Pub Date : 2024-11-07DOI: 10.1016/j.chembiol.2024.10.006
Alexandria N. Van Scoyk, Orlando Antelope, Donald E. Ayer, Randall T. Peterson, Anthony D. Pomicter, Shawn C. Owen, Michael W. Deininger
Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine toward maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple conditions, including cancer. Measuring sirtuins’ activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed “SIRTify”, a high-sensitivity assay for measuring sirtuin activity in vitro and in vivo. SIRTify is based on a split-version of the NanoLuc luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine-acylations and confirm the effects of sirtuin modulators. SIRTify quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.
{"title":"Bioluminescence assay of lysine deacylase sirtuin activity","authors":"Alexandria N. Van Scoyk, Orlando Antelope, Donald E. Ayer, Randall T. Peterson, Anthony D. Pomicter, Shawn C. Owen, Michael W. Deininger","doi":"10.1016/j.chembiol.2024.10.006","DOIUrl":"https://doi.org/10.1016/j.chembiol.2024.10.006","url":null,"abstract":"Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine toward maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple conditions, including cancer. Measuring sirtuins’ activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed “SIRT<em>ify</em>”, a high-sensitivity assay for measuring sirtuin activity <em>in vitro</em> and <em>in vivo</em>. SIRT<em>ify</em> is based on a split-version of the NanoLuc luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine-acylations and confirm the effects of sirtuin modulators. SIRT<em>ify</em> quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":null,"pages":null},"PeriodicalIF":8.6,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142594511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-04DOI: 10.1016/j.chembiol.2024.10.004
Mackenzie W. Krone, Craig M. Crews
Targeted protein degradation (TPD) has greatly advanced as a therapeutic strategy in the past two decades, and we are on the cusp of rationally designed protein degraders reaching clinical approval. Offering pharmacological advantages relative to occupancy-driven protein inhibition, chemical methods for regulating biomolecular proximity have provided opportunities to tackle disease-related targets that were undruggable. Despite the pre-clinical success of designed degraders and existence of clinical therapies that serendipitously utilize TPD, expansion of the TPD toolbox is necessary to identify and characterize the next generation of molecular degraders. Here we highlight three areas for continued growth in the field that should be prioritized: expansion of TPD platform with greater spatiotemporal precision, increased throughput of degrader synthesis, and optimization of cooperativity in chemically induced protein complexes. The future is bright for TPD in medicine, and we expect that innovative approaches will increase therapeutic applications of proximity-induced pharmacology.
{"title":"Next steps for targeted protein degradation","authors":"Mackenzie W. Krone, Craig M. Crews","doi":"10.1016/j.chembiol.2024.10.004","DOIUrl":"https://doi.org/10.1016/j.chembiol.2024.10.004","url":null,"abstract":"Targeted protein degradation (TPD) has greatly advanced as a therapeutic strategy in the past two decades, and we are on the cusp of rationally designed protein degraders reaching clinical approval. Offering pharmacological advantages relative to occupancy-driven protein inhibition, chemical methods for regulating biomolecular proximity have provided opportunities to tackle disease-related targets that were undruggable. Despite the pre-clinical success of designed degraders and existence of clinical therapies that serendipitously utilize TPD, expansion of the TPD toolbox is necessary to identify and characterize the next generation of molecular degraders. Here we highlight three areas for continued growth in the field that should be prioritized: expansion of TPD platform with greater spatiotemporal precision, increased throughput of degrader synthesis, and optimization of cooperativity in chemically induced protein complexes. The future is bright for TPD in medicine, and we expect that innovative approaches will increase therapeutic applications of proximity-induced pharmacology.","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":null,"pages":null},"PeriodicalIF":8.6,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142574666","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-01DOI: 10.1016/j.chembiol.2024.10.003
Hiroki Miura, Kang-Hsin Wang, Tomoki Inagaki, Frank Chuang, Michiko Shimoda, Chie Izumiya, Tadashi Watanabe, Ryan R. Davis, Clifford G. Tepper, Somayeh Komaki, Ken-ichi Nakajima, Ashish Kumar, Yoshihiro Izumiya
Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a latent infection, and viral genes are poised to be transcribed in the latent chromatin. In the poised chromatins, KSHV latency-associated nuclear antigen (LANA) interacts with cellular chromodomain-helicase-DNA-binding protein 4 (CHD4) and inhibits viral promoter activation. CHD4 is known to regulate cell differentiation by preventing enhancers from activating promoters. Here, we identified a putative CHD4 inhibitor peptide (VGN73) from the LANA sequence corresponding to the LANA-CHD4 interaction surface. The VGN73 interacts with CHD4 at its PHD domain with a dissociation constant (KD) of 14 nM. Pre-treatment with VGN73 enhanced monocyte differentiation into macrophages and globally altered the repertoire of activated genes in U937 cells. Furthermore, the introduction of the peptide into the cancer cells induced caspase-mediated CHD4 cleavage, triggered cell death, and inhibited tumor growth in a xenograft mouse model. The VGN73 may facilitate cell differentiation therapy.
