Role of the Bifunctional Aminoacyl-tRNA Synthetase EPRS in Human Disease

Aminoacyl-tRNA synthetases (AARS) are a class of enzymes that catalyze the charging of tRNAs with cognate amino acids, a critical step that contributes to the fidelity of protein synthesis. Many AARSs also possess noncanonical functions such as regulation of apoptosis, mRNA translation, and RNA splicing. Some AARSs have evolved new domains with no apparent connection to their charging functions. For example, WHEP domains were originally identified in tryptophanyl-tRNA synthetase (WRS), histidyl-tRNA synthetase (HRS), and glutamyl-prolyl-tRNA synthetase (EPRS). EPRS is a unique bifunctional AARS, found only in higher eukaryotes, and consists of glutamyl-tRNA synthetase (ERS) and prolyl-tRNA synthetase (PRS) joined by a non-catalytic linker containing three WHEP domains in humans. Two compound heterozygous point mutations within human ERS (P14R and E205G) have been identified in the genomes of two patients with type 1 diabetes and bone disease. However, the mechanism by which these mutations contribute to disease is unknown. Our goal is to determine whether the point mutations affect the canonical catalytic activity of EPRS responsible for tRNA charging or noncanonical functions. Both P14 and E205 are highly conserved residues located in the GST and catalytic domain, respectively. An ERS variant appended to 2.5 WHEP domains (ERS 2.5W) has been purified and shown to display robust tRNA binding and aminoacylation activity in vitro. The P14R and E205G single mutants display the same binding affinity for tRNAGlu as WT ERS 2.5W, suggesting that the observed defect is at the catalytic step. Whereas the ERS 2.5W P14R mutant has near wild-type (WT) aminoacylation activity, the ERS 2.5W E205G variant has a severe aminoacylation defect. Both mutations, however, lead to reduced amino acid activation. Together with a collaborator, we are currently characterizing the effect of these two mutations on cell proliferation and the integrated stress response. Taken together, this work has important implications for the understanding of AARS-related human disease mechanisms and development of new therapeutics.College of Arts & SciencesOffice of Undergraduate Research & Creative InquiryNo embargoAcademic Major: Biochemistr

Aminoacyl tRNA synthetase complex interacting multifunctional protein 1 simultaneously binds Glutamyl-Prolyl-tRNA synthetase and scaffold protein aminoacyl tRNA synthetase complex interacting multifunctional protein 3 of the multi-tRNA synthetase complex

Higher eukaryotes have developed extensive compartmentalization of amino acid (aa) - tRNA coupling through the formation of a multi-synthetase complex (MSC) that is composed of eight aa-tRNA synthetases (ARS) and three scaffold proteins: aminoacyl tRNA synthetase complex interacting multifunctional proteins (AIMP1, 2 and 3). Lower eukaryotes have a much smaller complex while yeast MSC consists of only two ARS (MetRS and GluRS) and one ARS cofactor 1 protein, Arc1p (Simos et al., 1996), the homolog of the mammalian AIMP1. Arc1p is reported to form a tripartite complex with GluRS and MetRS through association of the N-terminus GST-like domains (GST-L) of the three proteins (Koehler et al., 2013). Mammalian AIMP1 has no GST-L domain corresponding to Arc1p N-terminus. Instead, AIMP3, another scaffold protein of 18 kDa composed entirely of a GST-L domain, interacts with Methionyl-tRNA synthetase (MARS) (Quevillon et al., 1999) and Glutamyl-Prolyl-tRNA Synthetase (EPRS) (Cho et al., 2015). Here we report two new interactions between MSC members: AIMP1 binds to EPRS and AIMP1 binds to AIMP3. Interestingly, the interaction between AIMP1 and AIMP3 complex makes it the functional equivalent of a single Arc1p polypeptide in yeast. This interaction is not mapped to AIMP1 N-terminal coiled-coil domain, but rather requires an intact tertiary structure of the entire protein. Since AIMP1 also interacts with AIMP2, all three proteins appear to compose a core docking structure for the eight ARS in the MSC complex

