ABSTRACT
Chemical modifications of ribonucleotides significantly alter the physicochemical properties and functions of RNA. Initially perceived as static and essential marks in ribosomal RNA (rRNA) and transfer RNA (tRNA), recent discoveries unveiled a dynamic landscape of RNA modifications in messenger RNA (mRNA) and other regulatory RNAs. These findings spurred extensive efforts to map the distribution and function of RNA modifications, aiming to elucidate their distribution and functional significance in normal cellular homeostasis and pathological states. Significant dysregulation of RNA modifications is extensively documented in cancers, accentuating the potential of RNA-modifying enzymes as therapeutic targets. However, the essential role of several RNA-modifying enzymes in normal physiological functions raises concerns about potential side effects. A notable example is N-acetyltransferase 10 (NAT10), which is responsible for acetylating cytidines in RNA. While emerging evidence positions NAT10 as an oncogenic factor and a potential target in various cancer types, its essential role in normal cellular processes complicates the development of targeted therapies. This review aims to comprehensively analyze the essential and oncogenic properties of NAT10. We discuss its crucial role in normal cell biology and aging alongside its contribution to cancer development and progression. We advocate for agnostic approaches to disentangling the intertwined essential and oncogenic functions of RNA-modifying enzymes. Such approaches are crucial for understanding the full spectrum of RNA-modifying enzymes and imperative for designing effective and safe therapeutic strategies.
Subject(s)
N-Terminal Acetyltransferases , Neoplasms , RNA , Humans , N-Terminal Acetyltransferases/genetics , Neoplasms/genetics , RNA/genetics , RNA, Messenger , RNA, Ribosomal , RNA, Transfer/geneticsABSTRACT
Cell fate transition involves dynamic changes of gene regulatory network and chromatin landscape, requiring multiple levels of regulation, yet the cross-talk between epitranscriptomic modification and chromatin signaling remains largely unknown. Here, we uncover that suppression of N-acetyltransferase 10 (NAT10), the writer for mRNA N4-acetylcytidine (ac4C) modification, can notably affect human embryonic stem cell (hESC) lineage differentiation and pluripotent reprogramming. With integrative analysis, we identify that NAT10-mediated ac4C modification regulates the protein levels of a subset of its targets, which are strongly enriched for fate-instructive chromatin regulators, and among them, histone chaperone ANP32B is experimentally verified and functionally relevant. Furthermore, NAT10-ac4C-ANP32B axis can modulate the chromatin landscape of their downstream genes (e.g., key regulators of Wnt and TGFß pathways). Collectively, we show that NAT10 is an essential regulator of cellular plasticity, and its catalyzed mRNA cytidine acetylation represents a critical layer of epitranscriptomic modulation and uncover a previously unrecognized, direct cross-talk between epitranscriptomic modification and chromatin signaling during cell fate transitions.