{"title":"A LANA peptide inhibits tumor growth by inducing CHD4 protein cleavage and triggers cell death","authors":"Hiroki Miura, Kang-Hsin Wang, Tomoki Inagaki, Frank Chuang, Michiko Shimoda, Chie Izumiya, Tadashi Watanabe, Ryan R. Davis, Clifford G. Tepper, Somayeh Komaki, Ken-ichi Nakajima, Ashish Kumar, Yoshihiro Izumiya","doi":"10.1016/j.chembiol.2024.10.003","DOIUrl":"https://doi.org/10.1016/j.chembiol.2024.10.003","url":null,"abstract":"Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a latent infection, and viral genes are poised to be transcribed in the latent chromatin. In the poised chromatins, KSHV latency-associated nuclear antigen (LANA) interacts with cellular chromodomain-helicase-DNA-binding protein 4 (CHD4) and inhibits viral promoter activation. CHD4 is known to regulate cell differentiation by preventing enhancers from activating promoters. Here, we identified a putative CHD4 inhibitor peptide (VGN73) from the LANA sequence corresponding to the LANA-CHD4 interaction surface. The VGN73 interacts with CHD4 at its PHD domain with a dissociation constant (K<sub>D</sub>) of 14 nM. Pre-treatment with VGN73 enhanced monocyte differentiation into macrophages and globally altered the repertoire of activated genes in U937 cells. Furthermore, the introduction of the peptide into the cancer cells induced caspase-mediated CHD4 cleavage, triggered cell death, and inhibited tumor growth in a xenograft mouse model. The VGN73 may facilitate cell differentiation therapy.","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":null,"pages":null},"PeriodicalIF":8.6,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142562065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-31DOI: 10.1016/j.chembiol.2024.10.002
Yingchao Hu, Honghui Li, Xiangyu Zhang, Yuxian Song, Jun Liu, Jie Pu, Shuang Wen, Hongyang Xu, Hongliang Xin, Bingwei Wang, Shuo Yang
As an executor of pyroptosis, gasdermin D (GSDMD) plays a critical role in inflammatory diseases and cancer. Thus, GSDMD is currently being widely explored as a drug target. Existing inhibitors targeting GSDMD, such as necrosulfonamide, disulfiram, and fumarate, primarily prevent pyroptosis by modifying human/mouse C191/C192 in the N-terminal fragment of GSDMD. However, cysteine modification can prevent the function of important proteins or enzymes, thereby leading to adverse reactions. Here, we chose an alternative key intervention site for GSDMD activation, which is located at the oligomerization interface I of its pore-forming structure. Through high-throughput virtual and experimental screening and in combination with efficacy and pharmacological validation, we have identified two safe, specific “repurposed drugs” that potently suppress GSDMD-mediated pyroptosis. Moreover, the candidates exhibited synergistic therapeutic effects of “1 + 1>2” in murine sepsis and tumorigenesis models. These recently identified GSDMD inhibitors hold great promise for clinical translation in the development of anti-inflammatory and anti-cancer immunotherapies.