Nuclear Photosynthetic Gene Expression Is Synergistically Modulated by Rates of Protein Synthesis in Chloroplasts and Mitochondria

Arabidopsis thaliana mutants prors1-1 and -2 were identified on the basis of a decrease in effective photosystem II quantum yield. Mutations were localized to the 5'-untranslated region of the nuclear gene PROLYL-tRNA SYNTHETASE1 (PRORS1), which acts in both plastids and mitochondria. In prors1-1 and -2, PRORS1 expression is reduced, along with protein synthesis in both organelles. PRORS1 null alleles (prors1-3 and -4) result in embryo sac and embryo development arrest. In mutants with the leaky prors1-1 and -2 alleles, transcription of nuclear genes for proteins involved in photosynthetic light reactions is downregulated, whereas genes for other chloroplast proteins are upregulated. Downregulation of nuclear photosynthetic genes is not associated with a marked increase in the level of reactive oxygen species in leaves and persists in the dark, suggesting that the transcriptional response is light and photooxidative stress independent. The mrpl11 and prpl11 mutants are impaired in the mitochondrial and plastid ribosomal L11 proteins, respectively. The prpl11 mrpl11 double mutant, but neither of the single mutants, resulted in strong downregulation of nuclear photosynthetic genes, like that seen in leaky mutants for PRORS1, implying that, when organellar translation is perturbed, signals derived from both types of organelles cooperate in the regulation of nuclear photosynthetic gene expression

The Zinc-binding Domain of Mammalian prolyl-tRNA synthetase is Indispensable for Catalytic Activity and Organism Viability

Aminoacyl-tRNA synthetases (AARS) participate in decoding the genome by catalyzing conjugation of amino acids to their cognate tRNAs. During evolution, biochemical and environmental conditions markedly influenced the sequence and structure of the 20 AARSs, revealing adaptations dictating canonical and orthogonal activities. Here, we investigate the function of the appended Zn2+-binding domain (ZBD) in the bifunctional AARS, glutamyl-prolyl-tRNA synthetase (GluProRS). We developed GluProRS mutant mice by CRISPR-Cas9 with a deletion of 29 C-terminal amino acids, including two of four Zn2+-coordinating cysteines. Homozygous ZBD mutant mice die before embryonic day 12.5, but heterozygous mice are healthy. ZBD disruption profoundly reduces GluProRS canonical function by dual mechanisms: it induces rapid proteasomal degradation of the protein and inhibits ProRS aminoacylation activity, likely by suboptimal positioning of ATP in the spatially adjacent catalytic domain. Collectively, our studies reveal the ZBD as a critical determinant of ProRS activity and GluProRS stability in vitro and in vivo

Segmentally Variable Genes: A New Perspective on Adaptation

Genomic sequence variation is the hallmark of life and is key to understanding diversity and adaptation among the numerous microorganisms on earth. Analysis of the sequenced microbial genomes suggests that genes are evolving at many different rates. We have attempted to derive a new classification of genes into three broad categories: lineage-specific genes that evolve rapidly and appear unique to individual species or strains; highly conserved genes that frequently perform housekeeping functions; and partially variable genes that contain highly variable regions, at least 70 amino acids long, interspersed among well-conserved regions. The latter we term segmentally variable genes (SVGs), and we suggest that they are especially interesting targets for biochemical studies. Among these genes are ones necessary to deal with the environment, including genes involved in host–pathogen interactions, defense mechanisms, and intracellular responses to internal and environmental changes. For the most part, the detailed function of these variable regions remains unknown. We propose that they are likely to perform important binding functions responsible for protein–protein, protein–nucleic acid, or protein–small molecule interactions. Discerning their function and identifying their binding partners may offer biologists new insights into the basic mechanisms of adaptation, context-dependent evolution, and the interaction between microbes and their environment. Segmentally variable genes show a mosaic pattern of one or more rapidly evolving, variable regions. Discerning their function may provide new insights into the forces that shape genome diversity and adaptationNational Science Foundation (998088, 0239435

