptosis related proteins, referred to collectively as “PROAP” and individually as “PROAP- 1 ,” “PROAP-2,” “PROAP-3,” “PROAP-4,” “PROAP-5,” “PROAP-6” “PROAP-7,” “PROAP-8,” “PROAP-9,” “PROAP-10,” “PROAP-1 1,” “PROAP-12,” “PROAP-13,” “PROAP-14,” “PROAP-15,” “PROAP- 16,” “PROAP-17,” “PROAP-18,” and “PROAP-19.”
  • the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -19 and fragments thereof.
  • the invention also includes a polypeptide comprising an amino acid sequence that differs by one or more conservative amino acid substitutions from an amino acid sequence selected from the group consisting of SEQ ID NO: 1-19.
  • the invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO 1-19 and fragments thereof
  • the invention also provides an isolated and pu ⁇ fied polynucleotide encoding the polypeptide comp ⁇ sing an amino acid sequence selected from the group consisting of SEQ ID NO 1-19 and fragments thereof
  • the invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comp ⁇ sing an ammo acid sequence selected from the group consisting of SEQ ID NO 1-19 and fragments thereof
  • the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 1-19 and fragments thereof
  • the invention also provides an isolated and pu ⁇ fied polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the ammo acid sequence selected from the group consisting of SEQ ID NO 1-19 and fragments thereof
  • the invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comp ⁇ sing the steps of (a) hyb ⁇ dizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hyb ⁇ dization complex, and (b) detecting the hybridization complex, wherein the presence of the hyb ⁇ dization complex correlates with the presence of a polynucleotide in the sample
  • the method further comprises amplifying the polynucleotide prior to hyb ⁇ dization
  • the invention also provides an isolated and purified polynucleotide comp ⁇ sing a polynucleotide sequence selected from the group consisting of SEQ ID NO 20-38 and fragments thereof
  • the invention further provides an isolated and pu ⁇ fied polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID NO 20-38 and fragments thereof
  • the invention also provides an isolated and pu ⁇ fied polynucleotide having a sequence which is complementary to the polynucleotide comp ⁇ smg a polynucleotide sequence selected from the group consisting of SEQ ID NO 20-38 and fragments thereof
  • the invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an ammo acid sequence selected from the group consisting of SEQ ID NO 1-19
  • the expression vector is contained withm a host cell
  • the invention also provides a method for producing a polypeptide, the method comp ⁇ sing the steps of (a) cultu ⁇ ng the host cell containing an expression vector containing a polynucleotide of the invention under conditions suitable for the expression of the polypeptide, and (b) recove ⁇ ng the polypeptide from the host cell culture
  • the invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1-19 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein).
  • An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • antisense refers to any composition containing a nucleic acid sequence which is complementary to the "sense" strand of a specific nucleic acid sequence. Antisense molecules may be produced by any method including synthesis or transcription.
  • a fragment of SEQ ID NO:20-38 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:20-38, for example, as distinct from any other sequence in the same genome.
  • a fragment of SEQ ID NO:20-38 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:20-38 from related polynucleotide sequences.
  • similarity refers to a degree of complementarity. There may be partial similarity or complete similarity. The word “identity” may substitute for the word “similarity.”
  • a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency.
  • a substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency.
  • BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters. Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
  • Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the hydrophobicity and acidity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • Human artificial chromosomes are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance.
  • humanized antibody refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml denatured salmon sperm DNA.
  • stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • wash temperatures are selected to be about 5°C to 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, denatured salmon sperm DNA at about 100-200 ⁇ g/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or I ⁇ t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • a solid support e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed.
  • insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • microarray refers to an arrangement of distinct polynucleotides on a substrate.
  • element and “array element” in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
  • modulate refers to a change in the activity of PROAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of PROAP.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
  • the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • sample is used in its broadest sense.
  • a sample suspected of containing nucleic acids encoding PROAP, or fragments thereof, or PROAP itself may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an "allelic” (as defined above), “splice,” “species,” or “polymo ⁇ hic” variant.
