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SlMYB72 and SlMYB75 antagonistically regulate trichome formation via the MYB-bHLH-WD40 complex in tomato.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-13 DOI: 10.1016/j.jbc.2025.108313
Tiancheng Qi, Mengbo Wu, Sijie Wang, Ying Yuan, Xin Xu, Qiongdan Zhang, Yongfei Jian, Dan Qiu, Yulin Cheng, Baowen Huang, Zhengguo Li, Weiqing Zhang, Wei Deng
{"title":"SlMYB72 and SlMYB75 antagonistically regulate trichome formation via the MYB-bHLH-WD40 complex in tomato.","authors":"Tiancheng Qi, Mengbo Wu, Sijie Wang, Ying Yuan, Xin Xu, Qiongdan Zhang, Yongfei Jian, Dan Qiu, Yulin Cheng, Baowen Huang, Zhengguo Li, Weiqing Zhang, Wei Deng","doi":"10.1016/j.jbc.2025.108313","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108313","url":null,"abstract":"<p><p>Trichomes are specialized epidermal outgrowths serving as protective barriers for plants against various stresses such as herbivore attacks. MYB-bHLH-WD40 complex is of great significance for unicellular trichome formation in Arabidopsis, whereas its role in the formation of multicellular trichomes in tomato remains largely unknown. In the present study, we identified that the R2R3-type MYB transcription factor SlMYB72 promotes the formation of type II, V, and VI trichomes through inhibiting the expression of SlCycB2, a repressor of trichome initiation. SlMYB75 is a negative regulator of trichome formation and positively regulates SlCycB2 expression. Interaction analyses showed that SlMYB72 and SlMYB75 can form MYB-bHLH-WD40 complexes with SlbHLH150 and SlTTG1, respectively, through mutual interactions. Dual-luciferase assay demonstrated that the regulatory functions of SlMYB72 and SlMYB75 in SlCycB2 expression can be enhanced by their corresponding MYB-bHLH-WD40 complexes. Interestingly, yeast-three-hybrid assay indicated that SlMYB75 competes with SlMYB72 for SlbHLH150 and SlTTG1, and counterbalances the down-regulation of SlCycB2 expression controlled by SlMYB72 alone, which is further confirmed by genetic hybrid experiments. These results reveal that SlMYB72 and SlMYB75 antagonistically regulate trichome formation and SlCycB2 expression through MYB-bHLH-WD40 complexes. These findings provide a novel perspective and theoretical basis for the formation of multicellular trichome in tomatoes and the development of highly resistant plants.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108313"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143424793","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Anaplerosis by medium-chain fatty acids through complex interplay with glucose and glutamine metabolism.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-13 DOI: 10.1016/j.jbc.2025.108307
Hannah M German, Jolita Ciapaite, Nanda M Verhoeven-Duif, Judith J M Jans
{"title":"Anaplerosis by medium-chain fatty acids through complex interplay with glucose and glutamine metabolism.","authors":"Hannah M German, Jolita Ciapaite, Nanda M Verhoeven-Duif, Judith J M Jans","doi":"10.1016/j.jbc.2025.108307","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108307","url":null,"abstract":"<p><p>The constant replenishment of tricarboxylic acid (TCA) cycle intermediates, or anaplerosis, is crucial to ensure optimal TCA cycle activity in times of high biosynthetic demand. In inborn metabolic diseases, anaplerosis is often affected, leading to impaired TCA cycle flux and ATP production. In these cases, anaplerotic compounds can be a therapy option. Triheptanoin, a triglyceride containing three heptanoate chains, is thought to be anaplerotic through production of propionyl- and acetyl-CoA. However, the precise mechanism underlying its anaplerotic action remains poorly understood. In this study, we performed a comprehensive in vitro analysis of heptanoate metabolism and compared it to that of octanoate, an even-chain fatty acid which only provides acetyl-CoA. Using stable isotope tracing, we demonstrate that both heptanoate and octanoate contribute carbon to the TCA cycle in HEK293T cells, confirming direct anaplerosis. Furthermore, by using labeled glucose and glutamine, we show that heptanoate and octanoate decrease the contribution of glucose-derived carbon and increase the influx of glutamine-derived carbon into the TCA cycle. Our findings also point towards a change in redox homeostasis, indicated by an increased NAD<sup>+</sup>/NADH ratio, accompanied by a decreased lactate/pyruvate ratio and increased de novo serine biosynthesis. Taken together, these results highlight the broad metabolic effects of heptanoate and octanoate supplementation, suggesting that therapeutic efficacy may strongly depend on specific disease pathophysiology. Furthermore, they underline the need for careful selection of fatty acid compound and concentration to optimize anaplerotic action.