Subject(s)
Chromatin , N-Terminal Acetyltransferases , RNA, Messenger , Humans , Acetylation , Acetyltransferases/metabolism , Chromatin/genetics , Cytidine , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Cell Differentiation/geneticsABSTRACT
Epitranscriptomic RNA modifications can regulate the stability of mRNA and affect cellular and viral RNA functions. The N4-acetylcytidine (ac4C) modification in the RNA viral genome was recently found to promote viral replication; however, the mechanism by which RNA acetylation in the host mRNA regulates viral replication remains unclear. To help elucidate this mechanism, the roles of N-acetyltransferase 10 (NAT10) and ac4C during the infection and replication processes of the alphavirus, Sindbis virus (SINV), were investigated. Cellular NAT10 was upregulated, and ac4C modifications were promoted after alphavirus infection, while the loss of NAT10 or inhibition of its N-acetyltransferase activity reduced alphavirus replication. The NAT10 enhanced alphavirus replication as it helped to maintain the stability of lymphocyte antigen six family member E mRNA, which is a multifunctional interferon-stimulated gene that promotes alphavirus replication. The ac4C modification was thus found to have a non-conventional role in the virus life cycle through regulating host mRNA stability instead of viral mRNA, and its inhibition could be a potential target in the development of new alphavirus antivirals.IMPORTANCEThe role of N4-acetylcytidine (ac4C) modification in host mRNA and virus replication is not yet fully understood. In this study, the role of ac4C in the regulation of Sindbis virus (SINV), a prototype alphavirus infection, was investigated. SINV infection results in increased levels of N-acetyltransferase 10 (NAT10) and increases the ac4C modification level of cellular RNA. The NAT10 was found to positively regulate SINV infection in an N-acetyltransferase activity-dependent manner. Mechanistically, the NAT10 modifies lymphocyte antigen six family member E (LY6E) mRNA-the ac4C modification site within the 3'-untranslated region (UTR) of LY6E mRNA, which is essential for its translation and stability. The findings of this study demonstrate that NAT10 regulated mRNA stability and translation efficiency not only through the 5'-UTR or coding sequence but also via the 3'-UTR region. The ac4C modification of host mRNA stability instead of viral mRNA impacting the viral life cycle was thus identified, indicating that the inhibition of ac4C could be a potential target when developing alphavirus antivirals.
Subject(s)
Alphavirus Infections , Antigens, Surface , GPI-Linked Proteins , N-Terminal Acetyltransferases , Sindbis Virus , Virus Replication , Humans , Alphavirus Infections/genetics , Antigens, Surface/genetics , Cytidine/analogs & derivatives , GPI-Linked Proteins/genetics , RNA, Messenger/genetics , Sindbis Virus/physiology , Cell Line , N-Terminal Acetyltransferases/genetics , RNA StabilityABSTRACT
The quick progress of epigenetic study has kindled new hope for treating many cancers. When it comes to RNA epigenetics, the ac4C acetylation modification is showing promise, whereas N-acetyltransferase 10 plays a wide range of biological functions, has a significant impact on cellular life events, and is frequently highly expressed in many malignant tumors. N-acetyltransferase 10 is an acetyltransferase with important biological involvement in cellular processes and lifespan. Because it is highly expressed in many malignant tumors, it is considered a pro-carcinogenic gene. The review aims to introduce NAT10, summarize the effects of ac4C acetylation on tumor growth from multiple angles, and discuss the possible therapeutic targeting of NAT10 and the future directions of ac4C acetylation investigations.
Subject(s)
Neoplasms , RNA , Humans , Acetylation , Acetyltransferases , Carcinogenesis/genetics , Cell Transformation, Neoplastic , Neoplasms/genetics , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolismABSTRACT
Cell fate determination in mammalian development is complex and precisely controlled and accumulating evidence indicates that epigenetic mechanisms are crucially involved. N4-acetylcytidine (ac4C) is a recently identified modification of messenger RNA (mRNA); however, its functions are still elusive in mammalian. Here, we show that N-acetyltransferase 10 (NAT10)-mediated ac4C modification promotes ectoderm differentiation of human embryonic stem cells (hESCs) by acetylating nuclear receptor subfamily 2 group F member 1 (NR2F1) mRNA to enhance translation efficiency (TE). Acetylated RNA immunoprecipitation sequencing (acRIP-seq) revealed that levels of ac4C modification were higher in ectodermal neuroepithelial progenitor (NEP) cells than in hESCs or mesoendoderm cells. In addition, integrated analysis of acRIP-seq and ribosome profiling sequencing revealed that NAT10 catalysed ac4C modification to improve TE in NEP cells. RIP-qRT-PCR analysis identified an interaction between NAT10 and NR2F1 mRNA in NEP cells and NR2F1 accelerated the nucleus-to-cytoplasm translocation of yes-associated protein 1, which contributed to ectodermal differentiation of hESCs. Collectively, these findings point out the novel regulatory role of ac4C modification in the early ectodermal differentiation of hESCs and will provide a new strategy for the treatment of neuroectodermal defects diseases.