{"title":"Identification of two repurposed drugs targeting GSDMD oligomerization interface I to block pyroptosis","authors":"Yingchao Hu, Honghui Li, Xiangyu Zhang, Yuxian Song, Jun Liu, Jie Pu, Shuang Wen, Hongyang Xu, Hongliang Xin, Bingwei Wang, Shuo Yang","doi":"10.1016/j.chembiol.2024.10.002","DOIUrl":"https://doi.org/10.1016/j.chembiol.2024.10.002","url":null,"abstract":"As an executor of pyroptosis, gasdermin D (GSDMD) plays a critical role in inflammatory diseases and cancer. Thus, GSDMD is currently being widely explored as a drug target. Existing inhibitors targeting GSDMD, such as necrosulfonamide, disulfiram, and fumarate, primarily prevent pyroptosis by modifying human/mouse C191/C192 in the N-terminal fragment of GSDMD. However, cysteine modification can prevent the function of important proteins or enzymes, thereby leading to adverse reactions. Here, we chose an alternative key intervention site for GSDMD activation, which is located at the oligomerization interface I of its pore-forming structure. Through high-throughput virtual and experimental screening and in combination with efficacy and pharmacological validation, we have identified two safe, specific “repurposed drugs” that potently suppress GSDMD-mediated pyroptosis. Moreover, the candidates exhibited synergistic therapeutic effects of “1 + 1>2” in murine sepsis and tumorigenesis models. These recently identified GSDMD inhibitors hold great promise for clinical translation in the development of anti-inflammatory and anti-cancer immunotherapies.","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":null,"pages":null},"PeriodicalIF":8.6,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142556318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-28DOI: 10.1016/j.chembiol.2024.10.001
Viviane S. De Paula, Abhinav Dubey, Haribabu Arthanari, Nikolaos G. Sgourakis
CRISPR-Cas9 has revolutionized genome engineering applications by programming its single-guide RNA, where high specificity is required. However, the precise molecular mechanism underscoring discrimination between on/off-target DNA sequences, relative to the guide RNA template, remains elusive. Here, using methyl-based NMR to study multiple holoenzymes assembled in vitro, we elucidate a discrete protein conformational state which enables recognition of DNA mismatches at the protospacer adjacent motif (PAM)-distal end. Our results delineate an allosteric pathway connecting a dynamic conformational switch at the REC3 domain, with the sampling of a catalytically competent state by the HNH domain. Our NMR data show that HiFi Cas9 (R691A) increases the fidelity of DNA recognition by stabilizing this "surveillance state" for mismatched substrates, shifting the Cas9 conformational equilibrium away from the active state. These results establish a paradigm of substrate recognition through an allosteric protein-based switch, providing unique insights into the molecular mechanism which governs Cas9 selectivity.
CRISPR-Cas9 通过对需要高特异性的单导 RNA 进行编程，彻底改变了基因组工程应用。然而，相对于引导 RNA 模板而言，区分目标 DNA 序列的精确分子机制仍未确定。在这里，我们利用基于甲基的核磁共振技术研究了体外组装的多个全酶，阐明了一种离散的蛋白质构象状态，它能识别原间隔邻接基序（PAM）远端的 DNA 错配。我们的研究结果勾勒出了一条异构途径，它将 REC3 结构域的动态构象转换与 HNH 结构域的催化状态取样连接起来。我们的核磁共振数据显示，HiFi Cas9 (R691A)通过稳定这种针对不匹配底物的 "监视状态"，使 Cas9 的构象平衡偏离活性状态，从而提高了 DNA 识别的保真度。这些结果建立了一种通过基于异构蛋白的开关来识别底物的范例，为研究支配 Cas9 选择性的分子机制提供了独特的见解。
{"title":"Dynamic sampling of a surveillance state enables DNA proofreading by Cas9","authors":"Viviane S. De Paula, Abhinav Dubey, Haribabu Arthanari, Nikolaos G. Sgourakis","doi":"10.1016/j.chembiol.2024.10.001","DOIUrl":"https://doi.org/10.1016/j.chembiol.2024.10.001","url":null,"abstract":"CRISPR-Cas9 has revolutionized genome engineering applications by programming its single-guide RNA, where high specificity is required. However, the precise molecular mechanism underscoring discrimination between on/off-target DNA sequences, relative to the guide RNA template, remains elusive. Here, using methyl-based NMR to study multiple holoenzymes assembled <em>in vitro</em>, we elucidate a discrete protein conformational state which enables recognition of DNA mismatches at the protospacer adjacent motif (PAM)-distal end. Our results delineate an allosteric pathway connecting a dynamic conformational switch at the REC3 domain, with the sampling of a catalytically competent state by the HNH domain. Our NMR data show that HiFi Cas9 (R691A) increases the fidelity of DNA recognition by stabilizing this \"surveillance state\" for mismatched substrates, shifting the Cas9 conformational equilibrium away from the active state. These results establish a paradigm of substrate recognition through an allosteric protein-based switch, providing unique insights into the molecular mechanism which governs Cas9 selectivity.","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":null,"pages":null},"PeriodicalIF":8.6,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142519849","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-24DOI: 10.1016/j.chembiol.2024.09.010
Guilherme M. Lima, Zeinab Jame-Chenarboo, Mirat Sojitra, Susmita Sarkar, Eric J. Carpenter, Claire Y. Yang, Edward Schmidt, Justine Lai, Alexey Atrazhev, Danial Yazdan, Chuanhao Peng, Elizabeth A. Volker, Ray Ho, Gisele Monteiro, Raymond Lai, Lara K. Mahal, Matthew S. Macauley, Ratmir Derda
Selective detection of disease-associated changes in the glycocalyx is an emerging field in modern targeted therapies. Detecting minor glycan changes on the cell surface is a challenge exacerbated by the lack of correspondence between cellular DNA/RNA and glycan structures. We demonstrate that multivalent displays of lectins on DNA-barcoded phages—liquid lectin array (LiLA)—detect subtle differences in density of glycans on cells. LiLA constructs displaying 73 copies of diCBM40 (CBM) lectin per virion (φ-CBM73) exhibit non-linear ON/OFF-like recognition of sialoglycans on the surface of normal and cancer cells. A high-valency φ-CBM290 display, or soluble CBM protein, cannot amplify the subtle differences detected by φ-CBM73. Similarly, multivalent displays of CBM and Siglec-7 detect differences in the glycocalyx between stem-like and non-stem populations in cancer. Multivalent display of lectins offer in situ detection of minor differences in glycocalyx in cells both in vitro and in vivo not feasible to currently available technologies.
