ARD1 단백질과 산화·환원 민감성 전사인자 NRF2와의 상호작용이 대장암 진행에 미치는 영향 및 기전연구

학위논문(박사) -- 서울대학교대학원 : 약학대학 약학과, 2023. 2. 서영준.Nuclear factor erythroid-2-related factor 2 (NRF2)는 산화적 또는 친전자적 스트레스에 대항하는 항산화 효소들의 발현을 조절하는 전사인자로, 염증, 노화 및 암의 발생과 같은 다양한 병리적 현상으로부터 세포를 보호하는 것으로 알려져 있다. 그러나, 최근 여러 암세포에서 NRF2는 종양 증식 및 진행에 관여하며 항암제와 같은 외부 스트레스에 대한 보호기전으로 작용한다. 암세포에서 비정상적인 NRF2의 과발현은 NRF2의 대표적 음성조절자인 KEAP1의 비활성화나 NRF2의 자체의 체세포 변이를 통해 발생하는 것으로 알려져 있지만 NRF2의 지속적인 활성화를 담당하는 대안적인 (alternative) 기전에 관해서는 명확히 규명된 바가 없다. N-아세틸화 효소로 알려진 arrest defective1 protein (ARD1)은 세포 분열, 증식 및 발암기전에 관여하며 산화적 스트레스에 대한 세포내 보호작용에서 중요한 역할을 하는 것으로 알려져 있다. ARD1은 유방암, 전립선암, 폐암, 간암, 자궁경부암, 방광암, 대장암에서 높게 발현되어 있으며 ARD1의 발현이 높을수록 암 환자들의 낮은 생존율이 보고된 바 있다. 번역 후 변형 형태 중 하나인 아세틸화는 단백질의 안정화에 관여하며, NRF2 아미노산 염기서열 내에 아세틸화가 가능한 라이신 잔기가 있음에도 불구하고 아세틸화에 의한 NRF2의 안정화 기전 관한 연구는 크게 이루어진 바가 없다. 본 연구에서는 ARD1이 NRF2의 아세틸화를 유도함으로서 대장암의 진행과정에 관여하는 분자 기전에 관하여 알아보고자 하였다. 면역형광염색 기법을 통하여 인체대장암 조직을 염색해 보았을때 ARD1과 NRF2의 발현이 서로 positive한 상관 관계를 보였으며, ARD1 유전자 발현을 선택적으로 억제할 수 있는 siRNA를 인체 대장암 세포주에 주입하였을 때 NRF2의 mRNA에는 영향을 미치지 못하였으나 NRF2의 단백질이 유의적으로 감소되었다. 이는 ARD1이 NRF2의 신생 합성에 관여하는 것이 아닌 단백질 번역 후 변형에 관여함을 시사하였다. 또한, 이 두 단백질은 인간 대장암 세포인 HCT-116와 인간 대장 종양 조직에서 물리적으로 상호작용함을 관찰하였으며 NRF2의 serial deletion construct를 통하여 NRF2의 Neh1와 Neh3 domain이 두 단백질의 결합에 직접 관여함을 알 수 있었다. ARD1의 과발현시 NRF2의 아세틸화가 증가되었으며 in vitro acetylation assay와 질량분석법을 통해 ARD1이 NRF2를 직접 아세틸화시킬 수 있음을 증명하였다. ARD1의 아세틸화 효소활성이 NRF2의 단백질 안정화에 관여하는지 확인하고자 아세틸화 효소활성 기능이 손상된 ARD1 돌연변이를 통하여 NRF2 단백질의 반감기를 측정한 결과 ARD1을 통한 NRF2의 아세틸화가 단백질 안정화에 관여함을 확인할 수 있었다. 결론적으로, ARD1은 NRF2의 아세틸화를 통하여 단백질 안정화에 관여하며 인간 대장암 세포의 이동 및 증식과 같은 암의 진행과정 참여한다.Aberrant overactivation/overexpression of NRF2 is implicated in tumor progression, which has been largely attributed to its mutation as well as inactivation of the inhibitory protein, KEAP1. However, alternative mechanisms responsible for sustained activation of NRF2 are less understood. Here, I showed that ARD1 with the acetyltransferase activity is a new regulator of NRF2. Elevated levels of ARD1 and NRF2 were detected in human colon tumor tissues as well as human colon cancer cell