글루타밀-프롤릴 tRNA 합성효소의 폐 및 간 섬유화에서 STAT Signaling을 통한 Collagen과 Fibronectin의 발현 조절 연구

학위논문 (박사)-- 서울대학교 대학원 : 약학대학 약학과, 2019. 2. 이정원.섬유화란 염증성 및 대사성 질환에 의해 세포의 과분열과 장기의 기능부전이 일어나 세포외기질 (extracellular matrix, ECM) 이 비정상적으로 과다하게 축적되는 과정을 말한다. 최근 연구에 따르면 상산(常山, Dichroa febrifuga) 으로부터 분리된 화합물 febrifugine의 할로겐화 유도체인 halofuginone은 glutamyl-prolyly tRNA synthetase (EPRS)의 활성을 억제함으로써 섬유화를 저해한다고 밝혀졌다. 하지만 기존 연구에서는 halofuginone이 EPRS의 활성을 저해함에 있어서 EPRS의 번역적 활성에만 초점을 맞추었으며 in vivo 환경에서는 확인한 바 없으므로 EPRS를 타겟으로한 섬유화 치료제 개발을 위해서는 추가 연구가 필요한 상황이다. 본 연구에서는 폐 및 간의 섬유화 과정에서 ECM을 합성하는 EPRS의 비-번역적 기능을 연구하고 EPRS의 hetero knock-out 마우스를 이용하여 EPRS의 in vivo에서의 역할을 검증하고자 하였다. 우선 섬유화 과정 중 ECM 합성에 있어서 EPRS의 기능을 확인하고자 하였다. EPRS가 knock-down(KD) 되거나 overexpression 된 hepatic stellate cell (LX2 cell) 및 alveolar epithelial cell (A549 cell)에 TGFβ1을 처리하여 섬유화를 유도시킨 후 ECM의 발현을 살펴보았다. 그 결과 EPRS가 KD 된 세포에서는 Collagen I, Fibronectin, Laminin 과 같은 ECM의 발현이 감소했지만 PRS가 과발현된 세포에서는 ECM의 발현이 증가한 것을 관찰할 수 있었다. 이는 western blotting을 통한 단백질 수준뿐만 아니라 qPCR 방식을 이용한 mRNA level 모두 비슷한 양상을 관찰할 수 있었다. 또한, promoter luciferase assay를 통해 EPRS가 KD된 세포에서 collagen I 및 laminin γ2의 promoter가 down-regulation 되는 것을 확인할 수 있었으므로 EPRS는 ECM 발현의 전사적인 수준에서 수행함을 알 수 있었다. 다음으로 EPRS 가 매개된 ECM 의 합성 조절의 신호전달을 관찰하기 위하여 섬유화와 밀접한 관계가 있다고 알려진 STAT 분자들의 인산화 정도를 분석하였다. 그 결과 STAT6가 EPRS의 발현과 함께 EPRS가 과발현 되었을 때 인산화가 증가되고 EPRS가 KD 될 때 인산화가 저해 되는 것으로 관찰되었다. 또한 SMAD2 단백질이 과발현 될 경우 STAT이 activation되어 SMAD가 STAT의 상위 신호전달에 관여함을 알 수 있었다. Immunoprecipitation 방법을 이용한 실험 결과 TGFβ1 receptor, EPRS, Janus kinase 그리고 STAT6 사이에 단백질-단백질 결합이 이루어 짐으로써 신호전달이 이루어 짐을 확인할 수 있었다. CCl4 처리 혹은 bile duct ligation 방법을 통해 간 섬유화를 유도하거나 bleomycin을 처리하여 폐 섬유화를 유도한 동물 실험 결과 fibrotic septa 주변에 STAT6의 인산화와 ECM의 발현이 동시에 증가 한 반면, 그 현상이 Eprs-/+ 마우스에서는 정도가 낮음을 알 수 있었다. 따라서 본 연구 결과 섬유화 과정에서 EPRS는 TGFβ1의존적으로 세포신호전달에 의한 비-번역적 과정을 거처 ECM의 발현을 조절하는 것을 확인 할 수 있었다. EPRS 단백질은 향후 간 및 폐 섬유화 억제 치료제의 타겟으로 활용 될 수 있을 것으로 기대된다.Background: Fibrosis is characterized by the increased accumulation of extracellular matrix (ECM), which drives abnormal cell proliferation and progressive organ dysfunction in many inflammatory and metabolic diseases. Studies have shown that halofuginone, a racemic halogenated derivative of febrifugine, purified from Dichroa febrifuga, inhibits glutamyl-prolyl-tRNA-synthetase (EPRS)-mediated fibrosis. However, the mechanism by which this occurs was only focused on the translational function of EPRS and in vivo efficacies were not studied. Thus, in order to develop efficacious drugs targeting EPRS, more studies are needed. Methods: In this study, I explored the mechanistic aspects of how EPRS could develop hepatic and pulmonary fibrotic phenotypes in cells and animal models. CCl4 administration, bile duct ligation operation, and bleomycin administration were used in order to induce fibrosis in wild type (Eprs+/+) or Eprs-/+ C57B/L6 mice. Results: Treatment of transforming growth factor 1 (TGF1) up-regulated extracellular matrix proteins, including fibronectin and collagen I, in LX2 hepatic stellate cells and A549 alveolar epithelial cells. This effect was inhibited in EPRS-suppressed cells and enhanced in PRS-overexpressed cells. Using the promoter luciferase assay, TGF1-mediated COL1A1 (collagen I, 1 chain) and LAMC2 (laminin 2) transcription in LX2 