{"title":"The liquid lectin array detects compositional glycocalyx differences using multivalent DNA-encoded lectins on phage","authors":"Guilherme M. Lima, Zeinab Jame-Chenarboo, Mirat Sojitra, Susmita Sarkar, Eric J. Carpenter, Claire Y. Yang, Edward Schmidt, Justine Lai, Alexey Atrazhev, Danial Yazdan, Chuanhao Peng, Elizabeth A. Volker, Ray Ho, Gisele Monteiro, Raymond Lai, Lara K. Mahal, Matthew S. Macauley, Ratmir Derda","doi":"10.1016/j.chembiol.2024.09.010","DOIUrl":"https://doi.org/10.1016/j.chembiol.2024.09.010","url":null,"abstract":"Selective detection of disease-associated changes in the glycocalyx is an emerging field in modern targeted therapies. Detecting minor glycan changes on the cell surface is a challenge exacerbated by the lack of correspondence between cellular DNA/RNA and glycan structures. We demonstrate that multivalent displays of lectins on DNA-barcoded phages—liquid lectin array (LiLA)—detect subtle differences in density of glycans on cells. LiLA constructs displaying 73 copies of diCBM40 (CBM) lectin per virion (φ-CBM<sub>73</sub>) exhibit non-linear ON/OFF-like recognition of sialoglycans on the surface of normal and cancer cells. A high-valency φ-CBM<sub>290</sub> display, or soluble CBM protein, cannot amplify the subtle differences detected by φ-CBM<sub>73</sub>. Similarly, multivalent displays of CBM and Siglec-7 detect differences in the glycocalyx between stem-like and non-stem populations in cancer. Multivalent display of lectins offer <em>in situ</em> detection of minor differences in glycocalyx in cells both <em>in vitro</em> and <em>in vivo</em> not feasible to currently available technologies.","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":null,"pages":null},"PeriodicalIF":8.6,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142488902","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-23DOI: 10.1016/j.chembiol.2024.09.009
Lu Xiao, Linglan Fang, Wenrui Zhong, Eric T. Kool
RNAs fold into compact structures and undergo protein interactions in cells. These occluded environments can block reagents that probe the underlying RNAs. Probes that can analyze structure in crowded settings can shed light on RNA biology. Here, we employ 2′-OH-reactive probes that are small enough to access folded RNA structure underlying close molecular contacts within cells, providing considerably broader coverage for intracellular RNA structural analysis. The data are analyzed first with well-characterized human ribosomal RNAs and then applied transcriptome-wide to polyadenylated transcripts. The smallest probe acetylimidazole (AcIm) yields 80% greater structural coverage than larger conventional reagent NAIN3, providing enhanced structural information in hundreds of transcripts. The acetyl probe also provides superior signals for identifying m6A modification sites in transcripts, particularly in sites that are inaccessible to a standard probe. Our strategy enables profiling RNA infrastructure, enhancing analysis of transcriptome structure, modification, and intracellular interactions, especially in spatially crowded settings.