lines. Knockdown of both ARD1 and NRF2 in human colon cancer HCT-116 cells suppressed the oncogenicity of these cells. Furthermore, ARD1 knockdown in human colon cancer cells significantly reduced the protein levels of NRF2 without affecting its mRNA expression; however, silencing of NRF2 did not alter ARD1 protein expression. In addition, these two proteins were co-localized and physically interacted with each other both in human colon cancer cells and human colon tumor tissues. Mechanistically, ARD1 overexpression increased the acetylation levels of NRF2. Moreover, the in vitro acetylation assay and mass spectrometric analysis demonstrated that ARD1 directly acetylated NRF2. Ectopic expression of mutant forms of ARD1 with defective acetyltransferase activity reduced the half-life of NRF2. In conclusion, ARD1 may potentiate the oncogenic function of NRF2 in human colon cancer by acetylating and stabilizing this transcription factor.Chapter Ⅰ. ARD1-mediated Lysine Acetylation as a Novel Post-translational Modification of NRF2 1 1. Introduction 2 Ⅰ. NRF2 5 1. NRF2 protein 8 2. Phosorylation of NRF2 11 3 Ubiquitylation of NRF2. 20 4. Acetylation of NRF2 24 5. Deacetylation of NRF2 30 6. SUMOylation of NRF2 31 7. Glycation and de-glycation of NRF2 33 8. Methylation of NRF2 33 9. Concluding remarks 34 Ⅱ. ARD1 39 1. Discovery of ARD1 39 2. ARD1 isoforms 39 3. ARD1 as a lysine acetyltransferase 40 4. Concluding remarks 45 Ⅲ. References 47 STATEMENT OF PURPOSE 62 Chapter Ⅱ. ARD1 stabilizes NRF2 through direct interaction and promotes colon cancer progression 64 1. Introduction 65 2. Materials and Methods 68 3. Results 82 3.1 Correlation of NRF2 and ARD1 with CRC 82 3.2 Knockdown of NRF2 and ARD1 attenuates oncogenicity of CRC cells 88 3.3 ARD1 knockdown reduces the stability of NRF2 in human colon cancer cells 94 3.4 ARD1 physically interacts with NRF2 105 3.5 ARD1 decreases cellular responses to oxidative stress in HCT-116 cells 114 3.6 ARD1 knockdown reduces the stability of NRF2 in human colon cancer cells 116 4. Discussion 124 5. Conclusion 128 6. References 131 Abstract in Korean 136박