and A549 cells were down-regulated by EPRS suppression, suggesting that EPRS may play roles in ECM production at transcriptional levels. Furthermore, signal transducer and activator of transcription (STAT) signaling activation was involved in the effects of TGF1 on ECM expression in an EPRS-dependent manner. This was mediated via a protein-protein complex formation consisting of TGF1 receptor, EPRS, Janus kinases, and STATs. Additionally, ECM expression in fibrotic livers and lungs were overlapped with EPRS expression along fibrotic septa regions and was positively correlated with STAT6 activation in fibrosis mouse models. This was less obvious in livers and lungs of Eprs-/+ mice. Conclusion: These findings suggest that during fibrosis development, EPRS plays roles in non-translational processes of ECM expression via the TGF1/STAT signaling pathway. Therefore, EPRS can be used as a potential target to develop anti-fibrosis treatments.ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES vii LIST OF TABLES xi LIST OF ABBREBIATIONS xii BACKGROUND 1 ⅰ. Fibrosis 2 ⅱ. Hepatic fibrosis 4 ⅲ. Pulmonary fibrosis 8 ⅳ. TGFβ1 Pathway 11 ⅴ. EPRS 13 ⅵ. Halofuginone 22 CHAPTER 1. GLUTAMYL-PROLYL-TRNA SYNTHETASE INDUCES FIBROTIC EXTRACELLULAR MATRIX VIA BOTH TRANSCRIPTIONAL AND TRANSLATIONAL MECHANISMS 26 1.1. ABSTRACT 27 1.2. INTRODUCTION 28 1.3. MATERIALS AND METHODS 31 Reagents and plasmids 31 Cell culture 31 Western blot analysis 32 ECM deposition assay and collagen footprint assay 32 Quantitative reverse-transcription (qRT) PCR 33 Co-immunoprecipitation 33 Luciferase assay 34 Animal experiments 34 Liver organoid culture 35 Immunohistochemistry and staining 35 1.4. RESULTS 39 Inhibition or suppression of EPRS in LX2 HSCs decreased the production and deposition of ECMs under TGF1 signaling 39 EPRS-mediated transcriptional regulation of ECMs involved STAT6 activation 42 EPRS-mediated signaling occurred downstream of TGF1 44 In vivo liver tissues from fibrotic mice showed EPRS-dependent STAT6 phosphorylation and ECM production 45 Liver organoids in a three-dimensional (3D) Matrigel system revealed EPRS-dependent regulation of ECM induction 47 1.5. DISCUSSION 65 CHAPTER 2. GLUTAMYL-PROLYL-TRNA SYNTHETASE REGULATES EPITHELIAL EXPRESSION OF MESENCHYMAL MARKERS AND EXTRACELLULAR MATRIX PROTEINS: IMPLICATIONS FOR IDIOPATHIC PULMONARY FIBROSIS 69 2.1. ABSTRACT 70 2.2. INTRODUCTION 71 2.3. MATERIALS AND METHODS 73 Reagents and plasmids 73 Cell culture 73 Western blot analysis 74 qRT-PCR 74 Co-immunoprecipitation 74 Luciferase assay 75 Animal experiments 75 Immunohistochemistry and staining 76 Statistics 76 2.4. RESULTS 79 EPRS expression regulated ECM production in A549 alveolar type II cells upon TGF1 stimulation 79 Regulation of TGFβ1-induced ECM protein synthesis by EPRS occurred via STAT activation 80 TGFβ1-mediated SMAD3 phosphorylation upregulated phosphorylation of STAT6 depending on EPRS expression. 81 EPRS-mediated signaling in TGF1-treated cells involved the formation of a multi-protein complex consisting of STAT6 and TGF1R 81 Lung tissues from bleomycin-treated mice showed EPRS-dependent STAT6 phosphorylation and ECM protein production in vivo 82 2.5. DISCUSSION 93 CONCLUSIONS & PERSPECTIVES 97 REFERENCES 101 ABSTRACT IN KOREAN 112Docto

