Thesis advisor: Abhishek Chatterjee / Noncanonical amino acid (ncAA) mutagenesis provides powerful new ways to probe and manipulate protein function both in vitro and in living cells. Increasing the number of ncAAs that can be site-specifically encoded can greatly expand the scope of this promising technology. We aimed to address the challenges that limit the multisite ncAA incorporation technology in both Escherichia coli and mammalian cells. Our work has significantly expanded the scope of this technology through the development of mutually compatible suppression systems and the optimization of their expression. Using these platforms, we further demonstrate powerful new applications of dual-ncAA incorporation technology both in E. coli and mammalian cells. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

With the objective to functionalize proteins for the understanding of their biological roles and developing protein-based biosensors, I have been developing methods to synthesize proteins with defined modifications and applying them to study protein functional roles and generate proteins with new properties. These methods rely on the read-through of an in-frame stop codon in mRNA by a nonsense suppressor tRNA specifically acylated with a noncanoncial amino acid (NAA) by a unique aminoacyl-tRNA synthetase and the genetic incorporation of this NAA at the stop codon site. NAAs either provide chemical handles for site-specific manipulation or mimic the posttranslational modifications, which are critical for understanding cellular regulations and signal transduction. The pyrrolysine synthetase (PylRS) has been wildly used to incorporate NAAs into proteins in E. coli. Taking advantage of PylRS, I have developed method to genetically incorporate ketone-containing N--acetyl-L-lysine analog, 2-amino-8-oxononanoic acid (KetoK), into proteins for their site-specific modifications and used it to mimic the protein lysine acetylation process. I have also modified the ribosome in order to improve the amber suppression efficiency and therefore to achieve incorporation of multiple copies of NAA into one protein. By overexpressing a truncated ribosomal protein, L11C, I have demonstrated 5-fold increase of amber suppression level in E. coli, leading to higher expression levels for proteins incorporated with NAAs. I have also demonstrated this method can be applied successfully to incorporate at least 3 NAAs into one protein in E. coli. With the success of incorporating multiple NAAs into one protein, I have further introduced two distinct NAAs into one protein simultaneously. This is done by using a wild type or evolved PylRS-pylTUUA pair and an evolved M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS)-tRNACUA pair. By suppressing both UAG and UAA stop codons in one mRNA, a protein incorporated with two NAAs is synthesized with a decent yield. There is of great interest to incorporate new NAAs into proteins, which is done by library selection. By introducing both positive and negative selective markers into one plasmid, I have developed a one-plasmid selection method. In this method, the positive and negative selections are accomplished by in a single type of cells hosting a single selection plasmid.

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Thesis advisor: Abhishek Chatterjee / Thesis advisor: Eranthie Weerapana / Over the past twenty years, the site-specific incorporation of unnatural amino acids (UAAs) into a target protein through genetic code expansion (GCE) has emerged as one of the foremost technologies to selectively modify proteins in their native cellular context. This technology relies on engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that are orthogonal to the host cells’ endogenous aaRS/tRNA pairs. Traditionally, scientists look towards evolutionarily distant domains of life to identify orthogonal aaRS/tRNA pairs that can be further engineered for GCE applications in the host system. For example, bacterial aaRS/tRNA pairs are used for GCE in eukaryotes. The directed evolution of orthogonal aaRS/tRNA pairs for eukaryotic GCE has been less fortuitous due to the cumbersome nature of established yeast-based selection platforms. Recently, our lab circumvented this platform-based limitation by developing “altered translational machinery” (ATM) Escherichia coli strains that enabled the directed evolution of bacterial aaRS/tRNA pairs for eukaryotic GCE applications. In the ATM-tyrosyl (ATMY) E. coli strain, reintroduction of the E. coli tyrosyl-tRNA (tRNAEcTyrCUA) as a nonsense suppressor led to cross-reactivity with the endogenous E. coli glutaminyl-tRNA synthetase (EcGlnRS), restricting the activity range of aaRSs that could be selected, ultimately diminishing the scope of incorporable UAAs. To recover the dynamic range of this platform, cross-reactivity of the tRNAEcTyrCUA was eliminated through directed evolution of the tRNA acceptor stem. This new, orthogonal tRNA revealed weak mutant aaRSs whose suppression efficiencies were boosted through additional rounds of directed evolution. Improved aaRS mutants exhibited higher solubility, thermal stability, and suppression efficiency than their predecessor. While the newly engineered, orthogonal tRNAEcTyrCUA gave access to novel aaRS/tRNA pairs for eukaryotic GCE, some notable UAAs were still missing that could be incorporated with the archaeal Methanococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair in bacteria. Following a systematic investigation into the discrepancy between the E. coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA and MjTyrRS/tRNA pairs, we found that it can be partially attributed to the low structural robustness of the EcTyrRS. This limitation was overcome by rationally designing chimeric TyrRSs composed of EcTyrRS and a structural homologue from the thermophilic bacterium Geobacillus stearothermophilus. The chimeric scaffolds demonstrated enhanced stability, activity, and resilience to destabilizing active site mutations, offering a potentially more attractive scaffold for GCE. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Hypereosinophilic (HES) syndrome is an umbrella term encompassing several disease subsets that affects humans and veterinary species, ultimately resulting in >1,500 eosinophils/uL circulating in the blood documented over six-months. This eventually culminates in end-organ infiltration and increased patient morbidity and mortality. In mice where the gene Map3k14 encoding NF -kB inducing kinase (NIK) is knocked out, a HES-like syndrome develops that is dependent on Th2 cells and cytokines. NIK is the upstream regulator of the noncanonical NF-kB pathway and is involved in lymphoid organ development, B cell lymphopoiesis, and myelopoiesis. In addition to regulating the noncanonical NF-kB pathway, NIK is also involved in regulation of kB dimers of the canonical NF-kB pathway and can function independent of NF-kB signaling by regulating lipid and glucose metabolism, mitochondrial, and RIP1 binding to influence cell survival and death. Despite previous studies performed in the Nik-/- model, the mechanisms underlying eosinophil development, plasticity, and fitness in conjunction with the bone marrow and splenic microenvironments have not been fully elucidated. In the present work, we reviewed current data exploring the influence of the noncanonical NF-kB pathway and NIK specifically on the development of acute myeloid leukemias (AMLs) and Myelodysplastic Syndrome (MDS) with a focus on how these mechanisms might induce subvariants of HES. We next examined the effect of NIK loss on eosinophilopoiesis within hematopoietic tissues in vivo and in various cell culture environments in vitro via cytology, histology, flow cytometry, FACS, positive cell selection, MTT assay, BrDU assay, and protein microarray analysis. Overall, our findings suggest that NIK influences eosinophil maturation, proliferation, metabolism, survival, and potentially plasticity in vivo and in vitro under different environmental conditions and Th2 cytokine influence. NIK loss was also associated with altered free and bound TNFR1 levels on day 13 in vitro. TNFR1 acts upstream of RIP1 and suggests that these differences may be due to NF-kB independent functions of NIK. Overall, these results provide further insight into the potential mechanisms underlying eosinophilopoiesis in the Nik-/- murine model. This information may prove useful in discovering new treatment options underlying subvariants of HES in both human and veterinary patients. / Doctor of Philosophy / A less well-known albeit important white blood cell (WBC) is the eosinophil. It is essential for combating parasitic infections but is also involved in allergic responses. Hypereosinophilic Syndrome (HES) is an umbrella term encompassing a variety of diseases that affects human and veterinary patients. It results in an overproduction of eosinophils not associated with parasitic infections or allergic responses. Although several variants of the disease exist, diagnosing subsets of HES poses a diagnostic challenge and can impact patient care and prognosis long term. Mice carrying a genetic deletion (Map3k14) encoding a kinase known as NF-kB inducing kinase (NIK) develop HES-like syndrome; these mice are hereafter referred to as Nik-/- mice. HES-like syndrome in Nik-/- mice develops secondary to NIK loss in Th2 lymphocytes (another type of WBC). NIK is an upstream regulator of the noncanonical NF-kB pathway that influences WBC, lymph node, and spleen development. NIK also regulates canonical NF-kB molecules and can function independent of NF-kB signaling by impacting fat and glucose metabolism, binding to mitochondria, and interacting with a kinase known as RIP1 to regulate cell death or survival. Despite previous work studying HES-like syndrome in Nik-/- mice, the specifics of the bone marrow and eosinophil development in response to Th2 cells have not been fully characterized. In the present work, we reviewed data exploring the noncanonical NF-kB pathway and NIK specifically in the development of WBC cancers and how this might manifest as HES. We then studied eosinophil development in the bone marrow, spleen and in culture for both Nik-/- and wild-type mice by assessing cell and tissue morphology, cell surface marker expression, response to Th2 signaling molecules, as well as cell maturation, death, metabolism, proliferation, and cytokine production. Our findings suggest that NIK is essential for eosinophil growth and survival. We also noted differences in a molecule known as TNFR1 in Nik-/- cultures on day 13. This molecule acts upstream of RIP1, suggesting an NF-kB independent function of NIK in regulating eosinophil maturation in response to Th2 molecules. This information may prove useful in discovering new treatments for HES in human and veterinary patients.

