Protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction

Abstract

The invention relates to protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction. Using overlapping hexapeptides that encode for the entire amino acid sequences of the linker domains of human P-glycoprotein gene 1 and 3 (HP-gp1 and HP-gp3), a direct and specific binding between HP-gp1 and 3 linker domains and intracellular proteins was demonstrated. Three different stretches (617EKGIYFKLVTM627, (SEQ ID NO: 1) 658SRSSLIRKRSTRRSVRGSQA677 (SEQ ID NO: 2) and 694PVSFWRIMKLNLT706 (SEQ ID NO: 3) for HP-gp1 and 618LMKKEGVYFKLVNM631 (SEQ ID NO: 4), 648KAATRMAPNGWKSRLFRHSTQKNLKNS674 (SEQ ID NO: 5), and 695PVSFLKVLKLNKT707 (SEQ ID NO: 6) for HP-gp3) in linker domains bound to proteins with apparent molecular masses of ˜80 kDa, 57 kDa and 30 kDa. The binding of the 57 kDa protein was further characterized. Purification and partial N-terminal amino acid sequencing of the 57 kDa protein showed that it encodes the N-terminal amino acids of alpha and beta-tubulins. The method of the present invention was further validated with Annexin. The present invention thus demonstrates a novel concept whereby the interactions between two proteins are mediated by strings of few amino acids with high and repulsive binding energies, enabling the identification of high affinity binding sites between any interacting proteins.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10 / 010,310, filed Nov. 13, 2001, entitled, “Protein-Protein Interactions and Methods for Identifying Interacting Proteins and the Amino Acid Sequence at the Site of Interaction,” now U.S. Pat. No. 7,176,035, which claims the benefit of PCT Appl. No. PCT / CA00 / 0587, filed May 12, 2000, which in turns claims priority from U.S. Provisional Appl. No. 60 / 134,259, filed May 14, 1999, the entire contents of all of which are hereby incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates to proteonomics. More specifically, the invention relates to protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction. BACKGROUND OF THE INVENTION [0003] Specific protein-protein interactions are critical events in biological processes. Protein-protein interactions govern biological processes tha...

AI Technical Summary

Benefits of technology

"The present invention provides a method to identify protein-protein interaction domains and the amino acid sequences of these domains. This approach is based on the understanding of the principle of protein-protein interactions and allows for the identification of interacting proteins and the specific amino acid sequences of interactions in several days, as compared to several months using other methods. The invention also allows for the identification of proteins that interact with a therapeutically important protein and the isolation of lead drugs. The method can address three important steps in drug development: identifying the amino acid sequences of all interacting domains in target proteins, identifying all interacting proteins for drug development, and screening for specific drugs against each of the interacting domains in a target protein. The invention also provides a novel approach to identifying high affinity interacting domains and the amino acid sequences thereof."

Problems solved by technology

A major obstacle in drug development for the treatment of diseases has been the identification of target proteins and their functional sites.

In fact, most research and development (R&D) projects in pharmaceutical companies take several years to identify a valid target protein.

The selection of drugs that bind to and inhibit the functions of these proteins takes several years and is generally non-specific and random.

Such drugs thus often lead to major side-effects.

Therefore, it is not surprising that many R&D projects never lead to the development of specific drugs even after three to five years of intensive research efforts.

Images