{"title":"RNA infrastructure profiling illuminates transcriptome structure in crowded spaces","authors":"Lu Xiao, Linglan Fang, Wenrui Zhong, Eric T. Kool","doi":"10.1016/j.chembiol.2024.09.009","DOIUrl":"https://doi.org/10.1016/j.chembiol.2024.09.009","url":null,"abstract":"RNAs fold into compact structures and undergo protein interactions in cells. These occluded environments can block reagents that probe the underlying RNAs. Probes that can analyze structure in crowded settings can shed light on RNA biology. Here, we employ 2′-OH-reactive probes that are small enough to access folded RNA structure underlying close molecular contacts within cells, providing considerably broader coverage for intracellular RNA structural analysis. The data are analyzed first with well-characterized human ribosomal RNAs and then applied transcriptome-wide to polyadenylated transcripts. The smallest probe acetylimidazole (AcIm) yields 80% greater structural coverage than larger conventional reagent NAIN3, providing enhanced structural information in hundreds of transcripts. The acetyl probe also provides superior signals for identifying m<sup>6</sup>A modification sites in transcripts, particularly in sites that are inaccessible to a standard probe. Our strategy enables profiling RNA infrastructure, enhancing analysis of transcriptome structure, modification, and intracellular interactions, especially in spatially crowded settings.","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":null,"pages":null},"PeriodicalIF":8.6,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142487423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-22DOI: 10.1016/j.chembiol.2024.09.008
Kelly H. Sokol, Cameron J. Lee, Thomas J. Rogers, Althea Waldhart, Abigail E. Ellis, Sahithi Madireddy, Samuel R. Daniels, Rachel (Rae) J. House, Xinyu Ye, Mary Olesnavich, Amy Johnson, Benjamin R. Furness, Ryan D. Sheldon, Evan C. Lien
Ferroptosis is a form of cell death caused by lipid peroxidation that is emerging as a target for cancer therapy, highlighting the need to identify factors that govern ferroptosis susceptibility. Lipid peroxidation occurs primarily on phospholipids containing polyunsaturated fatty acids (PUFAs). Here, we show that even though extracellular lipid limitation reduces cellular PUFA levels, lipid-starved cancer cells are paradoxically more sensitive to ferroptosis. Using mass spectrometry-based lipidomics with stable isotope fatty acid labeling, we show that lipid limitation induces a fatty acid trafficking pathway in which PUFAs are liberated from triglycerides to synthesize highly unsaturated PUFAs such as arachidonic and adrenic acid. These PUFAs then accumulate in phospholipids, including ether phospholipids, to promote ferroptosis sensitivity. Therefore, PUFA levels within cancer cells do not necessarily correlate with ferroptosis susceptibility. Rather, how cancer cells respond to extracellular lipid levels by trafficking PUFAs into proper phospholipid pools contributes to their sensitivity to ferroptosis.
{"title":"Lipid availability influences ferroptosis sensitivity in cancer cells by regulating polyunsaturated fatty acid trafficking","authors":"Kelly H. Sokol, Cameron J. Lee, Thomas J. Rogers, Althea Waldhart, Abigail E. Ellis, Sahithi Madireddy, Samuel R. Daniels, Rachel (Rae) J. House, Xinyu Ye, Mary Olesnavich, Amy Johnson, Benjamin R. Furness, Ryan D. Sheldon, Evan C. Lien","doi":"10.1016/j.chembiol.2024.09.008","DOIUrl":"https://doi.org/10.1016/j.chembiol.2024.09.008","url":null,"abstract":"Ferroptosis is a form of cell death caused by lipid peroxidation that is emerging as a target for cancer therapy, highlighting the need to identify factors that govern ferroptosis susceptibility. Lipid peroxidation occurs primarily on phospholipids containing polyunsaturated fatty acids (PUFAs). Here, we show that even though extracellular lipid limitation reduces cellular PUFA levels, lipid-starved cancer cells are paradoxically more sensitive to ferroptosis. Using mass spectrometry-based lipidomics with stable isotope fatty acid labeling, we show that lipid limitation induces a fatty acid trafficking pathway in which PUFAs are liberated from triglycerides to synthesize highly unsaturated PUFAs such as arachidonic and adrenic acid. These PUFAs then accumulate in phospholipids, including ether phospholipids, to promote ferroptosis sensitivity. Therefore, PUFA levels within cancer cells do not necessarily correlate with ferroptosis susceptibility. Rather, how cancer cells respond to extracellular lipid levels by trafficking PUFAs into proper phospholipid pools contributes to their sensitivity to ferroptosis.","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":null,"pages":null},"PeriodicalIF":8.6,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142487024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-21DOI: 10.1016/j.chembiol.2024.09.007
Jujun Zhou, Qin Chen, Ren Ren, Jie Yang, Bigang Liu, John R. Horton, Caleb Chang, Chuxuan Li, Leora Maksoud, Yifei Yang, Dante Rotili, Abhinav K. Jain, Xing Zhang, Robert M. Blumenthal, Taiping Chen, Yang Gao, Sergio Valente, Antonello Mai, Xiaodong Cheng
DNA methylation, as exemplified by cytosine-C5 methylation in mammals and adenine-N6 methylation in bacteria, is a key epigenetic process. Developing non-nucleoside inhibitors to cause DNA hypomethylation is crucial for treating various conditions without the toxicities associated with existing cytidine-based hypomethylating agents. This study characterized fifteen quinoline-based analogs, particularly compounds with additions like a methylamine (9) or methylpiperazine (11), which demonstrate similar low micromolar inhibitory potency against human DNMT1 and Clostridioides difficile CamA. These compounds (9 and 11) intercalate into CamA-bound DNA via the minor groove, causing a conformational shift that moves the catalytic domain away from the DNA. This study adds to the limited examples of DNA methyltransferases being inhibited by non-nucleotide compounds through DNA intercalation. Additionally, some quinoline-based analogs inhibit other DNA-interacting enzymes, such as polymerases and base excision repair glycosylases. Finally, compound 11 elicits DNA damage response via p53 activation in cancer cells.
