Trypsin as a Biochemical Tool for the Characterization of Ras- Related Protein Structure and Function
- 1. Department of Chemistry and Biochemistry, University of Arkansas, USA
Bovine trypsin is a well-characterized serine protease used as a vital tool in biochemistry, particularly proteomics, as it cleaves peptides or proteins, with high specificity, at the carboxy-terminus of lysine (Lys) and arginine (Arg) amino acid residues. Moreover, the fragment peptides are typically in mass ranges that are amenable to detection by mass spectrometry techniques other than Matrix Assisted Laser Desorption Ionization (MALDI). In addition, Trypsin is found in the digestive system of many vertebrates, and is stable under diverse chemical conditions. The RAS (Rat Sarcoma) families of proteins are involved in several cell-signaling activities that are vital to processes such as cell proliferation and differentiation. If not properly regulated, these signaling processes can result in diseases such as cancer, underlying their importance as targets for drug discovery efforts to control Ras-stimulated abnormal cell signaling. The activity of Ras-related proteins is dictated by their nucleotide-bound state where the GTP-bound is active, and the GDP-bound state inactive. The regulation of this nucleotide-cycling activity is dictated by protein-protein interactions with effectors that may induce conformational changes. Here, we highlight recent advancements showing the use of trypsin in the characterization of Ras-related protein structure and function in cell signaling and regulation.
Muhoza D, Duverna E, Adams PD (2018) Trypsin as a Biochemical Tool for the Characterization of Ras-Related Protein Structure and Function. JSM Enzymol Protein Sci 3(1): 1011.
Bovine trypsin is a classical and well-characterized serine protease [1,2]. Trypsin furthermore is a widely used and vital tool in biochemistry, particularly proteomics, as it cleaves peptides or proteins, with high specificity, at the carboxy-terminus of lysine (Lys) and arginine (Arg) amino acid residues. This high specificity makes trypsin a valuable tool in proteomics studies, as many high-scale mass spectrometry-based proteomics studies are done through the use of trypsin to cleave protein mixtures into specific peptide fragments . In addition, the fragment peptides typically occupy mass ranges that are amenable to detection by mass spectrometry techniques other than Matrix Assisted Laser Desorption Ionization (MALDI) . Nevertheless, it should be noted that trypsin’s rate of cleavage is slowed when Lys or Arg is adjacent to acidic residues or disulfide bridges, and cleavage does not occur when Lys or Arg is followed by a proline (Pro) residue . Trypsin is naturally found in the digestive system of many vertebrates, and is stable under diverse chemical conditions . The high specificity of trypsin is also responsible for its efficient use in biochemical and structural biological approaches.
As an example, experiments using trypsin can be helpful for understanding Ras-related (Ras) proteins. These proteins are involved in various cell-signaling activities, and, if not properly regulated, aberrant cell-signaling can lead to cancers or other developmental pathologies . This underlies their importance as targets for drug discovery efforts to control Ras-stimulated abnormal cell signaling . Moreover, the frequency of Ras gene mutations in selected human cancers has continued to highlight why these proteins are important in cell transformation. The activity of Ras-related proteins is dictated by their nucleotidebound state where the GTP-bound is active, and the GDP-bound state inactive. The regulation of this nucleotide-cycling activity is dictated by protein-protein interactions with effectors , which often induce conformational changes in both the Rasrelated protein and effector proteins. Indeed, mutations leading to altered protein-protein interactions and/or abnormal function that result from conformational changes can be probed byproteolytic digestion using proteases such as trypsin. Here, we highlight recent studies showcasing the use of trypsin in the characterization of Ras-related protein structure and function in cell signaling and regulation.