Subject(s)
Human Embryonic Stem Cells , Animals , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ectoderm/metabolism , Cell Differentiation , Base Sequence , Mammals/metabolism , COUP Transcription Factor I/genetics , COUP Transcription Factor I/metabolism , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolismABSTRACT
N-acetyltransferase 10 (NAT10), a versatile enzyme, has gained considerable attention as a significant player in the complex realm of cancer biology. Its enigmatic role in tumorigenesis extends across a wide array of cellular processes, impacting cell growth, differentiation, survival, and genomic stability. Within the intricate network of oncogenic signaling, NAT10 emerges as a crucial agent in multiple cancer types, such as breast, lung, colorectal, and leukemia. This compelling research addresses the intricate complexity of the mechanistic role of NAT10 in cancer development. By elucidating its active participation in essential physiological processes, we investigate the regulatory role of NAT10 in cell cycle checkpoints, coordination of chromatin remodeling, and detailed modulation of the delicate balance between apoptosis and cell survival. Perturbations in NAT10 expression and function have been linked to oncogenesis, metastasis, and drug resistance in a variety of cancer types. Furthermore, the bewildering interactions between NAT10 and key oncogenic factors, such as p53 and c-Myc, are deciphered, providing profound insights into the molecular underpinnings of cancer pathogenesis. Equally intriguing, the paradoxical role of NAT10 as a potential tumor suppressor or oncogene is influenced by context-dependent factors and the cellular microenvironment. This study explores the fascinating interplay of genetic changes, epigenetic changes, and post-translational modifications that shape the dual character of NAT10, revealing the delicate balance between cancer initiation and suppression. Taken together, this overview delves deeply into the enigmatic role of NAT10 in cancer, elucidating its multifaceted roles and its complex interplay with oncogenic networks.
Subject(s)
N-Terminal Acetyltransferases , Neoplasms , Humans , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolism , N-Terminal Acetyltransferase E/genetics , N-Terminal Acetyltransferase E/metabolism , Neoplasms/genetics , Protein Processing, Post-Translational , Tumor MicroenvironmentABSTRACT
Protein N-terminal (Nt) acetylation is an essential post-translational process catalysed by N-acetyltransferases or N-terminal acetyltransferases (NATs). Over the past several decades, several types of NATs (NatA- NatH) have been identified along with their substrates, explaining their significance in eukaryotes. It affects protein stability, protein degradation, protein translocation, and protein-protein interaction. NATs have recently drawn attention as they are associated with the pathogenesis of human diseases. In particular, NAT-induced epigenetic modifications play an important role in the control of mitochondrial function, which may lead to inflammatory diseases. NatC knockdown causes a marked reduction in mitochondrial membrane proteins, impairing their functions, and NatA affects mitophagy via reduced phosphorylation and transcription of the autophagy receptor. However, the NAT-mediated mitochondrial epigenetic mechanisms involved in the inflammatory process remain unexplored. The current review will impart an overview of the biological functions and aberrations of various NAT, which may provide a novel therapeutic strategy for inflammatory disorders.
Subject(s)
N-Terminal Acetyltransferases , Protein Processing, Post-Translational , Humans , N-Terminal Acetyltransferases/genetics , Proteolysis , Inflammation/genetics , Acetylation , Acetyltransferases/genetics , Acetyltransferases/metabolismABSTRACT
Most proteins receive an acetyl group at the N terminus while in their nascency as the result of modification by co-translationally acting N-terminal acetyltransferases (NATs). The N-terminal acetyl group can influence several aspects of protein functionality. From studies of NAT-lacking cells, it is evident that several cellular processes are affected by this modification. More recently, an increasing number of genetic cases have demonstrated that N-terminal acetylation has crucial roles in human physiology and pathology. In this Cell Science at a Glance and the accompanying poster, we provide an overview of the human NAT enzymes and their properties, substrate coverage, cellular roles and connections to human disease.
