p53 regulatory protein called RB18A and uses thereof

Details p53 regulatory protein called RB18A and uses thereof p53 regulatory protein called RB18A and uses thereof

2000-08-14

2004-11-16

Huff, Sheela (Department: 1642)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S300000, C530S324000, C514S012200

Reexamination Certificate

active

06818744

ABSTRACT:

The present invention relates to a new protein called RB18A for “Recognized By PAb1801 moAntibody”, which is a p53 regulatory protein, to the nucleotide sequence encoding said protein, and to the diagnostic and therapeutic applications thereof.
The p53 protein plays an important and complex role in cells. In normal cells, wild-type p53 is involved in genome stability (Lane, 1992) and in reparation of DNA lesions (Kastan et al., 1991; Kastan et al., 1992; Fritsche et al., 1993). In tumor cells, overexpression of wild-type p53 induces, depending on cell type. a G1 cell-cycle growth arrest (Ginsberg et al., 1991; Mercer et al., 1991) or apoptosis in vitro (Yonish-Rouache et al., 1991; Johnson et al., 1993) and in vivo (Shaw et al., 1992; Radinsky et al., 1994). These functions of p53 are related to its property to transactivate (Kastan et al., 1992; Scharer and Iggo, 1992; El-Deiry et al., 1993) or to repress transcription of different genes (Ginsberg et al., 1991; Santhanam et al., 1991; Subler et al., 1992) and to inhibit cellular DNA replication (Miller et al., 1995; Cox et al., 1995).
The regulatory functions of p53 are associated to specific domains (Soussi et al., 1990). Its N-terminal domain acts as a trans-activation domain (Fields and Jang, 1990; O'Rourke et al., 1990; Raycroft et al., 1990). Its central domain contains a sequence-specific DNA binding site (Wang et al., 1993; Pavietich et al., 1993; Bargonetti et al., 1993), which interacts with two copies of the 10 bp sequence 5′-PuPuPuC(A/T)(T/A)GPyPyPy-3′, separated by up to 13 bp (El-Deiry et al., 1992; Funk et al., 1992). This DNA element is present in promotor of several genes including WAF1 (El-Deiry et al., 1993), GADD45 (Kastan et al., 1992), the muscle creatine kinase gene (Weintraub et al., 1991), and the MDM2 gene (Barak et al., 1993). Other domains modulate the specific interaction of p53 with DNA. Indeed, a tetramerisation domain localized within the C-terminal stabilizes this interaction (El-Deiry et al., 1992; Funk et al., 1992; Arrowsmith and Morin, 1996). In addition, the p53 C-terminal domain contains a negative autoregulation site of sequence-specific DNA binding (Hupp et al., 1992). The C-terminal domain also carries a nonspecific DNA binding activity (Wang et al., 1993). This property has been associated to a DNA annealing and strand-transfer activities of p53 (Reed et al., 1995).
Thus, proteins which interact with one of these p53 domains should regulate p53 functions. Different proteins have been described as interacting with p53: viral SV40 large T antigen (Lane and Crawford, 1979), adenovirus ElB 55K (Sarnow et al., 1982), EBV BZLFI (Zhang et al., 1994), cellular Hsp70 (Pinhasi-Kimhi, 1986), MDM2 (Mommand et al., 1992), TBP (Seto et al., 1992), WT1 (Maheswaran et al., 1993), the Epstein-Barr virus/C3d receptor (CR2, CD21) (Barel et al., 1989) and more recently PACT (Simons et al., 1997). WO 95/14777 also discloses p53-binding polypeptides, called WBPI and p53UBC. Furthermore, monoclonal antibodies (moAb) directed against specific p53 domains are also p53 binding proteins which allow to analyze the role of p53 domains and to distinguish between wild-type or mutant p53 forms. PAb1620 moAb specifically recognized a conformational epitope only expressed on wild-type p53 (Milner et al., 1987), while PAb240 moAb recognized a sequencial epitope localized in the central region of p53 and demasked only on mutated p53 (Gannon et al., 1990). Other anti-p53 moAb as PAb1801 (binding to aminoacids 46 to 55 of p53) and DO1 (binding to aminoacids 21 to 25 of p53) or PAb421 (binding to aminoacids 371 to 380 of p53) were found to be directed against either the N or the C terminal domains of the p53 protein, respectively (Legros et al., 1994). PAb421 moAb activates the sequence-specific DNA binding activity of p53, in a similar manner to phosphorylation or binding of bacterial Hsp70(dnak) on the p53 C-terminal domain (Hupp et al., 1992).
