Intracellular targeting of molecules via syndecan-4
The present invention provides methods for intracellular and/or nuclear targeting of an agent capable of specifically binding to syndecan-4. The present invention further provides methods for the modification of the intracellular and/or nuclear targeting of said agent, as well methods for identifying compounds capable of modifying the syndecan-4 delivery pathway. The present invention further provides experimental kits to perform the methods according to the invention.
1. A method for nuclear targeting of an agent capable of specifically binding to syndecan-4, comprising
selecting an agent of interest to be targeted into a cell nucleus,
contacting said agent with a cell that comprises syndecan-4 expressed on its cell membrane,
allowing binding between said agent and the extracellular domain of the syndecan-4 to take place,
allowing the internalization of said agent after formation of a complex by the interaction between said agent and said cell-surface syndecan-4, and
releasing the agent from the internalized extracellular domain of the syndecan-4 inside the cell membrane,
further comprising inducing phosphorylation of the cytoplasmic domain of syndecan-4 resulting in formation of a nuclear localization signal (NLS) on said expressed syndecan-4.
2. The method according to claim 1 , wherein the agent has been modified to be capable of binding to the extracellular domain of syndecan-4 before allowing said interaction.
3. The method according to claim 1 , wherein the syndecan-4 expressed on said cell membrane is structurally altered or has an altered expression pattern.
4. The method according to claim 3 , wherein the extracellular domain of the altered syndecan-4 is specifically modified to be capable of binding said agent.
5. The method according to claim 1 , wherein said contacting is carried out in vivo or in vitro and wherein the agent is released in the cell nucleus.
6. The method according to claim 1 , wherein said interaction is between one agent and one syndecan-4 molecule.
7. The method according to claim 1 , wherein said agent is attached or complexed with a moiety, which moiety binds with the extracellular domain of the syndecan-4 upon allowing said interaction.
8. The method according to claim 1 , wherein the NLS is generated or modulated at the Ser179 position of the cytoplasmic domain of syndecan-4.
9. The method of claim 1 , further comprising attaching a modified syndecan-4 specific NLS having the sequence of SEQ ID NOs: 2 or 3 to the agent, and introducing the agent-NLS complex or conjugate into said cell.
10. The method of claim 1 , further comprising altering a transmembrane protein not capable of entering the nucleus so as to enable it to target an agent into the nucleus of a cell, by introducing the syndecan-4 specific NLS having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 into the cytoplasmic domain of said transmembrane protein.
11. The method of claim 1 , further comprising entering of the syndecan-4 from endocytosed vesicles into the cytoplasm of the cell.