The development and application of mass-spectrometry-based tools to monitor proteome remodeling in microbes
Outside of controlled laboratory environments, cells are continually sensing and adapting to highly variable environmental conditions in an effort to maintain cellular homeostasis and to maximize fitness in each condition. Although specific stresses elicit distinct cellular responses, the reshaping...
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| Format: | Thesis |
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Massachusetts Institute of Technology
2024
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| Online Access: | https://hdl.handle.net/1721.1/157155 |
| _version_ | 1824458327450451968 |
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| author | Telusma, Bertina |
| author2 | Davis, Joseph H. |
| author_facet | Davis, Joseph H. Telusma, Bertina |
| author_sort | Telusma, Bertina |
| collection | MIT |
| description | Outside of controlled laboratory environments, cells are continually sensing and adapting to highly variable environmental conditions in an effort to maintain cellular homeostasis and to maximize fitness in each condition. Although specific stresses elicit distinct cellular responses, the reshaping of the proteome is a central element in most cellular adaptation. This dynamic proteome remodeling involves a highly orchestrated combination of regulated proteins synthesis, degradation, and modification, each contributing to the overall goal of matching the capacity of the expressed proteome to the demands of the sensed environment. Although each pathway will contribute, ultimately, whether cells mount a response primarily driven by synthesis or by degradation hinges on the nature and duration of the stress, as well as the cell type involved. Understanding the balance of these contributions has historically been challenging. As such, there is a need for approaches that can quantitatively resolve the contributions of protein synthesis and protein degradation pathways in a wide array of cellular and environmental contexts.
Quantitative proteomics via mass spectrometry stands out as a powerful tool for deciphering these questions, as it allows one to simultaneously monitor thousands of proteins. In this work, I leverage the power of quantitative proteomics coupled with metabolic labeling to investigate how microbes remodel their proteome during cellular adaptation. In chapter 2, I describe the development and characterization of these proteomic methods, including a detailed analysis of the variety of metabolic labeling schemes that can be employed in budding yeasts, which facilitate the bulk of my thesis work. In chapters 3 and 4, I apply these methods to the methylotrophic yeast, Komagataella phaffii, which grows robustly on a diverse set of carbon sources. As such, I use K. phaffii as a key case study to explore questions of cellular adaptation. I find that the K. phaffii expressed proteome varies greatly between cells grown in methanol, oleate, or glucose and, interestingly, that proteome remodeling strategies vary in a context-dependent manner. Specifically, I find that autophagic degradation drives proteome remodeling under nitrogen starvation conditions, with selective autophagic degradation of peroxisome supporting the cells transition from methanol media to glucose media. In contrast, I uncover that synthesis and growth-coupled dilution is the primary driver as K. phaffii adapts from methanol media to oleate media. Given the deep proteome coverage enabled by my methods, and my application of these methods in a wide variety of genetic backgrounds (6) and environmental conditions (5), these datasets also serve as a rich resource to identify conditions stimulating degradation of specific proteins, as well as the genetically defined pathways supporting these activities. Finally, in appendices 1 and 2, I highlight how these approaches can be applied across different microbial species to broadly characterize the proteomic consequences of nutrient and genetic perturbations. Overall, my work highlights how the development and application of powerful quantitative methods provide a global view of how proteome remodeling supports cellular adaptation, reveal deeper insights into pathways supporting turnover of specific proteins, and help to identify potential therapeutic targets to ameliorate protein-turnover related diseases. |
| first_indexed | 2025-02-19T04:24:08Z |
| format | Thesis |
| id | mit-1721.1/157155 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2025-02-19T04:24:08Z |