Proteolytic digestion assays have yielded low-resolution structural information on Ras proteins for many years. A 1989 study by Grant and colleagues showed how structural features of N-Ras could be affected by the binding of the guanine nucleotide and metal ions to the protein. Mg2+, Ca2+, K+ , GDP were added to apo-Ras construct(s). The proteins were then subjected to trypsin and chymotrypsin digestion to determine how the presence of Mg2+, Ca2+, K+ or GDP influenced protein resistance to hydrolysis . These results paved the way for more sophisticated structural biological, as well as proteomics studies on Ras proteins. Recently, proteomics studies of Ras isoforms expressed in colorectal and pancreatic cell lines by Mageean et al. , showed the biological contributions of the isoforms expressed in the cells. In these types of studies, trypsin digestion resulted in Ras peptides that were analyzed by the in-gel digestion approach (Figure 1). This approach allowed for an accurate measurement of endogenous protein copies of the RAS isoforms (K-Ras, H-Ras, and N-Ras). In a proteomics study by Gnad et al., (cellular phosphorylation, caused by constitutive KRas/ Cdc42 signaling or PAK4 ablation, was characterized by digesting protein lysates from NIH3T3 cell lines with trypsin. Analysis by mass spectrometry revealed phosphorylation sites that were in agreement with expected sites . Junemann J et al., described an MS-based approach to quantify the degree of glycosylation in Ras/Rho-related small GTPases generated by trypsin and chymotrypsin digestions. A multiplexed MRM (Multiple Reaction Monitoring-based) assay was used to characterize the degree of glycosylation of the small GTPases and allowed for the detection of glycosylated toxic proteins . In a 2012 study by Ferreira et al., 2-Dimensional Electrophoresis (2DE), semi-quantitative image analysis, in-gel trypsin digestion and mass spectrometry were employed to characterize proteome changes amongst Ras isoforms (H, K, N). The results of this proteomic characterization study demonstrated that loss of function for 2 of the isoforms (H- and N-) could provide credible information on differences in protein expression and suggest possible therapeutic targets toward these proteins . Studies examining the effect(s) of single-point mutations in importantbinding regions of Rasrelated proteins, using trypsin digestion, have also been useful in highlighting the role(s) of key residues. One such study by Shin et al., highlighted how GTPase and translation functions of the eukaryotic translation initiation factor 5B (eIF5B) could be affected by a Gly to Alamutation in one of the flexible “Switch” regions of the protein, the Switch2 region. WT eIF5B and eIF5BG479A were subjected to limited trypsin proteolysis. Results showed differences in the number of protein fragments produced by the trypsin cleavage of the G479A variant as compared to WT, suggesting that the G479A mutation fostered a conformational change in the Switch 2 region .
Ras-related proteins have been shown tooften undergo conformational changes that lead to aberrantcell signaling and hyperactivity. An approach to combat this has included the characterization of small molecule targets with the potential to alter Ras-stimulated abnormal cell-signaling . To this end, trypsin digestions have revealed important information on the influence small molecule targets can have on Ras-related protein conformation upon function. Recently, Patricelli et al., showed that a small molecule, ARS-853, could serve as a covalent inhibitor of a K-Ras variant, in a cellular environment, by binding to the GDPbound inactive form of the protein and preventing its activation via GDP dissociation. Trypsin digestions of a control wild type K-Ras peptide were compared to that of the variant K-RasG12C in the presence of ARS-853. Mass spectroscopy revealed differences in the fragments from the digestions of the G12C variant in comparison to wild type, suggesting that the small molecule engaged the K-Ras wild type and variant peptides differently . Moreover, the combination of stable isotope labeling with limited trypsin proteolysis has been used to monitor protein conformational changes upon the binding of small molecules . In these studies, the limited proteolysis was analyzed by mass spectrometry showing how this novel combination approach could be used to observe specific structural changes induced by small molecule binding.
In summary, the use of trypsin to cleave proteins into peptide fragments for characterization has been a robust approach applied to basic proteomics studies, andits specificity has made it important for these experiments as well . In addition, although there are more advanced techniques to probe protein conformational changes, such as spectroscopy and X-ray crystallography, trypsin digestion can be used as a quick and relatively inexpensive method to generate preliminary, lowresolution structural data on Ras-related proteins. As Ras-related protein structure and function continues to be an important aspect towards a better understanding of these proteins’ role(s) in stimulating various diseased states, approaches using proteolytic digestions with trypsin can be expected to continue to help uncover information on conformational changes that may be exploited for subsequent structure-based drug design.
