University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Spring 2010 Structural, Biochemical, and Cell Biological Characterization of Rab7 Mutants That Cause Peripheral Neuropathy Brett A. McCray University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Molecular and Cellular Neuroscience Commons Recommended Citation McCray, Brett A., "Structural, Biochemical, and Cell Biological Characterization of Rab7 Mutants That Cause Peripheral Neuropathy" (2010). Publicly Accessible Penn Dissertations. 145. https://repository.upenn.edu/edissertations/145 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/145 For more information, please contact [email protected]. Structural, Biochemical, and Cell Biological Characterization of Rab7 Mutants That Cause Peripheral Neuropathy Abstract Coordinated trafficking of intracellular vesicles is of critical importance for the maintenance of cellular health and homeostasis. Members of the Rab GTPase family serve as master regulators of vesicular trafficking, maturation, and fusion by reversibly associating with distinct target membranes and recruiting specific effector proteins. Rabs act as molecular switches by cycling between an active, GTP-bound form and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Rab7 specifically regulates the trafficking and maturation of vesicle populations that are involved in protein degradation including late endosomes, lysosomes, and autophagic vacuoles. Missense mutations of Rab7 cause a dominantly-inherited axonal degeneration known as Charcot-Marie-Tooth type 2B (CMT2B) through an unknown mechanism. Patients with CMT2B present with length-dependent degeneration of peripheral sensory and motor neurons that leads to weakness and profound sensory loss. To gain insight into the pathogenesis of CMT2B, we undertook extensive characterization of two disease-causing Rab7 mutants, L129F and V162M. We present the 2.8 Å crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. We further demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect as previously suggested, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Using an unbiased proteomics approach, we characterize effector interactions in wild-type and mutant Rab7 and identify several novel Rab7 interactors. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that increased activation of mutant Rab7 is counterbalanced by unregulated, GTP-hydrolysis-independent membrane cycling. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease by misregulation of native Rab7 activity. Future experiments will address the impact of Rab7 misregulation on neuronal trafficking and ophictr signaling. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Neuroscience First Advisor J. Paul Taylor Keywords rab7, charcot-marie-tooth, neurodegeneration, GTPase, peripheral neuropathy, neuropathy Subject Categories Molecular and Cellular Neuroscience This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/145 STRUCTURAL, BIOCHEMICAL, AND CELL BIOLOGICAL CHARACTERIZATION OF RAB7 MUTANTS THAT CAUSE PERIPHERAL NEUROPATHY Brett A. McCray A Dissertation In Neuroscience Presented to the Faculties of the University of Pennsylvania In Partial Fulfillment to the Requirements for the Degree of Doctor of Philosophy 2010 ____________________________ J. Paul Taylor, M.D., Ph.D. Dissertation Supervisor ____________________________ Rita Balice-Gordon, Ph.D. Graduate Group Chairperson Dissertation Committee Steven Scherer, M.D., Ph.D. Erika Holzbaur, Ph.D. Virginia M.-Y. Lee, Ph.D. Randall Pittman, Ph.D. STRUCTURAL, BIOCHEMICAL, AND CELL BIOLOGICAL CHARACTERIZATION OF RAB7 MUTANTS THAT CAUSE PERIPHERAL NEUROPATHY Copyright 2010 Brett A. McCray ABSTRACT STRUCTURAL, BIOCHEMICAL, AND CELL BIOLOGICAL CHARACTERIZATION OF RAB7 MUTANTS THAT CAUSE PERIPHERAL NEUROPATHY Brett A. McCray Supervisor: J. Paul Taylor Coordinated trafficking of intracellular vesicles is of critical importance for the maintenance of cellular health and homeostasis. Members of the Rab GTPase family serve as master regulators of vesicular trafficking, maturation, and fusion by reversibly associating with distinct target membranes and recruiting specific effector proteins. Rabs act as molecular switches by cycling between an active, GTP-bound form and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Rab7 specifically regulates the trafficking and maturation of vesicle populations that are involved in protein degradation including late endosomes, lysosomes, and autophagic vacuoles. Missense mutations of Rab7 cause a dominantly- inherited axonal degeneration known as Charcot-Marie-Tooth type 2B (CMT2B) through an unknown mechanism. Patients with CMT2B present with length-dependent degeneration of peripheral sensory and motor neurons that leads to weakness and profound sensory loss. To gain insight into the pathogenesis of CMT2B, we undertook iii extensive characterization of two disease-causing Rab7 mutants, L129F and V162M. We present the 2.8 Å crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. We further demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect as previously suggested, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Using an unbiased proteomics approach, we characterize effector interactions in wild-type and mutant Rab7 and identify several novel Rab7 interactors. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that increased activation of mutant Rab7 is counterbalanced by unregulated, GTP-hydrolysis-independent membrane cycling. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease by misregulation of native Rab7 activity. Future experiments will address the impact of Rab7 misregulation on neuronal trafficking and trophic signaling. iv TABLE OF CONTENTS Cover Page i Title Page ii Abstract iii Table of Contents v List of Tables viii List of Illustrations ix Chapter 1: Introduction Intracellular vesicular dynamics 2 Microtubules and vesicular trafficking 2 Specification of organelle identity 4 The endo-lysosomal pathway 6 The autophagy-lysosomal pathway 8 Rab GTPases 9 The Rab GTPase activity cycle 12 Rab7 and vesicular trafficking 17 Signaling endosomes and axonal transport 23 Retrograde axonal trafficking and disease 26 Charcot-Marie-Tooth disease 28 Charcot-Marie-Tooth disease type 2B 32 Chapter 2: Disease mutations in Rab7 result in unregulated nucleotide exchange and inappropriate activation Abstract 38 v Introduction 39 Materials and Methods 43 Results 55 Tables 73 Supplementary Data 75 Discussion 83 Chapter 3: Conclusions and Future Directions CMT2B disease mutations and misregulation of the Rab7 91 activity cycle Rab7 disease mutations and trafficking of signaling endosomes 93 Characterization of Rab7-effector interactions in neurons 98 Functional characterization of the Rab7-VapB interaction 100 Concluding remarks 104 Appendix 1: The role of autophagy in age-related neurodegeneration Abstract 111 Living may be hazardous to your health 112 Induction and maturation of autophagosomes 116 Regulation of autophagy 117 Multiple roles for autophagy: matters of life and death 118 Decline of autophagy with aging 120 Accumulation of autophagic vacuoles in patients and models of 122 neurodegeneration Autophagy is neuroprotective 124 vi Can autophagy be co-opted for therapeutic benefit? 127 Unanswered questions and future directions 128 Appendix 2: HDAC6 regulates the steady-state levels and turnover of polyglutamine-expanded androgen receptor Abstract 132 Introduction 134 Materials and Methods 136 Results and Discussion 139 References 143 vii LIST OF TABLES Chapter 3: Disease mutations in Rab7 result in unregulated nucleotide exchange and inappropriate activation Table 2.1. Data collection and refinement statistics 73 Table 2.2. Proteomics summary and statistics 74 viii LIST OF ILLUSTRATIONS Chapter 1: Introduction Fig. 1.1. Intersection of the endocytic and autophagic pathways 7 Fig. 1.2. Structure of the Rab GTPase Rab7