| publishDate | 2024 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/1571552024-10-10T03:16:20Z The development and application of mass-spectrometry-based tools to monitor proteome remodeling in microbes Telusma, Bertina Davis, Joseph H. Massachusetts Institute of Technology. Department of Biology Outside of controlled laboratory environments, cells are continually sensing and adapting to highly variable environmental conditions in an effort to maintain cellular homeostasis and to maximize fitness in each condition. Although specific stresses elicit distinct cellular responses, the reshaping of the proteome is a central element in most cellular adaptation. This dynamic proteome remodeling involves a highly orchestrated combination of regulated proteins synthesis, degradation, and modification, each contributing to the overall goal of matching the capacity of the expressed proteome to the demands of the sensed environment. Although each pathway will contribute, ultimately, whether cells mount a response primarily driven by synthesis or by degradation hinges on the nature and duration of the stress, as well as the cell type involved. Understanding the balance of these contributions has historically been challenging. As such, there is a need for approaches that can quantitatively resolve the contributions of protein synthesis and protein degradation pathways in a wide array of cellular and environmental contexts. Quantitative proteomics via mass spectrometry stands out as a powerful tool for deciphering these questions, as it allows one to simultaneously monitor thousands of proteins. In this work, I leverage the power of quantitative proteomics coupled with metabolic labeling to investigate how microbes remodel their proteome during cellular adaptation. In chapter 2, I describe the development and characterization of these proteomic methods, including a detailed analysis of the variety of metabolic labeling schemes that can be employed in budding yeasts, which facilitate the bulk of my thesis work. In chapters 3 and 4, I apply these methods to the methylotrophic yeast, Komagataella phaffii, which grows robustly on a diverse set of carbon sources. As such, I use K. phaffii as a key case study to explore questions of cellular adaptation. I find that the K. phaffii expressed proteome varies greatly between cells grown in methanol, oleate, or glucose and, interestingly, that proteome remodeling strategies vary in a context-dependent manner. Specifically, I find that autophagic degradation drives proteome remodeling under nitrogen starvation conditions, with selective autophagic degradation of peroxisome supporting the cells transition from methanol media to glucose media. In contrast, I uncover that synthesis and growth-coupled dilution is the primary driver as K. phaffii adapts from methanol media to oleate media. Given the deep proteome coverage enabled by my methods, and my application of these methods in a wide variety of genetic backgrounds (6) and environmental conditions (5), these datasets also serve as a rich resource to identify conditions stimulating degradation of specific proteins, as well as the genetically defined pathways supporting these activities. Finally, in appendices 1 and 2, I highlight how these approaches can be applied across different microbial species to broadly characterize the proteomic consequences of nutrient and genetic perturbations. Overall, my work highlights how the development and application of powerful quantitative methods provide a global view of how proteome remodeling supports cellular adaptation, reveal deeper insights into pathways supporting turnover of specific proteins, and help to identify potential therapeutic targets to ameliorate protein-turnover related diseases. Ph.D. 2024-10-09T18:25:01Z 2024-10-09T18:25:01Z 2024-09 2024-09-26T01:22:59.910Z Thesis https://hdl.handle.net/1721.1/157155 0000-0002-6804-9527 In Copyright - Educational Use Permitted Copyright retained by author(s) https://rightsstatements.org/page/InC-EDU/1.0/ application/pdf Massachusetts Institute of Technology |
| spellingShingle | Telusma, Bertina The development and application of mass-spectrometry-based tools to monitor proteome remodeling in microbes |
| title | The development and application of mass-spectrometry-based tools to monitor proteome remodeling in microbes |
| title_full | The development and application of mass-spectrometry-based tools to monitor proteome remodeling in microbes |
| title_fullStr | The development and application of mass-spectrometry-based tools to monitor proteome remodeling in microbes |
| title_full_unstemmed | The development and application of mass-spectrometry-based tools to monitor proteome remodeling in microbes |
| title_short | The development and application of mass-spectrometry-based tools to monitor proteome remodeling in microbes |
| title_sort | development and application of mass spectrometry based tools to monitor proteome remodeling in microbes |
| url | https://hdl.handle.net/1721.1/157155 |
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