Subject(s)
Acetyltransferases , N-Terminal Acetyltransferases , Humans , Acetylation , Acetyltransferases/genetics , Acetyltransferases/metabolism , N-Terminal Acetyltransferases/chemistry , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolism , Protein Processing, Post-Translational , Proteins/metabolismABSTRACT
Aberrant RNA modifications are frequently associated with cancers, while the underlying mechanisms and clinical significance remain poorly understood. Here, we find that the ac4C RNA acetyltransferase NAT10 is significantly upregulated in esophageal cancers (ESCAs) and associated with poor ESCA prognosis. In addition, using ESCA cell lines and mouse models, we confirm the critical functions of NAT10 in promoting ESCA tumorigenesis and progression in vitro and in vivo. Mechanistically, NAT10 depletion reduces the abundance of ac4C-modified tRNAs and decreases the translation efficiencies of mRNAs enriched for ac4C-modified tRNA-decoded codons. We further identify EGFR as a key downstream target that facilitates NAT10's oncogenic functions. In terms of clinical significance, we demonstrate that NAT10 depletion and gefitinib treatment synergistically inhibit ESCA progression in vitro and in vivo. Our data indicate the mechanisms underlying ESCA progression at the layer of mRNA translation control and provide molecular insights for the development of effective cancer therapeutic strategies.
Subject(s)
N-Terminal Acetyltransferases , Neoplasms , RNA, Transfer , Animals , Mice , ErbB Receptors/genetics , Gefitinib/pharmacology , Gefitinib/therapeutic use , Protein Biosynthesis , RNA, Transfer/genetics , Humans , Cell Line, Tumor , N-Terminal Acetyltransferases/genetics , Drug Resistance, NeoplasmABSTRACT
N4-acetylcytidine (ac4C) is a lately discovered nucleotide modification that has been shown to be closely implicated in cancer. N-acetyltransferase10(NAT10) acts as an enzyme that regulates mRNA acetylation modifications. Currently, the role of NAT10-mediated RNA acetylation modification in cervical cancer remains to be elucidated. On the basis of transcriptome analysis of TCGA and GEO open datasets (GSE52904, GSE29570, GSE122697), NAT10 is upregulated in cervical cancer tissues and correlated with poor prognosis. Knockdown of NAT10 suppressed the cell proliferation, invasion, and migration of cervical cancer cells. The in vivo oncogenic function of NAT10 was also confirmed in xenograft models. Combined RNA-seq and acRIP-seq analysis revealed HNRNPUL1 as the target of NAT10 in cervical cancer. NAT10 positively regulate HNRNPUL1 expression by promoting ac4C modification and stability of HNRNPUL1 mRNA. Furthermore, depletion of HNRNPUL1 suppressed the cell division, invasion, and migration of cervical cancer. HNRNPUL1 overexpression partially restored cellular function in cervical cancer cells with NAT10 knockdown. Thus, this study demonstrates that NAT10 contributes to cervical cancer progression by enhancing HNRNPUL1 mRNA stability via ac4C modification, and NAT10-ac4C-HNRNPUL1 axis might be a potential target for cervical cancer therapy.
Subject(s)
Uterine Cervical Neoplasms , Humans , Female , Uterine Cervical Neoplasms/genetics , Acetylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA Stability/genetics , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolismABSTRACT
Protein N-terminal (Nt) acetylation is one of the most abundant modifications in eukaryotes, covering ~50-80 % of the proteome, depending on species. Cells with defective Nt-acetylation display a wide array of phenotypes such as impaired growth, mating defects and increased stress sensitivity. However, the pleiotropic nature of these effects has hampered our understanding of the functional impact of protein Nt-acetylation. The main enzyme responsible for Nt-acetylation throughout the eukaryotic kingdom is the N-terminal acetyltransferase NatA. Here we employ a multi-dimensional proteomics approach to analyze Saccharomyces cerevisiae lacking NatA activity, which causes global proteome remodeling. Pulsed-SILAC experiments reveals that NatA-deficient strains consistently increase degradation of ribosomal proteins compared to wild type. Explaining this phenomenon, thermal proteome profiling uncovers decreased thermostability of ribosomes in NatA-knockouts. Our data are in agreement with a role for Nt-acetylation in promoting stability for parts of the proteome by enhancing the avidity of protein-protein interactions and folding.
