Renuka Shanmugam, Reuben Anderson, Anja H Schiemann, Evelyn Sattlegger
The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.
{"title":"Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation.","authors":"Renuka Shanmugam, Reuben Anderson, Anja H Schiemann, Evelyn Sattlegger","doi":"10.1042/BCJ20220531","DOIUrl":"10.1042/BCJ20220531","url":null,"abstract":"<p><p>The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11088878/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140027295","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Wilbourn, Barry, Nesbeth, Darren N., Wainwright, Linda J., Field, Mark C.
The authors of the original article “Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition” DOI: 10.1042/bj3320111: Wilbourn et al., Biochem. J.332, 111–118 (1998) would like to correct Figure 5 of this article. After publication, a reader identified that Figure 5 contained a duplicated Western blot image in panel ‘B’ between the “28” and “29” experimental groups. The authors confirmed that the “28” Western blot image was inadvertently duplicated and re-used for the
{"title":"Correction: Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition","authors":"Wilbourn, Barry, Nesbeth, Darren N., Wainwright, Linda J., Field, Mark C.","doi":"10.1042/bj3320111_cor","DOIUrl":"https://doi.org/10.1042/bj3320111_cor","url":null,"abstract":"The authors of the original article “Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition” DOI: 10.1042/bj3320111: Wilbourn et al., Biochem. J.332, 111–118 (1998) would like to correct Figure 5 of this article. After publication, a reader identified that Figure 5 contained a duplicated Western blot image in panel ‘B’ between the “28” and “29” experimental groups. The authors confirmed that the “28” Western blot image was inadvertently duplicated and re-used for the ","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140547508","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Kylie A Vestal, Chandramohan Kattamuri, Muhasin Koyiloth, Luisina Ongaro, James A Howard, Aimee M Deaton, Simina Ticau, Aditi Dubey, Daniel J Bernard, Thomas B Thompson
Activins are one of the three distinct subclasses within the greater Transforming growth factor β (TGFβ) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.
活性蛋白是转化生长因子β(TGFβ)超家族中三个不同的亚类之一。最初发现活化素是因为它们在生殖生物学中的关键作用,后来又证明它们能改变细胞的分化和增殖。目前,活化素亚类的成员包括活化素 A(ActA)、ActB、ActC、ActE,以及更远的成员 myostatin 和 GDF11。虽然大多数活化素亚类成员的生物学作用和信号传导机制已被深入研究,但 ActE 的信号传导潜力在很大程度上仍不为人所知。在这里,我们研究了同源二聚体 ActE 的信号能力。配体:受体复合物的分子建模显示,ActC 和 ActE 在 I 型和 II 型受体结合表位上具有高度相似性。ActE通过ALK7发出特异性信号,利用典型的活化素II型受体ActRIIA和ActRIIB,并且对细胞外拮抗剂follistatin和WFIKKN具有抗性。在成熟的小鼠脂肪细胞中,ActE 与 ActC 一样通过 ALK7 引起 SMAD2/3 反应。总之,我们的研究结果证实 ActE 是一种激活 I 型受体 ALK7 的特异性信号配体。
{"title":"Activin E is a transforming growth factor β ligand that signals specifically through activin receptor-like kinase 7.","authors":"Kylie A Vestal, Chandramohan Kattamuri, Muhasin Koyiloth, Luisina Ongaro, James A Howard, Aimee M Deaton, Simina Ticau, Aditi Dubey, Daniel J Bernard, Thomas B Thompson","doi":"10.1042/BCJ20230404","DOIUrl":"10.1042/BCJ20230404","url":null,"abstract":"<p><p>Activins are one of the three distinct subclasses within the greater Transforming growth factor β (TGFβ) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11088876/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140292629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The Editorial Office has been made aware of potential issues surrounding the scientific validity of this paper, hence has issued an expression of concern to notify readers whilst the Editorial Office investigates. It has been noted that there seems to be a partial duplication between Figure 4C PAR2-OE control panel and Figure 4E Si-NC MHY1485 panel, as well as a duplication between Figure 7B Sham and UUO+rapa panels.