Analysis of the Role of Aminoacyl tRNA Synthetase Genes in Global Protein Synthesis and mRNA Specific Regulation of Translation in Cancer Cells

Analysis of the Role of Aminoacyl tRNA Synthetase Genes in Global Protein Synthesis and mRNA Specific Regulation of Translation in Cancer Cells Elyse Nguyen, Depts. of Biology and Chemistry, Dipak Poria, & Esta Sterneck, with Dr. Sarah Williams, Dept. of Forensic Science Coordinated control of transcription and translation of gene expression impels cellular fate decision under different microenvironmental stresses. Cancer cells often usurp these regulatory machineries to adapt under microenvironmental stress or under therapeutic intervention. The transcription factor CEBPδ is induced by various stressors and mediates cellular adaptation and survival. RNA-seq analysis of a CEBPD-silenced human melanoma cell line, MB-435s, showed decreased expression of 12 aminoacyl-tRNA synthetase (aaRS) genes. Our group recently found that deletion of CEBPD by CRISPR/Cas9 (CEBPD-KO) compromised aminoacyl tRNA synthetase (aaRS) expression and global protein synthesis. However, despite this decrease in global protein production, the synthesis of certain proteins, such as ATF4, which promotes survival and/or death under stress conditions, is increased. Aminoacyl tRNA synthetases are essential enzymes in the process of protein synthesis which catalyze the addition of appropriate amino acid to its corresponding tRNA, and therefore act as a rate limiting step in cellular protein synthesis. In the current project, we sought to investigate the effect of silencing of specific aaRS genes, glutamyl-prolyl-tRNA synthetase (EPRS) and valyl-tRNA synthetase (VARS) on global protein translation and ATF4 expression. To address this question, we silenced the EPRS and VARS gene expression using two independent short-hairpin-RNA (shRNA) targeting two different regions of EPRS and VARS mRNAs in MB-435s cells. Silencing of EPRS gene showed compensatory upregulation of VARS and vice versa. Interestingly, our preliminary data suggested an upregulation of global protein synthesis after EPRS and VARS silencing in MB435s cells measured by puromycin pulse labelling. Ongoing experiments to validate the preliminary data and ATF4 expression will be discussed.https://scholarscompass.vcu.edu/uresposters/1326/thumbnail.jp

