Copyright (2014) By Rebecca L. Maglathlin ii Acknowledgements “In the discovery of secret things, and in the investigation of hidden causes, stronger reasons are obtained from sure experiments and demonstrated arguments than from probable conjectures and the opinions of philosophical speculators.” -William Gilbert, Loadstone and Magnetic Bodies, and on The Great Magnet of the Earth, translated from the 1600 edition of De Magnete by P. Fleury Mottelay (Bernard Quaritch, London, 1893) I would like to thank my mentor, Jack Taunton, for instilling in me that “good” is never enough and that greatness is just as much a matter of hard work and perseverance as it is a function of intelligence and insight. I would like to thank Jeff Johnson and Tasha Johnson for their work on the mass spectrometry in Chapter 2. I would also like to thank Gonzalo Ureta and Emma McCullagh of Sebastian Bernales’ Lab (Fundacion Ciencia de la Vida, Chile) for their work cited in Chapter 3. I would like to thank the members of the Taunton Lab, past and present, for their insights, expertise, friendship and general all around awesomeness. I would specifically like to thank Ville, Sarah and Andy for their guidance on this project and for their beautiful work cited herein. I would also like to thank Geoff Smith for his contribution of the STAT5 phosphorylation experiment in Chapter 2. I would like to thank my family for all their support and understanding. Finally, I would like to thank Peter for all of his help in harnessing the power of silicon. iii Part of this thesis is a reproduction of material in the publication process. Chapter 2 is reproduced in part with permission from: Maglathlin, R.L, Johnson, J., Krogan, N., Taunton, J. Proteomic profiling of a selective Sec61 inhibitor identifies a predictive model for rapid target identification. Manuscript in preparation. The mass spectrometry was completed by Jeff Johnson and Tasha Johnson (Krogan Lab, UCSF and Gladstone Institute). The STAT5 phosphorylation blot in Figure 2.7 was performed by Geoff Smith (Taunton Lab, UCSF). Chapter 3 is reproduced in part with permission from: Maglathlin, R.L., Ureta, G., McCullagh, E., Bernales, S., Taunton, J. Sec61α as a novel target in multiple myeloma. Manuscript in preparation. Experiments performed by Gonzolo Ureta are cited in the figure legends when necessary. iv To my family v Toward drugging the translocon: sequence determinants and cellular consequences of Sec61 inhibition Rebecca L. Maglathlin Abstract Secretory and membrane proteins (secretory proteins) are the conduits through which cells communicate. Membrane proteins make up 35% of the human genome and, though greater than 60% of drug targets are secretory proteins, they remain difficult to inhibit, as many lack traditional small molecule binding sites. In eukaryotes, the biogenesis of most secretory proteins begins with cotranslational translocation into the endoplasmic reticulum (ER). Cotransins are small molecule inhibitors of cotranslational translocation and therefore inhibitors of secretory protein expression. Cotransins inhibit translocation by binding directly to the Sec61 translocon and perturbing the interaction between Sec61α and the targeting sequence of the nascent chain (either an N-terminal signal peptide or the first transmembrane domain). Remarkably, cotransins inhibit translocation of only a small subset of secretory proteins, dependent on the sequence of the targeting signal. Here, we describe work toward both identifying the sequence determinants of cotransin selectivity and the broader cellular consequences of cotransin inhibition, including the discovery of Sec61α as a novel target in multiple myeloma. Using a quantitative membrane proteomics appraoch, we directly identify targets of a selective vi cotransin analog, CT8, in an unbiased manner. We show that CT8 inhibits only 25% of the identified secreted proteome. CT8-sensitivity of signal peptides correlates with the free energy of membrane integration suggesting that 1) signal peptides and transmembrane domains gate Sec61α via a similar mechanism and 2) cotransins interfere with this signal-partitioning step. Finally, we use this metric to predict the sensitivity of novel CT8-sensitive proteins based solely on primary amino acid sequence. To solidify Sec61α as a novel target for myeloma, we show that cotransin potently induces apoptosis in patient-derived CD138+ cells while leaving CD138- cells virtually unaffected. We begin to explore the myeloma-specific mechanism of CT8- induced apoptosis by showing that the CT8-sensitive ER-resident chaperone p58ipk is an essential protein in multiple myeloma cells. This study furnishes a method for predicting new cotransin-sensitive proteins, thereby allowing for the rapid