  • Polymo ⁇ hic variants also may encompass "single nucleotide polymo ⁇ hisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base.
  • SNPs single nucleotide polymo ⁇ hisms
  • the presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2 0 9 (May-07- 1999) set at default parameters
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides
  • the invention is based on the discovery of new human proliferation and apoptosis related proteins (PROAP), the polynucleotides encoding PROAP, and the use of these compositions for the diagnosis, treatment, or prevention of cell prohferative, immunological, and reproductive disorders
  • Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding PROAP
  • Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively
  • Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each PROAP were identified, and column 4 shows the cDNA libraries from which these clones were isolated
  • Column 5 shows Incyte clones and their corresponding cDNA libraries Clones for which cDNA hbranes are not indicated were de ⁇ ved from pooled cDNA hbranes
  • the Incyte clones m column 5 weie used to
  • Table 2 show various properties of each of the polypeptides of the invention
  • column 1 references the SEQ ID NO
  • column 2 shows the number of amino acid residues in each polypeptide
  • column 3 shows potential phosphorylation sites
  • column 4 shows potential glycosylation sites
  • column 5 shows the ammo acid residues comprising signature sequences and motifs
  • column 6 shows homologous sequences as identified by BLAST analysis
  • column 7 shows analytical methods used to identify each polypeptide through sequence homology and protein motifs
  • PROAP- 1 has chemical and structural simila ⁇ ty with mouse npdcf-1 (Gl 452276, SEQ ID NO 39) In particular, PROAP- 1 and npdcf- 1 share 66% identity and have similar isoelectnc points (7 5 and 7 2, respectively)
  • PROAP-2 has chemical and structural similarity with human EB1 (Gl 998357, SEQ ID NO 40) In particular, PROAP-2 and EB 1 share 64% identity and have similar isoelectnc points (5 3 and 4 9, respectively)
  • PROAP-3 has chemical and structural similanty with mouse serum deprivation response (sdr) protein (Gl 455719, SEQ ID NO 41) In particular, PROAP-3 is 86% identitical to sdr from residue Ml through V239 on sdr.
  • PROAP- 13 has chemical and structural similarity with human dimlp homolog (Gl 2565275; SEQ ID NO:42). In particular, PROAP-13 and Dimlp share 36% identity.
  • PROAP-14 has chemical and structural similarity with Fly FAS-associated factor (FFAF) from P. melanogaster (Gl 3688609; SEQ ID NO:43). In particular, PROAP- 14 and FFAF share 40% identity.
  • PROAP- 15 has chemical and structural similarity with cell death activator CIDE-B from M. musculus (Gl 3114594; SEQ ID NO:44). In particular, PROAP-15 and CIDE-B share 83% identity.
  • FIG. 4 lists diseases, disorders, or conditions associated with those tissues expressing PROAP as a fraction of total tissues expressing PROAP.
  • Column 5 lists the vectors used to subclone each cDNA library.
  • SEQ ID NO: 20 in reproductive, nervous, and cardiovascular tissues, of SEQ ID NO:21 in nervous tissue, of SEQ ID NO:22 in reproductive and gastrointestinal tissues, of SEQ ID NO:28, which is detected exclusively in a cDNA library derived from tibia meniscus tissue, of SEQ ID NO: 30, which is detected exclusively in a cDNA library derived from diseased liver, of SEQ ID NO:32 in brain tumor-associated tissues, of SEQ ID NO:33 in tumors of the breast and brain, and of SEQ ID NO: 34 in tumors of the breast and testicle.
  • the invention also encompasses polynucleotides which encode PROAP.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:20-38, which encodes PROAP.
  • nucleotide sequences which encode PROAP and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring PROAP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding PROAP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non- naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of DNA sequences which encode PROAP and PROAP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding PROAP or any fragment thereof.
  • Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Perkin- Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
  • sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Perkin-Elmer). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Perkin-Elmer), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short
  • the nucleic acid sequences encoding PROAP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.)
  • Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al.
  • primers may be designed using commercially available software, such as OLIGO 4 06 Pnmer Analysis software (National Biosciences, Plymouth MN) or another appropnate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C When screening for full-length cDNAs.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths
  • Output/light intensity may be converted to electrical signal using approp ⁇ ate software (e g , GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled Capillary electrophoresis
  • PROAP or fragments or functional equivalents thereof, in approp ⁇ ate host cells Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent ammo acid sequence may be produced and used to express PROAP
  • nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter PROAP-encodmg sequences for a variety of pu ⁇ oses including, but not limited to, modification of the cloning, processing, and/or expression of the gene product DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences
  • ohgonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth
  • the peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier ( 1990) Methods Enzymol. 182:392-421.)
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY.)
  • the nucleotide sequences encoding PROAP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding PROAP. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding PROAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding PROAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • the invention is not limited by the host cell employed.
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding PROAP.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding PROAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla C A) or PSPORT 1 plasmid (Life Technologies). Ligation of sequences encoding PROAP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
  • vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of PROAP may be used.
  • vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of PROAP.
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of PROAP. Transcription of sequences encoding PROAP may be driven viral promoters, e.g., the 35 S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-31 1). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
  • pathogen-mediated transfection See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding PROAP may be ligated into an adenovirus transc ⁇ ption translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses PROAP in host cells.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells SV40 or EBV- based vectors may also be used for high-level protein expression.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • plasmid HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycatiomc amino polymers, or vesicles) for therapeutic pu ⁇ oses (See, e.g., Harnngton, J J. et al ( 1997) Nat. Genet 15:345-355.)
  • sequences encoding PROAP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the pu ⁇ ose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques approp ⁇ ate to the cell type
  • Any number of selection systems may be used to recover transformed cell lines These include, but are not limited to, the he ⁇ es simplex virus thymidine kinase and adenme phosphonbosyltransferase genes, for use in tk and apr cells, respectively (See, e g., Wigler, M. et al. (1977) Cell 1 1:223-232; Lowy, I et al. (1980) Cell 22.817-823 ) Also, antimetabohte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the am oglycosides neomycin and G-418
  • als an ⁇ pat confer resistance to chlorsulfuron and phosphinotncin acetyltransferase, respectively.
  • host cells that contain the nucleic acid sequence encoding PROAP and that express PROAP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of PROAP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on PROAP is preferred, but a competitive binding assay may be employed.
  • assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunology. Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. ( 1998) Immunochemical Protocols, Humana Press, Totowa NJ.)
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding PROAP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding PROAP, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 an appropriate RNA polymerase
  • Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding PROAP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode PROAP may be designed to contain signal sequences which direct secretion of PROAP through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or “pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • nucleic acid sequences encoding PROAP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric PROAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of PROAP activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the PROAP encoding sequence and the heterologous protein sequence, so that PROAP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
  • synthesis of radiolabeled PROAP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
  • PROAP Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of PROAP and proliferation and apoptosis related proteins.
  • the expression of PROAP is closely associated with cancer, inflammation, and proliferating, reproductive, and developmental tissues. Therefore, PROAP appears to play a role in cell proliferative, immunological, and reproductive disorders.
  • PROAP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PROAP.
  • disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, ci ⁇ hosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung
  • a vector capable of expressing PROAP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PROAP including, but not limited to, those described above.
  • a pharmaceutical composition comprising a substantially purified PROAP in conjunction with a suitable pharmaceutical earner may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PROAP including, but not limited to, those provided above.
  • an agonist which modulates the activity of PROAP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PROAP including, but not limited to, those listed above.
  • Dragee cores may be used m conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arable, talc, polyvmylpy ⁇ ohdone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characte ⁇ ze the quantity of active compound, i e , dosage
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arable, talc, polyvmylpy ⁇ ohdone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characte ⁇ ze the quantity of active compound, i e , dosage
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pu ⁇ ose The determination of an effective dose is well withm the capability of those skilled in the art
  • Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
  • the cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above.
