UCSF UC San Francisco Electronic Theses and Dissertations Title Identification and exploration of apoptotic and caspase proteolytic substrates. Permalink https://escholarship.org/uc/item/86t0d35v Author Seaman, Julia Publication Date 2016 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California ii Dedication To my husband, Nate, and my family, Mom, Dad and Chris. iii Acknowledgements I am very grateful for the advice, assistance and support from the Wells Lab. My advisor, Jim Wells, has created a wonderful laboratory environment for collaborative research and friendships. The lab, and members in it, could not have survived without the innumerable skills of Marja Tarr. My graduate career has been greatly assisted by sharing the experiences with fellow students Ashley Smart and Charlie Morgan in the lab. It has been fun to explore science and solve the big and small problems with them and the other graduate students in lab- Justin Rettenmaier, Hai Tran, Sam Pollock, and Alex Martinko. I have relied heavily on everyone else who counts himself or herself as a Wellsome member. The postdocs- especially Kazu Shimbo, Nick Agard, Arun Wiita, Jason Porter, Juan Diaz, JT Koerber, Nathan Thomsen, Duy Ngyuen, Peter Lee, Min Zhuang, Zach Hill and Olivier Julien- have helped improved my scientific skills and graduate school experience throughout my time in the lab. Finally, my scientific work relies heavily on many members who came before me. UCSF has been a great environment to learn and explore during my PhD. I am very thankful my program, PSPG and its staff- Deana Kroetz, Debbie Acoba and Rebecca Brown. The PSPG program also introduced me to my classmates who have helped relieve the stress of science throughout the years. Additionally, working at UCSF has exposed me to a great many number of core facilities and collaborators who have contributed to all facets of my work. First, I would like to thank the Mass Spectrometry Facility and its team under Al Burlingame, especially Dave Maltby and Robert Chalkley. I have also have worked with the Preclinical Therapeutic Core under Byron Hann and Don Hom and the MMTI with Blake Aftab. Finally, I would like to thank, Seth Ginsberg and Louis Tharp of the Global Healthy Living Foundation, for their fellowship and guidance. iv Abstract Apoptosis, a type of programmed cell death, is a universal and essential cellular function. There are numerous homeostatic biological roles of apoptosis and many diseases have or cause mys- or dis- regulated apoptosis, most notably cancer’s escape from apoptotic signals. Apoptosis has many distinctive characteristics including membrane blebbing, chromatin condensation, and most importantly for these studies, the activation of a class of protease proteins called caspases. Caspases are cysteine aspartic proteases with a unique preference to cleave hundred of substrates after acidic residues, especially aspartate. These cleavage events can activate, modify, or inhibit the substrate’s function, which leads to the dismantling of the cell during apoptosis. This process is well conserved throughout metazoan evolution, with parallel pathways in coral, fish, flies, worms and mice. To study apoptotic protease activity, the Wells Lab has developed a proteomic-based technique. This technique is an unbiased positive enrichment labeling mass spectrometry protocol. While the protocol was developed for studying caspase substrates during apoptosis in human cell culture, it is very versatile, allowing for use in almost any protein sample like perturbed cellular lysates, different species and primary samples. Chapter 1 covers the protocol specifics and uses, summarizing a decade’s worth of optimization and application. The DegraBase is the compilation of 44 different experiments using the N-terminal labeling method to examine apoptotic proteolytic activity described in Chapter 2. While much of the experimentation work was completed before I started the project, I completed compilation and standardization of raw data, and worked with Emily Crawford on the analysis. This global analysis reveals there is a large increase in proteolytic activity after apoptotic induction, and caspases account for 25% of the newly created fragments. Within caspase substrates, there is no v single biological process or sub-cellular location that is targeted, as caspases appear to cleave substrates throughout all the different pathways of the cell. Additionally, this database is also a good resource for endogenous proteolysis, including free methionines, and signal and transis peptide processing. Analysis of the DegraBase also reveals evolutionary and biological discoveries. As the apoptotic pathway to activate caspases is highly conserved throughout