Evolution of acceptor stem tRNA recognition by class II prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases (AARS) are an essential family of enzymes that catalyze the attachment of amino acids to specific tRNAs during translation. Previously, we showed that base-specific recognition of the tRNAPro acceptor stem is critical for recognition by Escherichia coli prolyl-tRNA synthetase (ProRS), but not for human ProRS. To further delineate species-specific differences in acceptor stem recognition, atomic group mutagenesis was used to probe the role of sugar–phosphate backbone interactions in recognition of human tRNAPro. Incorporation of site-specific 2′-deoxynucleotides, as well as phosphorothioate and methylphosphonate modifications within the tRNA acceptor stem revealed an extensive network of interactions with specific functional groups proximal to the first base pair and the discriminator base. Backbone functional groups located at the base of the acceptor stem, especially the 2′-hydroxyl of A66, are also critical for aminoacylation catalytic efficiency by human ProRS. Therefore, in contrast to the bacterial system, backbone-specific interactions contribute significantly more to tRNA recognition by the human enzyme than base-specific interactions. Taken together with previous studies, these data show that ProRS-tRNA acceptor stem interactions have co-adapted through evolution from a mechanism involving ‘direct readout’ of nucleotide bases to one relying primarily on backbone-specific ‘indirect readout’

인간 세포질 aminoacyl-tRNA synthetases의 동적인 상호작용과 효소 활성에 대한 분석법 개발

학위논문 (박사) -- 서울대학교 대학원 : 약학대학 약학과, 2020. 8. 김성훈.To the best of our knowledge, protein synthesis (translation) is a universal process, which resides in all extant lifeforms. An aminoacyl-tRNA synthetase (ARS) takes a role in the very first step of the translation process; it catalyzes esterification (aminoacylation) of a specific amino acid on its cognate transfer RNAs (tRNAs) to make aminoacylated tRNAs (aminoacyl-tRNAs). The aminoacyl-tRNA delivers the amino acid to the ribosome which catalyzes the translation of a messenger RNA (mRNA) into a polypeptide chain. The cytoplasmic ARSs are differentially regulated in different species; they have gained additional domains and noncanonical functions throughout evolution, and the largest multi-tRNA synthetase complex (MSC) among the eukaryotes exists in higher eukaryotes, which is comprised of eight ARSs for eight or nine amino acids. Among them, the mammalian MSC is the most complexed one, which is composed of eight cytoplasmic ARSs for nine amino acids, and three scaffold proteins. Consequently, nearly half of the aminoacyl-tRNA efflux becomes concentrated at the MSC. Stable supply of the aminoacyl-tRNA to the ribosome is, therefore, considered