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108307"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Correction: KLF6 activates Sp1-mediated prolidase transcription during TGF-β1 signaling.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-13 DOI: 10.1016/j.jbc.2025.108214
Ireti Eni-Aganga, Zeljka Miletic Lanaghan, Farah Ismail, Olga Korolkova, Jeffery Shawn Goodwin, Muthukumar Balasubramaniam, Chandravanu Dash, Jui Pandhare
{"title":"Correction: KLF6 activates Sp1-mediated prolidase transcription during TGF-β1 signaling.","authors":"Ireti Eni-Aganga, Zeljka Miletic Lanaghan, Farah Ismail, Olga Korolkova, Jeffery Shawn Goodwin, Muthukumar Balasubramaniam, Chandravanu Dash, Jui Pandhare","doi":"10.1016/j.jbc.2025.108214","DOIUrl":"10.1016/j.jbc.2025.108214","url":null,"abstract":"","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"301 3","pages":"108214"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11871435/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425377","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Correction: Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-13 DOI: 10.1016/j.jbc.2025.108213
Prem Prakash, Purva Khodke, Muthukumar Balasubramaniam, Benem-Orom Davids, Thomas Hollis, Jamaine Davis, Bajarang Kumbhar, Chandravanu Dash
{"title":"Correction: Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties.","authors":"Prem Prakash, Purva Khodke, Muthukumar Balasubramaniam, Benem-Orom Davids, Thomas Hollis, Jamaine Davis, Bajarang Kumbhar, Chandravanu Dash","doi":"10.1016/j.jbc.2025.108213","DOIUrl":"10.1016/j.jbc.2025.108213","url":null,"abstract":"","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"301 3","pages":"108213"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11871443/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425383","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
GCIP and SIRT6 cooperatively suppress ITGAV gene expression by modulating c-myc transcription ability.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-13 DOI: 10.1016/j.jbc.2025.108314
Yi-Ching Huang, Tien-Ming Yuan, Bang-Hung Liu, Ruei-Yue Liang, Kai-Li Liu, Show-Mei Chuang
{"title":"GCIP and SIRT6 cooperatively suppress ITGAV gene expression by modulating c-myc transcription ability.","authors":"Yi-Ching Huang, Tien-Ming Yuan, Bang-Hung Liu, Ruei-Yue Liang, Kai-Li Liu, Show-Mei Chuang","doi":"10.1016/j.jbc.2025.108314","DOIUrl":"10.1016/j.jbc.2025.108314","url":null,"abstract":"<p><p>Grap2 and CyclinD1 interacting protein (GCIP) has been suggested to function as a tumor suppressor and acts as a transcriptional regulator that negatively controls cancer cell growth, invasion, and migration. Knockdown of GCIP reportedly enhances cancer cell migration and invasion, but no previous study has examined the mechanism(s) by which GCIP suppresses migration/invasion in cancer cells. Here, we report that cDNA microarray-based expression profiling of A549 cells without and with knockdown of GCIP reveals that the expression levels of ITGAV and ICAM-1 are negatively regulated by GCIP. In vitro co-immunoprecipitation and in vivo proximity ligation assays reveal that GCIP interacts with c-Myc. Sequence analyses reveal the presence of two c-Myc regulatory motifs (E-boxes) within the ITGAV promoter. Luciferase reporter and ChIP assays indicate that GCIP represses ITGAV transcription by interacting with c-Myc on the E-box binding sites of the ITGAV promoter region. Furthermore, GCIP interacts with SIRT6 in vitro and in vivo and cooperates with SIRT6, thereby linking its activity, to negatively regulate transcription at the E-box by modulating c-Myc transcription ability. Taken together, these findings contribute to our understanding of GCIP in tumorigenesis and identify a previously unrecognized function of GCIP: It can interact with c-Myc and SIRT6 at E-box binding sites of the ITGAV promoter region. Our data collectively reveal a regulatory network involving GCIP, SIRT6, c-Myc, and ITGAV, and suggest that the SIRT6-GCIP complex negatively regulates the oncogenic function of c-Myc in cell proliferation and migration.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108314"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425368","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Staufen2 dysregulation in neurodegenerative disease.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-13 DOI: 10.1016/j.jbc.2025.108316