Subject(s)
N-Terminal Acetyltransferases , Saccharomyces cerevisiae Proteins , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolism , Saccharomyces cerevisiae/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , N-Terminal Acetyltransferase A/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Proteome/metabolism , Acetylation , Acetyltransferases/genetics , Acetyltransferases/metabolism , N-Terminal Acetyltransferase E/metabolismABSTRACT
Homologous recombination (HR) is a highly accurate mechanism for repairing DNA double-strand breaks (DSBs) that arise from various genotoxic insults and blocked replication forks. Defects in HR and unscheduled HR can interfere with other cellular processes such as DNA replication and chromosome segregation, leading to genome instability and cell death. Therefore, the HR process has to be tightly controlled. Protein N-terminal acetylation is one of the most common modifications in eukaryotic organisms. Studies in budding yeast implicate a role for NatB acetyltransferase in HR repair, but precisely how this modification regulates HR repair and genome integrity is unknown. In this study, we show that cells lacking NatB, a dimeric complex composed of Nat3 and Mdm2, are sensitive to the DNA alkylating agent methyl methanesulfonate (MMS), and that overexpression of Rad51 suppresses the MMS sensitivity of nat3Δ cells. Nat3-deficient cells have increased levels of Rad52-yellow fluorescent protein foci and fail to repair DSBs after release from MMS exposure. We also found that Nat3 is required for HR-dependent gene conversion and gene targeting. Importantly, we observed that nat3Δ mutation partially suppressed MMS sensitivity in srs2Δ cells and the synthetic sickness of srs2Δ sgs1Δ cells. Altogether, our results indicate that NatB functions upstream of Srs2 to activate the Rad51-dependent HR pathway for DSB repair.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Acetyltransferases/genetics , DNA Repair , DNA-Binding Proteins/genetics , Homologous Recombination , Methyl Methanesulfonate/toxicity , N-Terminal Acetyltransferase B/genetics , N-Terminal Acetyltransferase B/metabolism , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Rad52 DNA Repair and Recombination Protein/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Parthanatos is a type of programmed cell death dependent on hyper-activation of poly (ADP-ribose) polymerase 1 (PARP-1). SIRT1 is a highly conserved nuclear deacetylase and often acts as an inhibitor of parthanatos by deacetylation of PARP1. Our previous study showed that deoxypodophyllotoxin (DPT), a natural compound isolated from the traditional herb Anthriscus sylvestris, triggered glioma cell death via parthanatos. In this study, we investigated the role of SIRT1 in DPT-induced human glioma cell parthanatos. We showed that DPT (450 nmol/L) activated both PARP1 and SIRT1, and induced parthanatos in U87 and U251 glioma cells. Activation of SIRT1 with SRT2183 (10 µmol/L) enhanced, while inhibition of SIRT1 with EX527 (200 µmol/L) or knockdown of SIRT1 attenuated DPT-induced PARP1 activation and glioma cell death. We demonstrated that DPT (450 nmol/L) significantly decreased intracellular NAD+ levels in U87 and U251 cells. Further decrease of NAD+ levels with FK866 (100 µmol/L) aggravated, but supplement of NAD+ (0.5, 2 mmol/L) attenuated DPT-induced PARP1 activation. We found that NAD+ depletion enhanced PARP1 activation via two ways: one was aggravating ROS-dependent DNA DSBs by upregulation of NADPH oxidase 2 (NOX2); the other was reinforcing PARP1 acetylation via increase of N-acetyltransferase 10 (NAT10) expression. We found that SIRT1 activity was improved when being phosphorylated by JNK at Ser27, the activated SIRT1 in reverse aggravated JNK activation via upregulating ROS-related ASK1 signaling, thus forming a positive feedback between JNK and SIRT1. Taken together, SIRT1 activated by JNK contributed to DPT-induced human glioma cell parthanatos via initiation of NAD+ depletion-dependent upregulation of NOX2 and NAT10.