Interaction between HIF-1α (ODD) and hARD1 does not induce acetylation and destabilization of HIF-1α

AbstractHypoxia inducible factor-1α (HIF-1α) is a central component of the cellular responses to hypoxia. Hypoxic conditions result in stabilization of HIF-1α and formation of the transcriptionally active HIF-1 complex. It was suggested that mammalian ARD1 acetylates HIF-1α and thereby enhances HIF-1α ubiquitination and degradation. Furthermore, ARD1 was proposed to be downregulated in hypoxia thus facilitating the stabilization of HIF-1α. Here we demonstrate that the level of human ARD1 (hARD1) protein is not decreased in hypoxia. Moreover, hARD1 does not acetylate and destabilize HIF-1α. However, we find that hARD1 specifically binds HIF-1α, suggesting a putative, still unclear, connection between these proteins

Epigenomic Regulation of Androgen Receptor Signaling: Potential Role in Prostate Cancer Therapy.

Androgen receptor (AR) signaling remains the major oncogenic pathway in prostate cancer (PCa). Androgen-deprivation therapy (ADT) is the principle treatment for locally advanced and metastatic disease. However, a significant number of patients acquire treatment resistance leading to castration resistant prostate cancer (CRPC). Epigenetics, the study of heritable and reversible changes in gene expression without alterations in DNA sequences, is a crucial regulatory step in AR signaling. We and others, recently described the technological advance Chem-seq, a method to identify the interaction between a drug and the genome. This has permitted better understanding of the underlying regulatory mechanisms of AR during carcinogenesis and revealed the importance of epigenetic modifiers. In screening for new epigenomic modifiying drugs, we identified SD-70, and found that this demethylase inhibitor is effective in CRPC cells in combination with current therapies. The aim of this review is to explore the role of epigenetic modifications as biomarkers for detection, prognosis, and risk evaluation of PCa. Furthermore, we also provide an update of the recent findings on the epigenetic key processes (DNA methylation, chromatin modifications and alterations in noncoding RNA profiles) involved in AR expression and their possible role as therapeutic targets

ARD1

Regeln zur Gerichtspraxis, Streitigkeiten und Erträge beim Jahrmark

Phosphorylation of ARD1 by IKK beta contributes to its destabilization and degradation

[[abstract]]I kappa B kinase beta (IKK beta), a major kinase downstream of various proinflammatory signals, mediates multiple cellular functions through phosphorylation and regulation of its substrates. On the basis of protein sequence analysis, we identified arrest-defective protein 1 (ARD1), a protein involved in apoptosis and cell proliferation processes in many human cancer cells, as a new IKK beta substrate. We provided evidence showing that ARD1 is indeed a bona. de substrate of IKK beta. IKK beta physically associated with ARD1 and phosphorylated it at Ser209. Phosphorylation by IKK beta destabilized ARD1 and induced its proteasome-mediated degradation. Impaired growth suppression was observed in ARD1 phosphorylation-mimic mutant (S209E)-transfected cells as compared with ARD1 non-phosphorylatable mutant (S209A)-transfected cells. Our findings of molecular interactions between ARD1 and IKK beta may enable further understanding of the upstream regulation mechanisms of ARD1 and of the diverse functions of IKK beta. (C) 2009 Elsevier Inc. All rights reserved

Characterization of hARD2, a processed hARD1 gene duplicate, encoding a human protein N-α-acetyltransferase

BACKGROUND: Protein acetylation is increasingly recognized as an important mechanism regulating a variety of cellular functions. Several human protein acetyltransferases have been characterized, most of them catalyzing ε-acetylation of histones and transcription factors. We recently described the human protein acetyltransferase hARD1 (human Arrest Defective 1). hARD1 interacts with NATH (N-Acetyl Transferase Human) forming a complex expressing protein N-terminal α-acetylation activity. RESULTS: We here describe a human protein, hARD2, with 81 % sequence identity to hARD1. The gene encoding hARD2 most likely originates from a eutherian mammal specific retrotransposition event. hARD2 mRNA and protein are expressed in several human cell lines. Immunoprecipitation experiments show that hARD2 protein potentially interacts with NATH, suggesting that hARD2-NATH complexes may be responsible for protein N-α-acetylation in human cells. In NB4 cells undergoing retinoic acid mediated differentiation, the level of endogenous hARD1 and NATH protein decreases while the level of hARD2 protein is stable. CONCLUSION: A human protein N-α-acetyltransferase is herein described. ARD2 potentially complements the functions of ARD1, adding more flexibility and complexity to protein N-α-acetylation in human cells as compared to lower organisms which only have one ARD

The acetyltransferase activity of San stabilizes the mitotic cohesin at the centromeres in a shugoshin-independent manner

Proper sister chromatid cohesion is critical for maintaining genetic stability. San is a putative acetyltransferase that is important for sister chromatid cohesion in Drosophila melanogaster, but not in budding yeast. We showed that San is critical for sister chromatid cohesion in HeLa cells, suggesting that this mechanism may be conserved in metazoans. Furthermore, although a small fraction of San interacts with the NatA complex, San appears to mediate cohesion independently. San exhibits acetyltransferase activity in vitro, and its activity is required for sister chromatid cohesion in vivo. In the absence of San, Sgo1 localizes correctly throughout the cell cycle. However, cohesin is no longer detected at the mitotic centromeres. Furthermore, San localizes to the cytoplasm in interphase cells; thus, it may not gain access to chromosomes until mitosis. Moreover, in San-depleted cells, further depletion of Plk1 rescues the cohesion along the chromosome arms, but not at the centromeres. Collectively, San may be specifically required for the maintenance of the centromeric cohesion in mitosis