Sharan Paul, Warunee Dansithong, Karla P Figueroa, Mandi Gandelman, Pravin Hivare, Daniel R Scoles, Stefan M Pulst
{"title":"Staufen2 dysregulation in neurodegenerative disease.","authors":"Sharan Paul, Warunee Dansithong, Karla P Figueroa, Mandi Gandelman, Pravin Hivare, Daniel R Scoles, Stefan M Pulst","doi":"10.1016/j.jbc.2025.108316","DOIUrl":"10.1016/j.jbc.2025.108316","url":null,"abstract":"<p><p>Staufen2 (STAU2) is an RNA-binding protein that controls mRNA trafficking and expression. Previously, we showed that its paralog, Staufen1 (STAU1), was overabundant in cellular and mouse models of neurodegenerative diseases and amyotrophic lateral sclerosis (ALS) patient spinal cord. Here, we investigated features of STAU2 that might parallel STAU1. STAU2 protein, but not mRNA, was overabundant in spinocerebellar ataxia type 2 (SCA2), ALS/frontotemporal dementia patient fibroblasts, ALS patient spinal cord tissues, and in central nervous system tissues from SCA2 and ALS animal models. Exogenous expression of STAU2 in human embryonic kidney 293 cells activated mechanistic target of rapamycin (mTOR) and stress granule formation. Targeting STAU2 by RNAi normalized mTOR in SCA2 and C9ORF72 cellular models. The microRNA miR-217, previously identified as downregulated in SCA2 mice, targets the STAU2 3'-UTR. We now demonstrate that exogenous expression of miR-217 significantly reduced STAU2 and mTOR levels in cellular models of neurodegenerative disease. These results suggest a functional link between STAU2 and mTOR signaling and identify a major role for miR-217 that could be exploited in therapeutic development.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108316"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143424802","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Heme delivery into soluble guanylyl cyclase requires a heme redox change and is regulated by NO and Hsp90 by distinct mechanisms.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-13 DOI: 10.1016/j.jbc.2025.108315
Yue Dai, Dennis J Stuehr
{"title":"Heme delivery into soluble guanylyl cyclase requires a heme redox change and is regulated by NO and Hsp90 by distinct mechanisms.","authors":"Yue Dai, Dennis J Stuehr","doi":"10.1016/j.jbc.2025.108315","DOIUrl":"10.1016/j.jbc.2025.108315","url":null,"abstract":"<p><p>Nitric oxide (NO) signaling often relies on it activating cGMP production by the heterodimeric enzyme soluble guanylyl cyclase (sGC). To mature to function, an sGCβ subunit must first incorporate heme and then form a heterodimer with a partner α subunit. Our previous studies in cells showed that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) supplies heme to the apo-sGCβ subunit, which is complexed with the cell chaperone Hsp90. Through its ATP hydrolysis, Hsp90 then promotes heme insertion into apo-sGCβ and consequent formation of a functional heterodimer. NO at physiologic levels somehow stimulates cell heme allocation into apo-sGCβ by this process. To gain insight, we utilized purified apo-sGCβ and GAPDH reporter proteins whose heme contents can be followed by fluorescence and determined the impact of Hsp90 and NO on heme transfer between them. Results show that heme transfer out of GAPDH and into apo-sGCβ is tightly coupled in all circumstances and is limited by the ability of the apo-sGCβ to incorporate the heme, which in turn relies on a ferric to ferrous heme transition taking place inside the sGCβ. Hsp90 can influence the heme transfer kinetics in a negative or positive manner through its conformational effects on apo-sGCβ, while NO speeds heme transfer by binding to the heme iron and thus speeding heme dissociation from GAPDH. Our findings provide new mechanistic understanding of sGC maturation and how Hsp90 and NO combine to dynamically regulate heme incorporation for sGC heterodimer formation and consequent cGMP production in biological settings.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108315"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425446","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Identifying the copper coordination environment between interacting neurodegenerative proteins: A new approach using pulsed EPR with 14N/15N isotopic labeling.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-13 DOI: 10.1016/j.jbc.2025.108311
