Summary: Protein sequencing arrays for mapping proteolytic
processing pathways and drug discovery at the proteomic level

By Dr. Edward Shipwash

Proteolytic processing is a very common and effective mechanism of physiological regulation in all organisms and viruses.  Processing is caused by cleavage of specific bonds in proteins or polypeptides by proteases enzymes and is detected using N- or C-terminal protein sequencing, which identifies the cleavage site.  Current end group sequencing instrument take a few hours to generate a sequence tag for a single protein sample.  This makes mapping proteolytic processing pathways at the proteomic level impossible.  Our technology will create a more favorable situation.  The repertoire of physiological processing is long and varied.  For example, proteolytic processing is the final step in the biosynthesis of a great variety of proteins.  Furthermore, regulated proteolytic processing generates active hormones, proteins, peptides and enzymes.  It regulates brain function, cardiovascular functions, liver functions, immune functions, blood coagulation, cell adhesion, cell migration, cell-cell communications, the assembly of the extracellular matrix, the assembly of phages and retro-viruses, reproduction, and developmental biology. 

Hence, it is not surprising that over 50 human diseases caused by defects in proteolytic enzymes have been discovered so far.  And still other diseases are caused by the unregulated expression of proteases.  These diseases include Cancers; Neurobiological disorders; Alzheimer’s Disease; Parkinson’s Disease; Liver Diseases; High Blood Pressure; Mental Retardation; Heart Diseases; Arthritis; Bone Diseases; Pancreatic Diseases; Autoimmune Diseases; Skin Diseases; Eye Diseases; Hemophilia; Diabetes; Muscular Dystrophy; Tourett Syndrome; Blood Coagulation Disorders; Obesity; Deafness; and Hypospermatogenesis.  Since the method used to identify proteolytic processing is protein end group sequencing, our company with its massively paralleled sequencing technology is in a unique position to map all of the proteolytic processing pathways in the proteome.  Each newly discovered proteolytic processing event discovered could be covered by a patent.  By mapping proteolytic processing pathways in diseased versus normal cells, we will uncover the molecular basis for many diseases.  This will provide the opportunity for rational drug design for each disease.  Knowing the cleavage sites as revealed by the protein sequencing will suggest the drug type to use.  Knowledge of the cleavage site should identify the protease type, and make protease inhibitors possible.  Other types of drugs are antibodies that bind to the cleave sites preventing unwanted cleavage.  Or synthetic peptides modeled on the cleavage sites that would inhibit the protease and prevent cleavage.  The proteomics industry has recognized the importance of proteolytic processing pathways and has defined two new subsets of proteomics, the degradome and the cryptome.  The degradome is the complete set of human proteases (over 560 are currently known).  The cryptome is the set of bioactive peptides (cryptins) that are liberated from proteins by proteolytic processing.  So proteomics recognizes the need to study all proteolytic processing events in normal and diseased cells, but they lack the technology to do so at the proteomic level.

By using our sequencing technologies, processing thousands of samples simultaneously in an industrial setting, we will be able to map all proteolytic processing pathways in normal and diseased cells making rational drug design possible. With the discovery of the molecular mechanism of a multitude of diseases, each one covered by a separate patent, our company will have sustainable revenue from both biomarkers used in disease diagnostics and following disease progression and drugs that cure diseases.  The market for protein-based drugs exceeded $10 billion in 2000, and protease inhibitors are well establish drugs that have been used to treat Alzheimer’s Disease, for example.  The goals of proteomics are: (1) Identifying all proteins made by a given cell in normal versus disease; (2) Determining the amount of each protein expressed in each cell type; (Determining the protein’s modifications and functions; (4) Determining the 3D structures of all proteins.  Our amino acid analysis and protein end group sequencing technologies can be applied to all of theses goals.  Our amino acid analysis and sequencing arrays can be used to identify proteins, identify modifications, and determine the amount of protein expressed in each cell type, and by making rapid protein domain mapping possible, accelerate the determination of 3D structures. 

Hence, AminoArrays is not only a proteomics company; it holds the potential to become a pharmaceuticals research and development company as well.  Furthermore mapping proteolytic processing pathways in the proteomes of human pathogens such as viruses, and pathogenic microorganisms and insect vectors will facilitate the production of new antibiotics, insecticides and pesticides.   

Proteolytic processing:  The arrows at left depict cleavage sites for proteases in the proteolytic processing of a protein substrate.  This processing will liberate the peptides, EDWARDSHIPWASHFRANCISRI, from the N-terminal end giving the new N-terminal sequence of the protein as CKEDWARDSHIP.  The proteolytic processing event activated the protein.  In some cases the liberated peptides function as bioactive peptides mediating various biological roles such as cell proliferation, cell-cell communication and blood vessel formation.  Protein N- terminal or C-terminal protein sequencing will reveal the cleavage sites for N-terminal or C-terminal proteolytic processing events, respectfully.  Protein sequencing will also identify the liberated peptides.  From the knowledge of the sequences of the cleavage sites, certain drugs such as protease inhibitors or antibodies that bind the cleavage sites will be developed using rational drug design.