Cythohesin-PH peptides that affect the ability of integrins...

Details Cythohesin-PH peptides that affect the ability of integrins... Cythohesin-PH peptides that affect the ability of integrins...

1996-09-05

2003-06-03

Gambel, Phillip (Department: 1644)

Chemistry: natural resins or derivatives; peptides or proteins;

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

C530S395000, C435S810000

Reexamination Certificate

active

06573362

ABSTRACT:

The present invention relates to the use of cytohesin-PH peptides to influence the ability of integrins to adhere.
BACKGROUND OF THE INVENTION
T-Lymphocyte activation is achieved by coordinated binding of adhesion molecule receptors and signal receptors which are then expressed on the surface of T cells when these receptors bind to their complementary receptors on the antigen-presenting cell. Besides the T-cell receptors (TCRs) and MHC (major histocompatibility class) class I or II proteins, which are always involved in leukocyte activation, various types of coreceptors also are necessary, such as the integrins, and the CD2, CD4 and CD8 molecules. The functional interaction between the TCRs and the T-lymphocyte coreceptors is dynamic in nature, that is, only the binding of a TCR to its target molecule brings about enhanced binding of the coreceptors to their complementary receptors.
The integrins are a large family of cell surface molecules. These molecules are heterodimers that comprise pairs of &agr; and &bgr; chains without disulfide linkages. Because there are several different &agr; and &bgr; chains, differences in ligand specificity are achieved by different combinations of the &agr; and &bgr; chains. The integrins are involved both in direct cell-cell interaction and in the binding of cells to the extracellular matrix.
Integrins that occur on non-activated lymphocytes are in a so-called “low-avidity state,” which is converted very rapidly by T-cell activation into a transient so-called “high-avidity state.” The mechanism of this so-called “inside-out signaling” has not yet been elucidated, however. Collins et al.
Curr. Opinion Imm.
6: 385-393 (1994).
According to the affinity modulation model on T-cell activation, there is a conformational change in the integrins which first makes the high-affinity ligand binding site accessible to the ligand. Possible molecular events bringing about the conformational change which are currently suggested are covalent modification (for example, phosphorylation) or binding of activator or repressor molecules to the cytoplasmic domain of the integrin &bgr; subunit, but there is no experimental evidence in favor of a particular mechanism. Diamond and Springer,
Curr. Biol.
4: 506-517 (1994).
Another type of signal protein is the hsec7hom (human SEC7 homolog) protein, which is mainly expressed in natural killer cells and cytotoxic T cells. This protein was thought to be the human homolog of the SEC7 protein from
S. cerevisiae
. However, because of the (i) great difference in the molecular weights of SEC7 and hsec7hom, (ii) the sequence similarity that is limited to a relatively short section, and (iii) the specific expression of hsec7hom, it is now thought that hsec7hom does not belong to the SEC7 protein family. See Liu and Pohaidak,
Biochimica et Biophysica Acta
1132: 75-78 (1992)) For these reason, the hsec7hom protein will be referred to as “cytohesin-1.”
Cytohesin-1 contains two regions which are homologous with domains of other proteins:
1. SEC7 domain: this domain contains about 200 amino acids and is only known to be found in a few other proteins. One of these proteins is the SEC7 protein, which is involved in secretion in yeasts. Another protein that possesses this domain is EMB30, which is involved in embryogenesis in Arabidopsis. Shevell et al.,
Cell
77: 1051-1062 (1994).
2. PH domain (Pleckstrin homology domain): this domain is about 100 (±25) amino acids long and has been found in a number of proteins, many of which play a part in the signal transduction. The three-dimensional structure of some PH domains has been elucidated. These domains are able to function as ligand-binding domains. Tsukada et al.,
Proc. Nat'l Acad. Sci USA
91: 11256-60 (1994). Although it has been shown that the heterotrimeric G proteins can interact with PH domains, no exact physiological function for PH domains has been previously found. Birney,
TIBS
19: 349-353 (1994). Because the C-terminus of the PH domain has not been conclusively determined, larger amino acid sequences can be employed to ensure that the entire PH domain is present.
The PH domain is of major importance with regard to the present invention because of its ability to interact with the integrins.
The integrins are found on leukocyte surfaces and are involved in the inflammation process. Within minutes after receiving an inflammatory stimulus, the integrins acquire, through signal transduction pathway(s), the ability to attach to cell-surface and extracellular ligands. In some cases, the activation is transient, which means that the integrins quickly lose the ability to adhere. The dynamic cycling between adhesive and non-adhesive states endows a cell with the ability to rapidly regulate adhesion to ligands on apposing cell surfaces and matrices. This ability may be implicated in cell movement, which requires a rapid flux of adhesive interactions.
The function of integrin adhesion was initially documented in experiments that interfered with integrin function by using antibodies of peptide antagonists. The physiology of integrins has been assessed by the investigation of natural or induced genetic mutations of individual subunits. These mutations result in a variety of pathological sequelae.
Integrin-mediated adhesions has functional roles in a wide variety of biological and pathological settings, including hemostasis, inflammation, and tumor metastasis and development. For example, in primary hemostasis, platelet attachment to blood vessel walls, and aggregation at the site of injury are mediated by the integrins. Adhesion and signal transduction by integrins are essential elements of a sequence of intracellular interactions leading to antigen-specific activation of T-lymphocytes.
In inflammation, integrins mediate the critical attachment-strengthening step in the adhesion cascade, which permits leukocytes to move from the vasculature, across the endothelium lining blood vessels, and into the parenchyma. The subsequent migration of cells through the parenchyma depends upon the transient nature of integrin adhesiveness. This migration also may depend upon a sequence of attachment and detachment of ligand(s) by rapidly activated and inactivated integrin subpopulations, which are located and the leading and trailing edges of the migrating cells.
Because the vast array of functions performed by the integrins, these molecules are implicated in a large number of disease states. Accordingly, there is a need for methods of influencing the ability of integrins to adhere. This need is satisfied by the present invention.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide methods of influencing the ability of integrins to adhere.
It is another object of the present invention to provide methods of influencing the ability of integrins to adhere by employing cytohesin-PH.
It is still another object of the present invention to administer proteins that contain the cytohesin-PH peptide to patients to treat diseases and otherwise improve the physical condition.
It is yet another object of the present invention to provide assays, including those to screen drugs, using proteins that contain the cytohesin-PH peptide.
In accomplishing these and other objects, there is provided, in accordance with one aspect of the invention, the use of a cytohesin-PH peptide, in particular as shown in
FIG. 2
(SEQ ID NO: 12) or parts of the sequence shown therein, such as amino-acid positions 258 to 398, (residues 258 to 398 of SEQ ID NO: 12) to regulate the T-lymphocyte activation.
The invention furthermore relates to the use of a DNA coding for a cytohesin-PH peptide, in particular as shown in
FIG. 2
(SEQ ID NO: 11) or parts of the sequence shown therein, such as nucleotide positions 841 to 1263, (bases 841 to 1263 of SEQ ID NO:11) for expression of the peptide.
The invention furthermore relates to the use of a DNA whose sequence is degenerate (often referred to as codon/anticodon wobble) with respect to the sequence of the DNA mentioned above in accordance with the nature

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