  • Hybridization probes derived from SEQ ID NO:20-38 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • An array analogous to a dot or slot blot may also be used to a ⁇ ange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical a ⁇ ay may be produced by hand or using available methods and machines and contain any appropriate number of elements.
  • nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microa ⁇ ay may be assessed through analysis of the scanned images.
  • Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microa ⁇ ay. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are a ⁇ anged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al.
  • PROAP expression and purification of PROAP is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express PROAP upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
  • An assay for PROAP activity measures cell proliferation as the amount of newly initiated DNA synthesis in Swiss mouse 3T3 cells.
  • a plasmid containing polynucleotides encoding PROAP is transfected into quiescent 3T3 cultured cells using methods well known in the art. The transiently transfected cells are then incubated in the presence of [ 3 H]thymidine, a radioactive DNA precursor. . Where applicable, varying amounts of PROAP ligand are added to the transfected cells. Inco ⁇ oration of [ H]thymidine into acid-precipitable DNA is measured over an appropriate time interval, and the amount inco ⁇ orated is directly proportional to the amount of newly synthesized DNA.
  • An alternative assay for PROAP activity measures the induction of apoptosis when PROAP is expressed at physiologically elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include pCMV SPORT (Life Technologies, Gaithersburg, MD) and pCR 3.1 (Invitrogen, Carlsbad, CA, both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP) (Clontech, Palo Alto, CA), CD64, or a CD64-GFP fusion protein.
  • Flow cytometry an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate their apoptotic state.
  • PROAP activity may be measured by the induction of growth a ⁇ est when PROAP is expressed at physiologically elevated levels in transformed mammalian cell lines.
  • PROAP cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression, and these constructs are stably transfected into a transformed cell line, such as NIH 3T6 or C6, using methods known in the art.
  • An additional plasmid, containing sequences which encode a selectable marker, such as hygromycin resistance, are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
  • Cells expressing PROAP are compared with control cells, either non-transfected or transfected with vector alone, for characteristics associated with growth a ⁇ est. Such characteristics can include, but are not limited to, a reduction in [ 3 H]-thymidine inco ⁇ oration into newly synthesized DNA, lower doubling and generation times, and decreased culture saturation density.
  • an assay for PROAP activity uses radiolabeled nucleotides, such as
  • [ ⁇ 32 P]ATP to measure either the inco ⁇ oration of radiolabel into DNA during DNA synthesis, or fragmentation of DNA that accompanies apoptosis.
  • Mammalian cells are transfected with plasmid containing cDNA encoding PROAP by methods well known in the art. Cells are then incubated with radiolabeled nucleotide for various lengths of time. Chromosomal DNA is collected, and radioactivity detected using a scintillation counter. Inco ⁇ oration of radiolabel into chromosomal DNA is proportional to the degree of stimulation of the cell cycle.
  • chromosomal DNA is collected as above, and analyzed using polyacrylamide gel electrophoresis, by methods well known in the art. Fragmentation of DNA is quantified by comparison to untransfected control cells, and is proportional to the apoptotic activity of PROAP.
  • PROAP function is assessed by expressing the sequences encoding PROAP at physiologically elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation.
  • a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
  • PROAP The influence of PROAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding PROAP and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding PROAP and other genes of interest can be analyzed by northern analysis or microa ⁇ ay techniques. XII. Production of PROAP Specific Antibodies
  • PAGE polyacrylamide gel electrophoresis
  • the PROAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a co ⁇ esponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
  • ohgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
  • ABI 431 A peptide synthesizer Perkin-Elmer
  • KLH Sigma- Aldrich, St. Louis MO
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oligopeptide- KLH complex in complete Freund's adjuvant.
  • Resulting antisera are tested for antipeptide and anti- PROAP activity by, for example, binding the peptide or PROAP to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG. XIII.
