Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-10-15
2002-07-09
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S188000
Reexamination Certificate
active
06416989
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel
Candida albicans
gene encoding a polypeptide which is a member of the histidine kinase family. More specifically, isolated nucleic acid molecules are provided encoding a
Candida albicans
polypeptide named Histidine Kinase-1 (CaHK-1). CaHK-1 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to methods for testing compounds for ability to inhibit CaHK-1, an enzyme which is active in phosphorylating host cell proteins to render the host cell susceptible to invasion by
Candida albicans.
BACKGROUND OF THE INVENTION
All cells must sense changes in their environment and respond appropriately. In this regard, the two-component signal transduction regulatory system was initially described in prokaryotic organisms where it is thought to play a function in chemotaxis, osmoregulation, sporulation, host-pathogen interactions and response to carbon, nitrogen and phosphate availability. In these microorganisms, the prototypical two-component regulator system is comprised of two proteins, a histidine protein kinase (also called a sensor protein and usually cell membrane-bound) and a response regulator (or effector protein), which is associated with an internal response. The sensor kinase, when activated by a signal, autophosphorylates a histidine residue using ATP as a phosphodonor; the histidine is a part of a conserved block of residues, typically referred to as the H-box. Subsequently, the phosphorylated sensor kinase serves as a phosphodonor to a conserved aspartate residue in the response regulator. This phosphorylation modulates the activity of the effector protein to elicit an adaptive response to the stimulus (reviewed in Hoch and Silhavy,
Two
-
component signal transduction
, ASM Press. Washington, D.C. USA (1995)).
Although the general sequence of events and the number of proteins involved is similar for all of these organisms, each pathway exhibits some variation on the basic scheme (Appleby et al.,
Signal transduction via the multi
-
step phosphorelay: not necessarily a road less traveled
, Cell 86, 845-848 (1996)). For instance, in
Bordetella pertussis
, the BvgS-BvgA two-component modulates the transcriptional control of several virulence factors. Although there are two proteins, four phosphorylation events occur in sequence, creating a four-step His-Asp-His-Asp phosphorelay (Uhl and Miller;
Integration of multiple domains in a two
-
component sensor protein; the Bordetella pertussis BvgAS phosphorelay
, EMBO J. 15, 1028-1036 (1996)). A similar mechanism has been the plant pathogenic bacterium,
Pseudomonas syringae.
Homologous pathways have recently been identified in several eukaryotic organisms, including,
Saccharomyces cerevisiae, Dictyostelium discoideum, Neurospora crassa
and
Arabidopsis thaliana
. In
S. cerevisiae
the phosphorelay through a two-component signal pathway is composed of three proteins. An Sln1p transmembrane protein serves as a sensor protein, which after autophosphorylation of a histidine residue and transfer to an aspartate in the same protein, phosphorylates a histidine residue of a second protein (Ypd1p). Ypd1p is a small cytoplasmic protein, which functions much like a sensor protein and, in turn, it phosphorylates a third protein effector in the relay system (Ssk1p). The activation of a downstream MAP kinase cascade is dependent upon the phosphorylation of Ssk1p. In cells which are grown under low osmotic conditions, phosphorylated Ssk1p does not activate the Map kinase pathway. However, under conditions of hyperosmolarity, phosphotransfer among the two-component does not occur. Consequently, the MAP kinase pathway and the transcription of genes involved in glycerol metabolism occur. This pathway, referred to as the HOG pathway (High Osmolarity Glycerol Response), thus provides a phosphorylated effector molecule which is inactive in environmentally stressed conditions. In
D. discoideum
two different histidine kinases (DhkA and DokA) have been described. DhkA modulates the transcriptional regulation of prestalk gene expression and the control of the terminal differentiation pathway. DokA is involved, like Sln1p in
S. cerevisiae
, in the osmoregulatory pathway. In
N. crassa
, a two-component histidine kinase (Nik-1) has been reported to be involved in hyphal development and osmosensitivity. Finally, in
A. thaliana
the product of the ETR1 gene may be involved in an early step in ethylene signal transduction through phosphorylation, as in the prokaryotic two-component systems. Thus, there is a need for the discovery of proteins responsible for causing diseases resulting from infection with pathogenic fungil because such proteins may be used in the development of treatments for such diseases.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a portion of the CaHK-1 polypeptide having the amino acid sequence shown in
FIGS. 2A-B
(SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone deposited as plasmid DNA as ATCC Deposit Number 209504 on Nov. 26, 1997. The nucleotide sequence was determined by sequencing the deposited cloned DNA, which is shown in
FIGS. 2A-D
(SEQ ID NO:1), and contains an open reading frame encoding a complete polypeptide of 971 amino acid residues, including an initiation codon encoding an N-terminal methionine at nucleotide positions 181 to 183. Nucleic acid molecules of the invention include those encoding the complete amino acid sequence excepting the N-terminal methionine shown in
FIGS. 2A-D
(SEQ ID NO:1), or the complete amino acid sequence excepting the N-terminal methionine encoded by the cloned DNA in ATCC Deposit Number 209504, which molecules also can encode additional amino acids fused to the N-terminus of the CaHK-1 amino acid sequence.
The invention further provides isolated nucleic acid molecules comprising a polynucleotide encoding a full length CaHK-1 polypeptide having the complete amino acid sequence shown in
FIGS. 5A-J
(SEQ ID NO:4) or the complete amino acid sequence encoded by the cDNA clones deposited as plasmid DNA as ATCC Deposit Numbers 209504 and 209505 deposited Nov. 26, 1997. The nucleotide sequence was determined by sequencing the deposited cloned DNA, which is shown in
FIGS. 5A-J
(SEQ ID NO:3), and contains an open reading frame encoding a complete polypeptide of 2471 amino acid residues, including an initiation codon encoding an N-terminal methionine at nucleotide positions 1117 to 1119. Nucleic acid molecules of the invention include those encoding a complete amino acid sequence excepting the N-terminal methionine shown in
FIG. 5A-J
(SEQ ID NO:3), or the partial amino acid sequence excepting the N-terminal methionine encoded by the cloned DNA in ATCC Deposit Numbers 209504 and 209505, which molecules also can encode additional amino acids fused to the N-terminus of the CaHK-1 amino acid sequence.
The CaHK-1 proteins of the present invention share sequence homology with the translation products of the mRNA for two component histidine kinases from several prokaryotes and eukaryotes (FIG.
3
), including the following conserved domains: (a) the predicted sensor domain (residues 482 to 721 in
FIGS. 2A-D
(SEQ ID NO:2) or residues 1982 to 2221 in
FIGS. 5A-J
) (SEQ ID NO:4); and (b) the predicted response regulator domain domain (residues 834 to 971 in
FIGS. 2A-D
(SEQ ID NO:2) or residues 2334 to 2471 in
FIGS. 5A-J
) (SEQ ID NO:4). Two component histidine kinases are thought to be important in virulence. The homology between CaHK-1 and other histidine kinases (
FIG. 3
) indicates that CaHK-1 may also be involved in virulence of
Candida albicans.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a full-length CaHK-1 polypeptide having the complete amino acid sequences in
FIGS. 2A-D
(
Abad Antonio Jose C.
Calderone Richard A.
Choi Gil H.
Human Genome Sciences Inc.
Human Genome Sciences Inc.
Prouty Rebecca E.
Steadman David J.
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