Background microRNA-23b (miR-23b) is a multiple functional miRNA. We hypothesize that miR-23b plays a role in the pathogenesis of sepsis. Our study investigated the effect of miR-23b on sepsis-induced immunosuppression. Methods Mice were treated with miR-23b inhibitors by tail vein injection 2 days after cecal ligation puncture (CLP)-induced sepsis. Apoptosis in spleens and apoptotic signals were investigated, and survival was monitored. T-cell immunoreactivities were examined during late sepsis. Nuclear factor B (NF-B)-inducing kinase (NIK), tumor necrosis factor (TNF)-receptor associated factor 1 (TRAF1), and X-linked inhibitor of apoptosis protein (XIAP), the putative targets of miR-23b, were identified by a dual-luciferase reporter assay. Results miR-23b expression is upregulated and sustained during sepsis. The activation of the TLR4/TLR9/p38 MAPK/STAT3 signal pathway contributes to the production of miR-23b in CLP-induced sepsis. miR-23b inhibitor decreased the number of spleen cells positive by terminal deoxynucleotidyl transferase dUTP nick-end labeling and improved survival. miR-23b inhibitor restored the immunoreactivity by alleviating the development of T-cell exhaustion and producing smaller amounts of immunosuppressive interleukin 10 and interleukin 4 during late sepsis. We demonstrated that miR-23b mediated immunosuppression during late sepsis by inhibiting the noncanonical NF-B signal and promoting the proapoptotic signal pathway by targeting NIK, TRAF1, and XIAP. Conclusions Inhibition of miR-23b reduces late-sepsis-induced immunosuppression and improves survival. miR-23b might be a target for immunosuppression.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第18384号 / エネ博第296号 / 新制||エネ||61(附属図書館) / 31242 / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 岸本 泰明, 教授 中村 祐司, 教授 松田 一成 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

Thesis advisor: Abhishek Chatterjee / The emergence of genetic code expansion (GCE) technology, which enables sitespecific incorporation of unnatural amino acids (UAAs) into proteins, has facilitated powerful new ways to probe and engineer protein structure and function. Using engineered orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs that suppress repurposed nonsense codons, a variety of structurally diverse UAAs have been incorporated into proteins in living cells. This technology offers tremendous potential for deciphering the complex biology of eukaryotes, but its scope in eukaryotic systems remains restricted due to several technical limitations. For example, development of the engineered tRNA/aaRS pairs for eukaryotic GCE traditionally relied on a eukaryotic cell-based directed evolution system, which are significantly less efficient relative to bacteria-based engineering platforms. The work described in this thesis establishes a new paradigm in GCE through the development of a novel class of universal tRNA/aaRS pairs, which can be used for ncAA incorporation in both E. coli and eukaryotes. We achieve this by developing engineered strains of E. coli, where one of its endogenous tRNA/aaRS pair is functionally replaced with an evolutionarily distant counterpart. The liberated pair can then be used for GCE in the resulting altered translational machinery (ATM) strain, as well as any eukaryote. Using this strategy, we have been able to genetically encode new bioconjugation chemistries, post-translational modifications, and facilitate the incorporation of multiple, distinct ncAAs into a single protein. The ATM technology holds enormous promise for significantly expanding the scope of the GCE technology in both bacteria and eukaryotes. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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