The authors of the present invention have now identified a new cellular protein, with an apparent molecular weight of 205 kDa. This protein was called RB18A for “Recognized By PAb1801 moAb”, as isolated after immunological screening of a cDNA expression library using the specific anti-p53 moAb (monoclonal antibody), PAb1801. Although no significant homology has been found with p53 at the level of nucleotide or deduced-protein sequence, RB18A protein shared some antigenic epitopes with p53 as recognized by different anti-p53 moAbs. Furthermore, RB18A protein shares some identical functional properties with the p53 protein, i.e. DNA-binding, homo-oligomerization, binding to p53 and activation of the sequence specific DNA binding function of p53.
In the instant application,
SEQ ID n
o
1 represents the cDNA sequence encoding the human RB18A protein.
SEQ ID n
o
2 represents the aminoacid sequence of the human RB18A protein.
The fragments of the human RB18A protein of interest are the following:
the aminoacid fragment from aminoacid 436 to aminoacid 1566, encoded by the nucleotide fragment from nucleotide 1541 to nucleotide 4933;
the aminoacid fragment from aminoacid 436 to aminoacid 1228, encoded by the nucleotide fragment from nucleotide 1541 to nucleotide 3919;
the aminoacid fragment from aminoacid 436 to aminoacid 927, encoded by the nucleotide fragment from nucleotide 1541 to nucleotide 3014;
the aminoacid fragment from aminoacid 1537 to aminoacid 1566, encoded by the nucleotide fragment from nucleotide 4846 to nucleotide 4933;
the aminoacid fragment from aminoacid 1234 to aminoacid 1566, encoded by the nucleotide fragment from nucleotide 3935 to nucleotide 4933;
the aminoacid fragment from aminoacid 1234 to aminoacid 1406, encoded by the nucleotide fragment from nucleotide 3935 to nucleotide 4453, which represents the p53 binding and homo-oligomerization domains;
the aminoacid fragment from aminoacid 927 to aminoacid 1406, encoded by the nucleotide fragment from nucleotide 3014 to nucleotide 4453, which represents the DNA binding domain.
A subject of the present invention is thus an isolated nucleic acid comprising a sequence selected from the group consisting of a fragment from nucleotide 1541 to nucleotide 4933 of SEQ ID n
o
1, a fragment from nucleotide 1541 to nucleotide 3919 of SEQ ID n
o
1, a fragment from nucdeotide 1541 to nucleotide 3014 of SEQ ID n
o
1, a fragment from nucleotide 4846 to nucleotide 4933 of SEQ ID n
o
1, a fragment from nucleotide 3935 to nucleotide 4933 of SEQ ID n
o
1, a fragment from nucleotide 3935 to nucleotide 4453 of SEQ ID n
o
1, a fragment from nucleotide 3014 to nucleotide 4453 of SEQ ID n
o
1, and a homologous nucleic acid sequence thereof.
More particularly the present invention relates to an isolated nucleic acid having a sequence selected from SEQ ID n
o
1, and a homologous nucleic acid sequence thereof.
“A homologous nucleotide sequence” is understood as meaning a sequence which differs from the sequences to which it refers by mutation, insertion, deletion or substitution of one or more bases.
Preferably, such homologous sequences show at least 70% of homology, preferably 80% of homology, more preferably 90% of homology with SEQ ID n
o
1 or fragments thereof, as above defined.
A polynucleotide of the invention, having a homologous sequence, hybridizes to the sequences to which it refers, preferably under stringent conditions. Parameters that define the conditions of stringency depend upon the temperature at which 50% of annealed strands separate (T
m
).
For sequences comprising more than 30 nucleotides, T
m
is calculated as follows:
T
m
=81.5+0.41(%
G+C
)+16.6 Log (positive ion concentration)−0.63(% formamide)−(600/polynucleotide size in base pairs)(Sambrook et al, 1989).
For sequences comprising less than 30 nucleotides, T
m
is calculated as follows:
T
m
=4(
G+C
)+2(
A+T
).
Under appropriate stringent conditions avoiding the hybridization of non specific sequences, hybridization temperature is around from. 5° C. to 30° C., preferably from 5° C. to 10° C. below th

Affiliated with

Also associated with

Rating

p53 regulatory protein called RB18A and uses thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with p53 regulatory protein called RB18A and uses thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and p53 regulatory protein called RB18A and uses thereof will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3363578