Subject(s)
Glioma , Parthanatos , Sirtuin 1 , Humans , Glioma/drug therapy , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolism , NAD/metabolism , NADPH Oxidase 2/metabolism , Parthanatos/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Reactive Oxygen Species/metabolism , Sirtuin 1/metabolism , Up-RegulationABSTRACT
N4-acetylcytidine (ac4C) is one type of RNA modification found in eukaryotes. RNA acetylation modifications are gradually expanding in oncology. However, the role of RNA acetylation modifications in colorectal cancer and its association with colorectal cancer microsatellite status remain unclear. Using public databases and in vitro experiments, we verified the expression and biological function of NAT10, as the key RNA acetylation modification enzyme, in colorectal cancer. The results showed that NAT10 was highly expressed in colorectal cancer, and significantly promoted colorectal cancer cell proliferation. NAT10 was also involved in several aspects of cell homoeostasis such as ion transport, calcium-dependent phospholipid binding, and RNA stability. NAT10 expression positively correlated with immune infiltration in colorectal cancer. We further constructed a risk regression model for mRNA acetylation in colorectal cancer using acetylation-related differential genes. We found that tumour immune infiltration, microsatellite instability (MSI) proportion, tumour immune mutation burden, and patient response to immunotherapy were positively correlated with risk scores. For the first time, our study showed that the level of mRNA acetylation modification level is elevated in colorectal cancer and positively correlates with immune infiltration and microsatellite status of patients. Based on our findings, NAT10 may be a new target for colorectal cancer treatment.
Subject(s)
Colonic Neoplasms , Colorectal Neoplasms , Humans , RNA, Messenger/genetics , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolism , Acetylation , DNA Methylation , Colonic Neoplasms/genetics , Colorectal Neoplasms/genetics , Microsatellite RepeatsABSTRACT
Epitranscriptomic RNA modifications constitute a critical gene regulatory component that can affect cancer progression. Among these, the RNA N4-acetylcytidine (ac4C) modification, which is mediated by the ac4C writer N-acetyltransferase 10 (NAT10), regulates the stabilization of mRNA. Here, we identified that the ac4C modification is induced upon cisplatin treatment and correlates with chemoresistance in bladder cancer. Both in vitro and in vivo, NAT10 promoted cisplatin chemoresistance in bladder cancer cells by enhancing DNA damage repair (DDR). Mechanistically, NAT10 bound and stabilized AHNAK mRNA by protecting it from exonucleases, and AHNAK-mediated DDR was required for NAT10-induced cisplatin resistance. Clinically, NAT10 overexpression was associated with chemoresistance, recurrence, and worse clinical outcome in patients with bladder cancer. Cisplatin-induced NFκB signaling activation was required for the upregulation of NAT10 expression, and NFκB p65 directly bound to the NAT10 promoter to activate transcription. Moreover, pharmacological inhibition of NAT10 with Remodelin sensitized bladder cancer organoids and mouse xenografts to cisplatin. Overall, the present study uncovered a mechanism of NAT10-mediated mRNA stabilization in bladder cancer, laying the foundation for NAT10 as a therapeutic target to overcome cisplatin resistance in bladder cancer. SIGNIFICANCE: The mRNA ac4C writer NAT10 stimulates DNA damage repair to promote cisplatin chemoresistance in bladder cancer, identifying NAT10 inhibition as a potential therapeutic approach to enhance cisplatin sensitivity.