Amanda Smart, Kevin Singewald, Zikri Hasanbasri, R David Britt, Glenn L Millhauser
{"title":"Identifying the copper coordination environment between interacting neurodegenerative proteins: A new approach using pulsed EPR with <sup>14</sup>N/<sup>15</sup>N isotopic labeling.","authors":"Amanda Smart, Kevin Singewald, Zikri Hasanbasri, R David Britt, Glenn L Millhauser","doi":"10.1016/j.jbc.2025.108311","DOIUrl":"10.1016/j.jbc.2025.108311","url":null,"abstract":"<p><p>The trafficking and aggregation of neurodegenerative proteins often involve the interaction between intrinsically disordered domains, stabilized by the inclusion of physiological metal ions such as copper or zinc. Characterizing the metal ion coordination environment is critical for assessing the stability and organization of these relevant protein-protein interactions but is challenging given the lack of regular molecular order or global structure. The cellular prion protein (PrP<sup>C</sup>) binds both monomers and aggregates of Alzheimer's amyloid-beta (Aβ), promoting Aβ internalization and aberrant signaling, respectively. Both proteins bind Cu<sup>2+</sup> with high affinity, opening the potential for copper to form an intermolecular bridge. We describe here a novel approach utilizing multiple EPR experiments to investigate the simultaneous Cu<sup>2+</sup> coordination of PrP<sup>C</sup> and Aβ in a 1:1:1 mixture. Uniformly <sup>15</sup>N-labeled PrP<sup>C</sup> is used in conjunction with natural abundance <sup>14</sup>N Aβ, the combination of which leads to distinct energy manifolds for paramagnetic Cu<sup>2+</sup> and is resolved by the pulsed EPR experiments ESEEM and HYSCORE. We develop acquisition parameters to simultaneously optimize <sup>14</sup>N (I = 1) and <sup>15</sup>N (I = ½) pulsed EPR signals and we also advance the theory of ESEEM and HYSCORE to quantitatively describe multiple <sup>15</sup>N imidazole coordination. This unique approach provides compelling evidence of a copper-stabilized ternary complex, with equatorial Cu<sup>2+</sup> coordination formed by one histidine imidazole from Aβ and three from PrP. Moreover, the methodologies developed here provide a framework for assessing the copper environment in other interacting neurodegenerative proteins.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108311"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425387","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
G protein-coupled estrogen receptor deficiency exacerbates demyelination through microglial ferroptosis.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-13 DOI: 10.1016/j.jbc.2025.108312
Xiaojuan Mi, Junjie Li, Ziqi Feng, Yanbo Liu, Chun Zhang, Yu Shao, Ting Wang, Zhilun Yang, Haowen Lv, Juan Liu
{"title":"G protein-coupled estrogen receptor deficiency exacerbates demyelination through microglial ferroptosis.","authors":"Xiaojuan Mi, Junjie Li, Ziqi Feng, Yanbo Liu, Chun Zhang, Yu Shao, Ting Wang, Zhilun Yang, Haowen Lv, Juan Liu","doi":"10.1016/j.jbc.2025.108312","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108312","url":null,"abstract":"<p><p>Microglial activation is the initial pathological event that occurs in demyelination, a prevalent feature in various neurological diseases. G protein-coupled estrogen receptor (GPER1), which is highly expressed in microglia, has been reported to reduce myelin damage. However, the precise molecular mechanisms involved remain unclear. In this study, the cuprizone (CPZ) -induced demyelination model was used to investigate the relationship between GPER1 and myelin sheath injury and its mechanism. The results demonstrated that GPER1 deficiency exacerbated cognitive impairment in mice. Along with more severe myelin damage as well as fewer oligodendrocytes. Moreover, GPER1 deficiency not only directly reduced the number of microglia in CPZ mice, but also caused iron ions overload in microglia of myelin debris induced in vitro. Transcriptomic, molecular biological, and morphological analyses revealed that microglial ferroptosis caused by GPER1 deficiency contributes to the reduction of microglia number. In summary, these findings revealed that GPER1 can regulate demyelination through ferroptosis of microglia.