  • Purification of Naturally Occurring PROAP Using Specific Antibodies Naturally occurring or recombinant PROAP is substantially purified by immunoaffinity chromatography using antibodies specific for PROAP.
  • An immunoaffinity column is constructed by covalently coupling anti-PROAP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • PROAP Media containing PROAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PROAP (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/PROAP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and PROAP is collected.
  • a buffer of pH 2 to pH 3 or a high concentration of a chaotrope, such as urea or thiocyanate ion
  • AB1/PARACEL FDF A Fast Data Finder useful in comparing and annotating Perkin-Elmer Applied Biosystems, Mismatch ⁇ 50% amino acid or nucleic acid sequences. Foster City, CA; Paracel Inc., Pasadena, CA.
  • ABI AutoAssembler A program that assembles nucleic acid sequences. Perkin-Elmer Applied Biosystems, Foster City, CA.

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Abstract

L'invention concerne des protéines associées à la prolifération humaine et à l'apoptose (PROAP) et des polynucléotides qui identifient et codent PROAP. L'invention concerne également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. L'invention concerne en outre des méthodes pour diagnostiquer, traiter ou prévenir des troubles liés à l'expression de PROAP.
PCT/US1999/024511 1998-10-20 1999-10-19 Proteines associees a la proliferation et a l'apoptose WO2000023589A2 (fr)

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WO2002033071A1 (fr) * 2000-10-17 2002-04-25 Takeda Chemical Industries, Ltd. Polypeptides analogues a la survivine et leurs adn
WO2002057444A1 (fr) * 2001-01-22 2002-07-25 Shinae Kondoh Genes associes a l'apoptose humaine et proteines produites associees a l'apoptose humaine
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WO2023178241A3 (fr) * 2022-03-16 2023-12-07 The Regents Of The University Of Colorado A Body Corporate Procédés et compositions pour perturber des agrégats de tau à l'aide de séquences de répétition de polysérine ciblant des protéines exogènes

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EP1103608A1 (fr) * 1999-11-25 2001-05-30 Sanofi-Synthelabo Protéine de type CIDE , son isolation et ses applications
WO2002033071A1 (fr) * 2000-10-17 2002-04-25 Takeda Chemical Industries, Ltd. Polypeptides analogues a la survivine et leurs adn
WO2002057444A1 (fr) * 2001-01-22 2002-07-25 Shinae Kondoh Genes associes a l'apoptose humaine et proteines produites associees a l'apoptose humaine
WO2002062958A2 (fr) * 2001-02-08 2002-08-15 Tularik Inc. Prc17: gene du cancer amplifie
WO2002062958A3 (fr) * 2001-02-08 2003-04-10 Tularik Inc Prc17: gene du cancer amplifie
EP1379540A4 (fr) * 2001-04-19 2005-11-16 Bristol Myers Squibb Co Polynucleotides et polypeptides associes au chemin nf-kb
EP1365242A1 (fr) * 2002-05-21 2003-11-26 MTM Laboratories AG Composés pour le diagnostique et le traitement de lésions colorectales
WO2008099189A2 (fr) * 2007-02-16 2008-08-21 Crusade Laboratories Limited Virus de l'herpès simplex et procédés de réplication virale
WO2008099189A3 (fr) * 2007-02-16 2009-01-15 Crusade Lab Ltd Virus de l'herpès simplex et procédés de réplication virale
US8163292B2 (en) 2007-02-16 2012-04-24 Virttu Biologics Limited Herpes simplex viruses and methods of viral replication
US8969063B2 (en) 2007-02-16 2015-03-03 Virttu Biologics Limited Herpes simplex viruses and methods of viral replication
WO2023178241A3 (fr) * 2022-03-16 2023-12-07 The Regents Of The University Of Colorado A Body Corporate Procédés et compositions pour perturber des agrégats de tau à l'aide de séquences de répétition de polysérine ciblant des protéines exogènes

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