Proteins are essential components in all living forms, and interrogation of their cellular activities is critical for studying various types of disease. At the same time, by harnessing their unique attributes, utilization of proteins as isolated molecules emerged as a powerful approach for medicinal and material applications (e.g. therapeutic proteins). The Ohata lab examines selective chemical reactions on proteins, which serve as “chemical tools” for both studying natural functions of proteins in living systems and creating new protein-based medicine or materials.
1) Site-specific labeling of natural proteins
A chemical reaction of natural proteins or protein modification is increasingly studied in diverse research fields spanning cell biology, biochemistry, medicinal science, and material chemistry. Understanding of protein modification in nature and development of artificial chemical modification technologies are of great interest, but realization of selective chemistry on the large biomolecules remains a formidable challenge. Such a selective chemistry would be helpful for site-specific protein modification for improvement of potency/efficacy of therapeutic proteins1,2 and surface immobilization of enzymes.3 We seek to develop new selective chemistry for site-specific modification of natural proteins.
2) Chemical reactions for detection of post-translational modifications (PTMs)
A myriad of cellular events are precisely regulated by consecutive post-translational modification (PTM) processes on proteins, and their dysregulation leads to crucial diseases such as cancer (e.g. aberrant phosphorylation) and neurodegenerative disorders (e.g. excessive cysteine oxidation).4 Because of the complexity of PTMs including various reaction types, spatial/temporal effects, and heterogeneity, there still remain significant needs for new detection techniques. Chemical labeling of PTMs on proteins would be a powerful approach but is significantly limited due to difficulty in creation of a molecular probe under challenging design criteria such as fast kinetics and mild reaction conditions in aqueous media. We seek to develop new chemical tools to detect PTM on native proteins.
PTM status of particular proteins could be closely associated with serious diseases such as Parkinson’s disease (DJ-1 protein)5 as well as congenital disorders (transferrin),6 and development of analytical tools for the molecular information is of great importance. However, the functional group-selective (i.e. chemoselective) chemical labeling would not exclusively target such a disease-related protein in the presence of various biomolecules. Proximity-ligation assay (PLA) is an antibody-based assay for detection of a protein of interest with PTM by DNA hybridization and polymerization processes.7 Thus, the combination of the chemical labeling and PLA enables us to study the molecular events on a protein of interest through imaging8 or polymerase chain reaction (PCR)-based analysis.9
3) Creation of unconventional biological medium
Current biological science relies heavily on aqueous media. However, degradation of biomolecules stored in aqueous media is an inevitable issue because of protein aggregation through hydrophobic interactions, enzymatic hydrolysis (e.g. protease self-digestion and DNA/RNA degradation by restriction enzymes10), and bacteria contamination. We seek to develop new biological media to address the issues of aqueous media.
(1) Beck, A.; Goetsch, L.; Dumontet, C.; Corvaïa, N. Strategies and Challenges for the next Generation of Antibody-Drug Conjugates. Nat Rev Drug Discov 2017, 16, 315.
(2) Ohata, J.; Ball, Z. T. A Hexa-Rhodium Metallopeptide Catalyst for Site-Specific Functionalization of Natural Antibodies. J. Am. Chem. Soc. 2017, 139, 12617.
(3) Datta, S.; Christena, L. R.; Rajaram, Y. R. S. Enzyme Immobilization: An Overview on Techniques and Support Materials. 3 Biotech 2013, 3, 1.
(4) Karve, T. M.; Cheema, A. K. Small Changes Huge Impact: The Role of Protein Posttranslational Modifications in Cellular Homeostasis and Disease. J Amino Acids 2011, 207691.
(5) Abu Bakar, N.; Lefeber, D. J.; van Scherpenzeel, M. Clinical Glycomics for the Diagnosis of Congenital Disorders of Glycosylation. J. Inherit. Metab. Dis. 2018, 41, 499.
(6) Canet-Avilés, R. M.; Wilson, M. A.; Miller, D. W.; Ahmad, R.; McLendon, C.; Bandyopadhyay, S.; Baptista, M. J.; Ringe, D.; Petsko, G. A.; Cookson, M. R. The Parkinson’s Disease Protein DJ-1 Is Neuroprotective Due to Cysteine-Sulfinic Acid-Driven Mitochondrial Localization. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9103.
(7) Tsutsumi, R.; Harizanova, J.; Stockert, R.; Schröder, K.; Bastiaens, P. I. H.; Neel, B. G. Assay to Visualize Specific Protein Oxidation Reveals Spatio-Temporal Regulation of SHP2. Nat Commun 2017, 8, 1.
(8) Ohata, J.; Krishnamoorthy, L.; Gonzalez, M. A.; Xiao, T.; Iovan, D. A.; Toste, F. D.; Miller, E. W.; Chang, C. J. An Activity-Based Methionine Bioconjugation Approach To Developing Proximity-Activated Imaging Reporters. ACS Cent. Sci. 2020, 6, 32.
(9) Robinson, P. V.; Tsai, C.; de Groot, A. E.; McKechnie, J. L.; Bertozzi, C. R. Glyco-Seek: Ultrasensitive Detection of Protein-Specific Glycosylation by Proximity Ligation Polymerase Chain Reaction. J. Am. Chem. Soc. 2016, 138, 10722.
(10) Cesare, A. J.; Heaphy, C. M.; O’Sullivan, R. J. Visualization of Telomere Integrity and Function In Vitro and In Vivo Using Immunofluorescence Techniques. Current Protocols in Cytometry 2015, 73, 12.40.1.