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108312"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425367","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
A CRISPR-edited isoform of the AMPK kinase LKB1 improves the response to cisplatin in A549 lung cancer cells.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-13 DOI: 10.1016/j.jbc.2025.108308
Matheus Brandemarte Severino, Ana Paula Morelli, Isadora Carolina Betim Pavan, Mariana Camargo Silva Mancini, Mariana Marcela Góis, Rafael Junqueira Borges, Renata Rosseto Braga, Luiz Guilherme Salvino da Silva, Nathalia Quintero-Ruiz, Maíra Maftoum Costa, Wesley de Lima Oliveira, Rosângela Maria Neves Bezerra, Eduardo Rochete Ropelle, Fernando Moreira Simabuco
{"title":"A CRISPR-edited isoform of the AMPK kinase LKB1 improves the response to cisplatin in A549 lung cancer cells.","authors":"Matheus Brandemarte Severino, Ana Paula Morelli, Isadora Carolina Betim Pavan, Mariana Camargo Silva Mancini, Mariana Marcela Góis, Rafael Junqueira Borges, Renata Rosseto Braga, Luiz Guilherme Salvino da Silva, Nathalia Quintero-Ruiz, Maíra Maftoum Costa, Wesley de Lima Oliveira, Rosângela Maria Neves Bezerra, Eduardo Rochete Ropelle, Fernando Moreira Simabuco","doi":"10.1016/j.jbc.2025.108308","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108308","url":null,"abstract":"<p><p>Lung cancer presents the highest mortality rate in the world when compared to other cancer types and often presents chemotherapy resistance to cisplatin. The A549 non-small cell lung cancer (NSCLC) line is widely used as a model for lung adenocarcinoma studies since it presents a high proliferative rate and a nonsense mutation in the STK11 gene. The LKB1 protein, encoded by the STK11 gene, is one of the major regulators of cellular metabolism through AMPK activation under nutrient deprivation. Mutation in the STK11 gene in A549 cells potentiates cancer hallmarks, such as deregulation of cellular metabolism, aside from the Warburg effect, mTOR activation, autophagy inhibition, and NRF2 and redox activation. In this study, we investigated the integration of these pathways associated with the metabolism regulation by LKB1/AMPK to improve cisplatin response in the A549 cell line. We first used the CRISPR/Cas9 system to generate cell lines with a CRISPR-edited LKB1 isoform (called Super LKB1), achieved through the introduction of a +1 adenine insertion in the first exon of the STK11 gene after NHEJ mediated repair. This insertion led to the expression of a higher molecular weight protein containing an alternative exon described in the Peutz-Jeghers Syndrome (PJS). Through metabolic regulation by Super LKB1 expression and AMPK activation, we found an increase in autophagy flux (LC3 GFP/RFP p<0.05), as well as a reduction in the phosphorylation of mTORC1 downstream targets (S6K2 phospho-Serine 423; p<0.05; and S6 ribosomal protein phospho-Serine 240/244; p<0.03). The NRF2 protein exhibited increased levels and more nuclear localization in A549 WT (wild-type) cells compared to the edited cells (p<0.01). We also observed lower levels of H<sub>2</sub>O<sub>2</sub> in the WT A549 cells, as a possible result of NRF2 activation, and a higher requirement of cisplatin to achieve the IC<sub>50</sub> (WT: 10 μM; c2SL+: 5.5 μM; c3SL+: 6 μM). The data presented here suggests that the regulation of molecular pathways by the novel Super LKB1 in A549 cells related to metabolism, mTORC1, and autophagy promotes a better response of lung cancer cells to cisplatin. This NHEJ-CRISPR-based approach may be potentially used for lung cancer gene therapy.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108308"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425348","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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