This publication was supported by grants from the Arkansas Biosciences Institute, the Arkansas Breast Cancer Research Program and the University of Arkansas for Medical Sciences.
1. Bender ML, Kezdy FJ, Gunter CR. The Anatomy of an Enzymatic Catalysis. -Chymotrypsin. J Amer Chem Soci. 1964; 86: 3714-3721.
2. Krieger M, Kay LM, Stroud RM. Structure and specific binding of trypsin: comparison of inhibited derivatives and a model for substrate binding. J Mol Biol. 1974; 83: 209-230.
3. Olsen JV, Ong SE, Mann M. Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics. 2004; 3: 608-614.
4. Trypsin- I.U.B.:188.8.131.52 2017.
5. Brown WE, Wold F. Alkyl isocyanates as active-site-specific reagents for serine proteases. Reaction properties. Biochemistry. 1973; 12: 828-834.
6. Smith MJ, Neel BG, Ikura M. NMR-based functional profiling of RASopathies and oncogenic RAS mutations. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110: 4574-4579.
7. Johnson CW, Mattos C. The allosteric switch and conformational states in Ras GTPase affected by small molecules. Enzymes. 2013; 33 Pt A: 41-67.
8. Hong-Kai Z, Jie C. Research Advances in the Role of RAS-GTPase-activating Proteins in Tumors. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2015; 37:364-369.
9. Grand RJ, Grant ML. Conformational changes occurring in N-ras p21 in response to binding of guanine nucleotide and metal ions probed by proteolysis performed under controlled conditions. FEBS Lett. 1989; 253: 281-286.
10. Mageean CJ, Griffiths JR, Smith DL, Clague MJ, Prior IA. Absolute Quantification of Endogenous Ras Isoform Abundance. PLoS One. 2015; 10: e0142674.
11. Gnad F, Young A, Zhou W, Lyle K, Ong CC, Stokes MP, et al. Systems-wide analysis of K-Ras, Cdc42, and PAK4 signaling by quantitative phosphoproteomics. Mol Cell Proteomics. 2013; 12: 2070-2080.
12. Junemann J, Lammerhirt CM, Polten F, Just I, Gerhard R, Genth H, et al. Quantification of small GTPase glucosylation by clostridial glucosylating toxins using multiplexed MRM analysis. Proteomics. 2017; 17.
13. Ferreira L, Fuentes-Calvo I, Munoz-Felix JM, Muniz-Martin C, Sanchez-Juanes F, Raposo C, et al. Functional specific roles of H-ras and N-ras. A proteomic approach using knockout cell lines. Electrophoresis. 2012; 33: 1385-1396.
14. Shin BS, Acker MG, Maag D, Kim JR, Lorsch JR, Dever TE. Intragenic suppressor mutations restore GTPase and translation functions of a eukaryotic initiation factor 5B switch II mutant. Mol Cell Biol. 2007; 27: 1677-1685.
15. Muhoza D, Adams PD. Two Small Molecules, ZCL278 and AZA197 Show Promise in Influencing Protein Interactions Involving the Ras-Related Protein Cell division cycle 42 [Cdc42] to Modulate Its Oncogenic Potential. Open Journal of Biophysics. 2017; 7: 71-81.
16. Patricelli MP, Janes MR, Li LS, Hansen R, Peters U, Kessler LV, et al. Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State. Cancer Discov. 2016; 6: 316-329.
17. Di Michele M, Stes E, Vandermarliere E, Arora R, Astorga-Wells J, Vandenbussche J, et al. Limited Proteolysis Combined with Stable Isotope Labeling Reveals Conformational Changes in Protein (Pseudo) kinases upon Binding Small Molecules. J Proteome Res. 2015; 14: 4179-4193.