to be a major role of the mammalian MSC. Furthermore, the mammalian MSC also serves as a reservoir for releasable ARSs or scaffold proteins to support the noncanonical functions of them. In part I, a split-luciferase complementation system was applied to investigate the configuration of the MSC in live mammalian cells. Multiplex interconnections between the components of the MSC were simplified into binary protein-protein interactions, and pairwise comparison of the interactions reconstituted a framework that is consistent with previous in vitro studies. Reversibility of the split-luciferase reporter binding demonstrated convertible organization of the mammalian MSC, including interferon gamma (IFNγ)-stimulated glutamyl-prolyl-tRNA synthetase 1 (EPRS1) release, as well as the cooperation with the ribosome bridged by the tRNAs. The cell-based analysis provided an improved understanding of the flexible framework of the mammalian MSC in physiological conditions. On the other hand, abnormality of the aminoacylation has been implicated in a wide variety of cancer pathologies. The ARSs exist in large excess in cancer cells due to their increased demand for the protein synthesis. Meanwhile, most other translation apparatuses are quantitatively limited. There has been no report for mutations of the ARSs that demonstrate constitutive activity of the aminoacylation; the hyperactivity of the ARSs may disrupt stable association of the MSC. Hence, interference of the aminoacylation activity is expected to be independent of genotype variation and may not develop drug resistance. In part II, a high-throughput screen (HTS) platform was established to find the mammalian ARS inhibitors. The ARSs of rabbit reticulocyte closely resemble both the individual and complexed structures of human ones. Therefore, an in vitro translation system with the rabbit-reticulocyte lysate may predispose active compounds to be readily applicable for mankind. The assay was further validated for identifying familiar translational inhibitors from a pilot screen, such as emetine, proving its suitability for the purpose. Having demonstrated excellent quality control (QC) parameters and reproducibility, it is proven ready for further HTS campaign with large molecular entities.단백질 합성 (번역)은 모든 형태의 생명이 가지고 있는 공통적인 특성이다. 아미노산-운반RNA 연결효소 (aminoacyl-tRNA synthetase; ARS)는 단백질 합성 과정에서 가장 첫 번째 단계를 담당하고 있다. 아미노산-운반RNA 연결효소는 운반RNA (transfer RNA; tRNA)와 상보적인 아미노산 (amino acid) 사이의 에스터화 반응 (esterification/aminoacylation)을 촉매하여 아미노산-운반RNA 중합체 (aminoacyl-tRNA)로 연결한다. 생성된 아미노산-운반RNA 중합체는 리보솜 (ribosome)으로 전달되어 전령RNA (messenger RNA; mRNA)를 펩타이트 중합체 (polypeptide)로 번역하는 과정의 재료로 사용된다. 세포질 아미노산-운반RNA 연결효소 (cytoplasmic ARS)는 종에 따라 세포내에서 제어되는 방식이 다르다. 이 효소들은 진화과정 동안 추가적인 단백질 도메인 (protein domain)과 새로운 기능들을 획득해왔다. 