DNA 甲基化是一个关键的表观遗传过程，例如哺乳动物中的胞嘧啶-C5 甲基化和细菌中的腺嘌呤-N6 甲基化。开发非核苷类抑制剂来引起 DNA 低甲基化，对于治疗各种疾病而不产生现有的基于胞嘧啶的低甲基化药物的毒性至关重要。本研究鉴定了 15 种喹啉类类似物，特别是添加了甲胺（9）或甲基哌嗪（11）的化合物，它们对人类 DNMT1 和艰难梭菌 CamA 具有类似的低微摩尔抑制效力。这些化合物（9 和 11）通过小沟插层到与 CamA 结合的 DNA 中，引起构象转变，使催化结构域远离 DNA。这项研究增加了非核苷酸化合物通过 DNA 插层抑制 DNA 甲基转移酶的有限实例。此外，一些喹啉类似物还能抑制其他与 DNA 有相互作用的酶，如聚合酶和碱基切除修复糖基酶。最后，化合物 11 可通过激活癌细胞中的 p53 引起 DNA 损伤反应。
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Pub Date : 2024-10-17DOI: 10.1016/j.chembiol.2024.09.003
Abnormal accumulation of hyperphosphorylated tau (pTau) is a major cause of neurodegeneration in Alzheimer’s disease (AD) and related tauopathies. Therefore, reducing pTau holds therapeutic promise for these diseases. Here, we developed a chimeric peptide, named D20, for selective facilitation of tau dephosphorylation by recruiting protein phosphatase 1 (PP1) to tau. PP1 is one of the active phosphatases that dephosphorylates tau. In both cultured primary hippocampal neurons and mouse models for AD or related tauopathies, we demonstrated that single-dose D20 treatment significantly reduced pTau by dephosphorylation at multiple AD-related sites and total tau (tTau) levels were also decreased. Multiple-dose administration of D20 through tail vein injection in 3xTg AD mice effectively ameliorated tau-associated pathologies with improved cognitive functions. Importantly, at therapeutic doses, D20 did not cause detectable toxicity in cultured neurons, neural cells, or peripheral organs in mice. These results suggest that D20 is a promising drug candidate for AD and related tauopathies.
高磷酸化 tau（pTau）的异常积累是阿尔茨海默病（AD）和相关 tau 病神经变性的主要原因。因此，降低 pTau 有望治疗这些疾病。在这里，我们开发了一种名为D20的嵌合肽，通过将蛋白磷酸酶1（PP1）招募到tau上，选择性地促进tau去磷酸化。PP1是使tau去磷酸化的活性磷酸酶之一。我们在培养的原代海马神经元和AD或相关tau病小鼠模型中证实，单剂量D20治疗可通过在多个AD相关位点去磷酸化而显著降低pTau，总tau（tTau）水平也会降低。在 3xTg AD 小鼠中通过尾静脉注射多剂量 D20 能有效改善与 tau 相关的病理现象，并改善认知功能。重要的是，在治疗剂量下，D20 不会对小鼠的培养神经元、神经细胞或外周器官产生可检测到的毒性。这些结果表明，D20是一种很有前景的候选药物，可用于治疗AD和相关的tau病症。
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