Subject(s)
Cisplatin , Urinary Bladder Neoplasms , Humans , Animals , Mice , Cisplatin/pharmacology , Cisplatin/therapeutic use , Drug Resistance, Neoplasm/genetics , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , RNA, Messenger/genetics , DNA Repair , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolismABSTRACT
Posttranslational modifications add tremendous complexity to proteomes; however, gaps remain in knowledge regarding the function and regulatory mechanism of newly discovered lysine acylation modifications. Here, we compared a panel of non-histone lysine acylation patterns in metastasis models and clinical samples, and focused on 2-hydroxyisobutyrylation (Khib) due to its significant upregulation in cancer metastases. By the integration of systemic Khib proteome profiling in 20 paired primary esophageal tumor and metastatic tumor tissues with CRISPR/Cas9 functional screening, we identified N-acetyltransferase 10 (NAT10) as a substrate for Khib modification. We further showed that Khib modification at lysine 823 in NAT10 functionally contribute to metastasis. Mechanistically, NAT10 Khib modification enhances its interaction with deubiquitinase USP39, resulting in increased NAT10 protein stability. NAT10 in turn promotes metastasis by increasing NOTCH3 mRNA stability in an N4-acetylcytidine-dependent manner. Furthermore, we discovered a lead compound #7586-3507 that inhibited NAT10 Khib modification and showed efficacy in tumor models in vivo at a low concentration. Together, our findings bridge newly identified lysine acylation modifications with RNA modifications, thus providing novel insights into epigenetic regulation in human cancer. We propose that pharmacological inhibition of NAT10 K823 Khib modification constitutes a potential anti-metastasis strategy.
Subject(s)
Lysine , Neoplasms , Humans , Lysine/metabolism , Epigenesis, Genetic , Acylation , Protein Processing, Post-Translational , Acetyltransferases/metabolism , Neoplasms/genetics , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolism , Ubiquitin-Specific Proteases/geneticsABSTRACT
N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30Δ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.
Subject(s)
N-Terminal Acetyltransferases , Saccharomyces cerevisiae , Humans , Acetylation , Chromatography, Liquid , Conserved Sequence , Genetic Complementation Test , Methionine/metabolism , N-Terminal Acetyltransferase C/genetics , N-Terminal Acetyltransferase C/metabolism , N-Terminal Acetyltransferase E , N-Terminal Acetyltransferases/deficiency , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolism , Phenotype , Protein Processing, Post-Translational , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/metabolism , Substrate SpecificityABSTRACT
BACKGROUND: N-acetyltransferase 10 (NAT10) is the only enzyme known to mediate the N4-acetylcytidine (ac4C) modification of mRNA and is crucial for mRNA stability and translation efficiency. However, its role in cancer development and prognosis has not yet been explored. This study aimed to examine the possible role of NAT10 in colon cancer. METHODS: The expression levels of NAT10 were evaluated by immunohistochemical analyses with a colon cancer tissue microarray, and its prognostic value in patients was further analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to analyze NAT10 expression in harvested colon cancer tissues and cell lines. Stable NAT10-knockdown and NAT10-overexpressing colon cancer cell lines were constructed using lentivirus. The biological functions of NAT10 in colon cancer cell lines were analyzed in vitro by Cell Counting Kit-8 (CCK-8), wound healing, Transwell, cell cycle, and ferroptosis assays. Xenograft models were used to analyze the effect of NAT10 on the tumorigenesis and metastasis of colon cancer cells in vivo. Dot blotting, acetylated RNA immunoprecipitation-qPCR, and RNA stability analyses were performed to explore the mechanism by which NAT10 functions in colon cancer progression. RESULTS: NAT10 was upregulated in colon cancer tissues and various colon cancer cell lines. This increased NAT10 expression was associated with shorter patient survival. Knockdown of NAT10 in two colon cancer cell lines (HT-29 and LoVo) impaired the proliferation, migration, invasion, tumor formation and metastasis of these cells, whereas overexpression of NAT10 promoted these abilities. Further analysis revealed that NAT10 exerted a strong effect on the mRNA stability and expression of ferroptosis suppressor protein 1 (FSP1) in HT-29 and LoVo cells. In these cells, FSP1 mRNA was found to be modified by ac4C acetylation, and this epigenetic modification was associated with the inhibition of ferroptosis. CONCLUSIONS: Our study revealed that NAT10 plays a critical role in colon cancer development by affecting FSP1 mRNA stability and ferroptosis, suggesting that NAT10 could be a novel prognostic and therapeutic target in colon cancer.