또한 고등 진핵생물 (higher eukaryote)에는 진핵생물 (eukaryote) 중에서 가장 큰 아미노산-운반RNA 연결효소 복합체 (multi-tRNA synthetase complex; MSC)가 존재하며, 이 복합체는 8종류의 아미노산-운반RNA 연결효소가 8 내지는 9종류의 아미노산을 담당한다. 이 중에서 포유동물의 아미노산-운반RNA 연결효소 복합체가 가장 복잡한 형태인데, 8종류의 세포질 아미노산-운반RNA 연결효소가 9종류의 아미노산을 담당하며, 추가적으로 3종류의 뼈대 단백질 (scaffold protein)이 존재한다. 이처럼 포유동물의 세포 내에서는 아미노산-운반RNA 중합체 이동의 절반 정도가 아미노산-운반RNA 연결효소 복합체에서 시작되고 있다. 따라서 아미노산-운반RNA 중합체를 리보솜으로 안정적으로 공급하는 것이 포유동물 아미노산-운반RNA 연결효소 복합체의 가장 중요한 역할 중의 하나일 것이라 예상된다. 한편으로, 몇몇의 세포질 아미노산-운반RNA 연결효소들과 뼈대 단백질들은 복합체에서 벗어나 새로운 기능을 수행하기 때문에, 아미노산-운반RNA 연결효소 복합체는 이들을 위한 저장고 (reservoir)가 되기도 한다. 본 논문의 첫 번째 부분에서는 분할 루시퍼레이즈 상보 시스템 (split-luciferase complementation system)을 사용하여 포유동물 세포 내에서 아미노산-운반RNA 연결효소 복합체의 구성을 탐색하였다. 구성요소들 간의 복합적인 상호연결은 두 단백질 간의 상호작용 (binary protein-protein interaction; binary PPI)의 합으로 단순화시켰고, 그들간의 쌍별 비교 (pairwise comparison; 구성 요소들의 서로 다른 모든 조합을 비교하는 방식)를 통하여 기존의 생체 외 연구 (in vitro studies)에 상응하는 복합체의 골조 (framework)를 유추해낼 수 있었다. 그리고 분할 루시퍼레이즈 리포터 (split-luciferase reporter) 간의 결합이 가역적이라는 점을 이용하여 인터페론감마 (interferon gamma; IFNγ)에 의한 글루타민-프롤린-운반RNA 연결효소 (glutamyl-prolyl-tRNA synthetase 1; EPRS1)의 방출이나, 운반RNA를 매개로 한 리보솜과 아미노산-운반RNA 연결효소 복합체의 협업과 같은 여러 자극들에 의한 복합체 내의 역동적인 구조 변화를 관찰하였다. 본 연구는 이와 같이 세포를 기반으로 한 분석법을 바탕으로 생리적 환경 (physiological condition)에서 포유동물 아미노산-운반RNA 연결효소 복합체의 골조가 유동적으로 변화하고 있음을 밝혀낼 수 있었다. 한편, 정상적이지 못한 아미노산-운반RNA 연결반응의 존재는 여러 종류의 암에서 잘 알려져 있다. 아미노산-운반RNA 연결효소는 암세포에서 과량으로 존재하며, 암이 진행되면서 늘어난 단백질 합성 요구량을 충족시킨다. 이는 대부분의 다른 단백질 합성 요소들이 암세포에서 과량으로 존재하지 않는다는 사실과 대비된다. 또한 아미노산-운반RNA 연결효소의 활성을 지속적으로 유지시키는 돌연변이는 지금까지 알려져 있지 않다. 이는 효소활성이 비정상적으로 높아진 아미노산-운반RNA 연결효소는 아미노산-운반RNA 연결효소 복합체의 형성과 유지를 저해하기 때문일 것이다. 따라서 아미노산-운반RNA 연결반응을 저해하는 치료법은 환자 개개인의 유전체 다양성 (genotype variation)에 상관없이 효과를 낼 수 있고, 약물 저항성도 나타나지 않을 것이라 기대된다. 본 논문의 두 번째 부분에서는 고속 대량 스크리닝 플랫폼 (high-throughput screening platform)을 구축하여 포유동물 아미노산-운반RNA 연결효소의 저해제를 찾고자 하였다. 이 시스템에서 사용된 토끼의 망상적혈구 용해물 (rabbit-reticulocyte lysate) 내의 아미노산-운반RNA 연결효소들은 단독 또는 결합 구조가 인간의 효소나 그 복합체와 매우 가깝게 닮아있기 때문에 찾아낸 화합물이 인간에게 바로 적용될 수 있는 가능성을 높여준다. 이 시스템은 본 연구에서 수행된 선행 스크리닝 (pilot screening)에서 에메틴 (emetine)과 같이 잘 알려진 단백질 합성 저해제를 찾아내었을 뿐만 아니라, 훌륭한 품질관리 매개변수 (quality control parameters; QC parameters)와 결과의 반복성을 보여주었다. 따라서 이 시스템은 추후 대량 화합물 라이브러리를 타겟으로 한 고속 대량 스크리닝에 활용이 용이할 것으로 기대된다.Introduction 1 Part I. Cell-based analysis of pairwise interactions between the components of the multi-tRNA synthetase complex 11 Graphical abstract 11 Highlights 12 Introduction 13 Materials and methods 18 Results 24 Discussion 34 Figures and table 37 Part II. High-throughput screen for protein synthesis inhibitors targeting aminoacyl-tRNA synthetases 89 Graphical abstract 89 Highlights 90 Introduction 91 Materials and methods 95 Results 98 Discussion 106 Figures and tables 107 Glossary 123 Abbreviations 124 References 128 국문초록 150Docto