metazoans, we wanted to investigate the conservation of caspase substrates. In Chapter 3, the comparison of caspase substrates in worm, fly, mouse and human reveals a hierarchal structure. My contribution includes the murine dataset and comparison analysis in collaboration with Emily Crawford. We found caspase cleavage is highly conserved at the pathway level, while individual targets and sites are not as well conserved the more distant the animals. The unbiased and large size of the DegraBase also reveals broader caspase activity than previously described. Caspases had been assumed to have absolute specificity for aspartate and no activity for glutamate. However, with the large dataset, in Chapter 4, I reveal significant activity after glutamate and even potential activity after phosphoserine. This activity is verified through biochemical assays and x-ray crystallography, and expands the number of apoptotic caspase substrates by 15%. As many chemotherapeutics induce apoptosis, apoptotic, especially caspase, proteolytic substrates may be good biomarkers of treatment efficacy. The development of mouse models and a positive control are discussed in Chapter 5. These models utilize the DegraBase and labeling technology to identify peptides enriched in the blood and tumor specific to treatment responding mice as potential biomarkers. vi Table of Contents Chapter 1 1 Global analysis of cellular proteolysis by selective enzymatic labeling of protein N-termini References 40 Chapter 2 44 The DegraBase: a database of proteolysis in healthy and apoptotic human cells References 77 Chapter 3 86 Conservation of caspase substrates across metazoans suggests hierarchical importance of signaling pathways over specific targets and cleavage site motifs in apoptosis. References 118 Chapter 4 123 Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues References 154 Chapter 5 169 Initial results and development of tools to identify apoptotic as biomarkers of chemotherapeutic efficacy References 204 vii List of Tables Chapter 1 Table 1 6 A summary of current methods for proteolytic cleavage site and substrate identification. Chapter 2 Table 1 71 P1’ amino acid frequency and Arg/N-End Rule Pathway half-lives Supplementary Tables 85 Chapter 3 Table 1 99 Summary of results Table 2 111 Fly and worm substrates with no human ortholog Supplementary Tables 122 Chapter 4 Table 1 128 Enrichment of P1 position amino acids Table 2 129 High conservation in mouse and human of P1 aspartate and glutamate cleavage sites and protein targets Table 3 134 Summary of kinetic values for cleavage of P1 aspartate and glutamate by caspases-3 and -7 Table 4 139 Enrichment of annotated phosphorylation sites observed cleaved Table 5 143 Summary of kinetic values for cleavage after aspartate, glutamate or phospho-serine Supplementary Tables 158 Chapter 5 Table 1 180 Proteins with peptides that were enriched for Study I Table 2 184 Proteins with peptides that were enriched for Study II Table 3 190 Enriched peptides Table 4 191 Proteins with peptides that were enriched for Study IV viii List of Figures Chapter 1 Figure 1 9 An overview of the subtiligase N-terminal labeling method Figure 2 11 A schematic difference between Forward and Reverse experiments Figure 3 16 Monitoring Apoptosis and Proteomic Distribution of Cleavage Substrates Figure 4 28 Selected Reaction Monitoring (SRM) for proteolytic substrate quantification Figure 5 31 Monitoring kinetics of recombinant caspase cleavage Figure 6 33 Plasma N-terminomics Figure 7 37 Sequence features of identified N-termini Chapter 2 Figure 1 48 Experimental schema, database design and database summary Figure 2 56 Venn diagrams show a larger overlap of unique protein and peptide identifications between untreated, apoptotic and apoptotic caspase-cleaved datasets Figure 3 59 IceLogo diagrams of untreated, apoptotic and caspase-cleaved datasets Figure 4 60 Proteolytic processing within datasets Figure 5 62 IceLogos for processing around the initiator methionine Figure 6 65 Mitochondrial and signal peptides within the untreated dataset Figure 7 68 Filled logos for endoproteolysis occurring at residue 66 or above Supplementary Figures 80 Chapter 3 Figure 1 90 Experimental scheme Figure 2 95 IceLogos and Secondary Structures for Human, Mouse, Fly and Worm cleavages Figure 3 98 Data analysis pipeline Figure 4 101 Comparison of sites and sequences ix Figure 5 107 Conservation hierarchy of caspase substrates Supplementary Figure 121 Chapter 4 Figure 1 132 IceLogos of aspartate and glutamate at P1 Figure 2 137 Crystal stuctures of caspase -3 and -7 with glutamate Figure 3 140 Cleavage of P1-phosphoserine by caspase-3 Figure 4 144 P1 aspartate and glutamate cleavage sites are not strongly