identification of clinically relevant arenas in which to begin testing these compounds. Within the context of multiple myeloma, we show that Sec61α is a viable therapeutic target and that cotransins represent a promising lead for further development. Using the information uncovered here, it will be possible to design new analogs to more specifically target proteins relevant to the anti-cancer properties of cotransins. vii Table of Contents Chapter 1: Introduction 1 1.1 Abstract 2 1.2 Secreted and membrane proteins in human disease 3 1.3 Biogenesis of secreted and membrane proteins 3 1.4 Substrate recognition by the Sec61 translocon 5 1.5 Cotransins are substrate selective inhibitors of Sec61α 10 1.6 Multiple myeloma and protein secretion 12 1.7 Conclusions 15 1.8 References 15 Chapter 2: Sequence determinants of Sec61α inhibition by cotransins 23 2.1 Abstract 24 2.2 Introduction 24 2.3 Proteomic analysis of CT8-treated cells 27 2.4 Cellular validation of CT8-sensitive proteins 36 2.5 Validation of CT8-sensitive proteins in cotranslational translocation assays 38 2.6 Validation of TRPML1, the first identified CT8-sensitive multi-spanning integral membrane protein 40 2.7 Measuring hydrophobicity of Sec61-targeting signals 43 2.8 CT8-sensitive signals tend to have higher calculated free energies of integration (ΔGcalc) 50 2.9 ΔGsub can predict sensitivity to CT8 63 2.10 Discussion 69 viii 2.11 Experimental procedures 73 2.12 References 88 Chapter 3: Sec61α as a novel therapeutic target in multiple myeloma 95 3.1 Abstract 96 3.2 Introduction 96 3.3 CT8 specifically induces apoptosis in multiple myeloma cells 98 3.4 CT8 inhibits the expression of ER chaperones ERdj3 and p58ipk 103 3.5 CT8 does not robustly induce the UPR in myeloma cells 107 3.6 R66I Sec61α mutant partially rescues proliferation of myeloma cells treated with CT8 112 3.7 Discussion 117 3.8 Experimental procedures 121 3.9 References 130 Chapter 4: Conclusions and future directions 137 4.1 Conclusions and future directions 138 4.2 Experimental procedures 145 4.3 References 147 Appendix A: List of drug targets 150 Appendix B: Source code for fasta_segment.py 180 Appendix C: List of plasmids 183 ix Appendix D: Primers for in vitro transcription templates 185 Appendix E: List of all proteins with signal peptides in the top quartile of ΔGsub 187 Publishing Agreement 224 x List of Tables Table 2.1 CT8-sensitive proteins 33-36 Table 2.2 Amino acid values for the Kyte-Doolittle and Hopp-Woods 43 hydrophobicity scales Table 2.3 Summary of hydrophobicity values used to describe Sec61-targeting 49 signals Table 2.4 Type II membrane proteins identified by SILAC membrane proteomics 52-53 Table 2.5 Signal peptide-containing proteins identified by SILAC mass spectrometry and used for enrichment analysis 54-59 Table 4.1 Drug targets from Okada et al. with ΔGsub in the top 10% 139 xi List of Figures Figure 1.1 Overview of cotranslational translocation 4 Figure 1.2 Crystal structure of bacterial SecY 7 Figure 1.3 Structure of SecY in complex with a nascent chain 9 Figure 1.4 Cotransins inhibit gating of the Sec61 translocon 10 Figure 2.1 SILAC workflow 27 Figure 2.2 CT8-sensitive proteins are primarily Sec61-dependent 30 Figure 2.3 Calculation of the L/H ratio for a theoretical protein with an EC50 = 100 nM 31 Figure 2.4 CT8 inhibits a subset of secretory proteins 32 Figure 2.5 Cellular validation of CT8-sensitive proteins identified by mass spectrometry 36 Figure 2.6 CT8 inhibits membrane localization of Jak3 37 Figure 2.7 CT8 inhibits STAT5 phosphorylation 38 Figure 2.8 Validation of CT8-sensitive proteins in cell-free cotranslational translocation assays 40 Figure 2.9 CT8 inhibits TRPML1 expression 41 Figure 2.10 Mutation of TMD1 in TRPML1 results in resistance to CT8 42 Figure 2.11 Hydropathy plots for TRPML1 45 Figure 2.12 Experimental design for determining ΔGapp 48 Figure 2.13 CT8-sensitive type II signal anchors tend to have higher ΔGcalc values 50 Figure 2.14 CT8-sensitive signal peptides have significantly higher ΔGavg values 61 Figure 2.15 ΔGsub separates sensitive and resistant signal peptide populations better than ΔGavg 63 Figure 2.16 Ranking by ΔGsub significantly enriches for CT8-senstive proteins 64 xii Figure 2.17 CT8 inhibition of integrin αV expression drives loss of integrin β5 66 Figure 2.18 Distribution of ∆Gsub for SILAC dataset matches that of the secretome 67 Figure 2.19 ∆Gsub ranking of proteins predicted to be either sensitive or resistant to CT8 67 Figure 2.20 ∆Gsub of signal peptides can predict sensitivity to CT8 68 Figure 3.1 CT8 inhibits cell proliferation and induces cell death in myeloma cells 99 Figure 3.2 CT8 induces apoptosis in the myeloma cell line JJN-3 100 Figure 3.3 CT8 induces apoptosis in patient-derived primary CD138+ Myeloma cells