Subject(s)
Apoptosis Regulatory Proteins , Colonic Neoplasms , Ferroptosis , Mitochondrial Proteins , N-Terminal Acetyltransferases , Humans , Acetylation , Colonic Neoplasms/genetics , Ferroptosis/genetics , Neoplastic Processes , RNA, Messenger/genetics , N-Terminal Acetyltransferases/genetics , Apoptosis Regulatory Proteins/genetics , Mitochondrial Proteins/geneticsABSTRACT
Naa50 is the catalytic subunit of N-terminal acetyltransferase complex E, which plays an important role in regulating plant development, endoplasmic reticulum stress and immune responses in Arabidopsis. In this study, the complete genomic sequence (but not the coding sequence) of Naa50 rescued the phenotype of Naa50 deletion mutants. Naa50 expression was noted in whole roots except for central root cap cells. The deletion of intron 1 resulted in a loss of Naa50 expression in the root meristem zone and in the epidermis, cortex and endodermis of the elongation zone and mature zone, while the deletion of intron 2 decreased Naa50 expression in the epidermis, cortex and endodermis of the root elongation zone and mature zone. The native Naa50 promoter together with introns 1 and 2 promotes the expression of Naa50 in sepal vascular bundles, filaments, pollen and stigmas; however, neither intron has positive effect on Naa50 expression in mature rosette leaves. The results of this study show that introns 1 and 2 in the Naa50 gene function as enhancers to promote the tissue-specific expression of Naa50.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant , Introns/genetics , Meristem/metabolism , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolism , Plant Roots/metabolismABSTRACT
BACKGROUND: Although a substantial increase in the survival of patients with other cancers has been observed in recent decades, pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest diseases. No effective screening approach exists. METHODS: Differential exosomal long noncoding RNAs (lncRNAs) isolated from the serum of patients with PDAC and healthy individuals were profiled to screen for potential markers in liquid biopsies. The functions of LINC00623 in PDAC cell proliferation, migration and invasion were confirmed through in vivo and in vitro assays. RNA pulldown, RNA immunoprecipitation (RIP) and coimmunoprecipitation (Co-IP) assays and rescue experiments were performed to explore the molecular mechanisms of the LINC00623/NAT10 signaling axis in PDAC progression. RESULTS: A novel lncRNA, LINC00623, was identified, and its diagnostic value was confirmed, as it could discriminate patients with PDAC from patients with benign pancreatic neoplasms and healthy individuals. Moreover, LINC00623 was shown to promote the tumorigenicity and migratory capacity of PDAC cells in vitro and in vivo. Mechanistically, LINC00623 bound to N-acetyltransferase 10 (NAT10) and blocked its ubiquitination-dependent degradation by recruiting the deubiquitinase USP39. As a key regulator of N4-acetylcytidine (ac4C) modification of mRNA, NAT10 was demonstrated to maintain the stability of oncogenic mRNAs and promote their translation efficiency through ac4C modification. CONCLUSIONS: Our data revealed the role of LINC00623/NAT10 signaling axis in PDAC progression, showing that it is a potential biomarker and therapeutic target for PDAC.