{"title":"Expression of Concern: Protease-activated receptor-2 promotes kidney tubular epithelial inflammation by inhibiting autophagy via the PI3K/Akt/mTOR signalling pathway","authors":"Du, Chunyang, Zhang, Tao, Xiao, Xia, Shi, Yonghong, Duan, Huijun, Ren, Yunzhuo","doi":"10.1042/bcj20170272_eoc","DOIUrl":"https://doi.org/10.1042/bcj20170272_eoc","url":null,"abstract":"The Editorial Office has been made aware of potential issues surrounding the scientific validity of this paper, hence has issued an expression of concern to notify readers whilst the Editorial Office investigates. It has been noted that there seems to be a partial duplication between Figure 4C PAR2-OE control panel and Figure 4E Si-NC MHY1485 panel, as well as a duplication between Figure 7B Sham and UUO+rapa panels.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140545046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Respiratory complex I is a redox-driven proton pump. Several high-resolution structures of complex I have been determined providing important information about the putative proton transfer paths and conformational transitions that may occur during catalysis. However, how redox energy is coupled to the pumping of protons remains unclear. In this article, we review biochemical, structural and molecular simulation data on complex I and discuss several coupling models, including the key unresolved mechanistic questions. Focusing both on the quinone-reductase domain as well as the proton-pumping membrane-bound domain of complex I, we discuss a molecular mechanism of proton pumping that satisfies most experimental and theoretical constraints. We suggest that protonation reactions play an important role not only in catalysis, but also in the physiologically-relevant active/deactive transition of complex I.
呼吸复合体 I 是一种氧化还原驱动的质子泵。目前已经确定了多个 I 号复合体的高分辨率结构,提供了有关催化过程中可能发生的质子传递路径和构象转变的重要信息。然而,氧化还原能量如何与质子泵结合仍不清楚。在本文中,我们回顾了复合物 I 的生化、结构和分子模拟数据,并讨论了几种耦合模型,包括尚未解决的关键机制问题。我们以醌还原酶结构域和复合物 I 的质子泵膜结合结构域为重点,讨论了一种满足大多数实验和理论约束条件的质子泵的分子机制。我们认为质子化反应不仅在催化过程中起着重要作用,而且在与生理相关的复合物 I 的活性/非活性转换过程中也起着重要作用。
{"title":"Long-range electron proton coupling in respiratory complex I — insights from molecular simulations of the quinone chamber and antiporter-like subunits","authors":"Djurabekova, Amina, Lasham, Jonathan, Zdorevskyi, Oleksii, Zickermann, Volker, Sharma, Vivek","doi":"10.1042/bcj20240009","DOIUrl":"https://doi.org/10.1042/bcj20240009","url":null,"abstract":"Respiratory complex I is a redox-driven proton pump. Several high-resolution structures of complex I have been determined providing important information about the putative proton transfer paths and conformational transitions that may occur during catalysis. However, how redox energy is coupled to the pumping of protons remains unclear. In this article, we review biochemical, structural and molecular simulation data on complex I and discuss several coupling models, including the key unresolved mechanistic questions. Focusing both on the quinone-reductase domain as well as the proton-pumping membrane-bound domain of complex I, we discuss a molecular mechanism of proton pumping that satisfies most experimental and theoretical constraints. We suggest that protonation reactions play an important role not only in catalysis, but also in the physiologically-relevant active/deactive transition of complex I.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140349186","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Foster, Benjamin M., Wang, Zijuan, Schmidt, Christine K.
Maintaining stability of the genome requires dedicated DNA repair and signalling processes that are essential for the faithful duplication and propagation of chromosomes. These DNA damage response (DDR) mechanisms counteract the potentially mutagenic impact of daily genotoxic stresses from both exogenous and endogenous sources. Inherent to these DNA repair pathways is the activity of protein factors that instigate repair processes in response to DNA lesions. The regulation, coordination, and orchestration of these DDR factors is carried out, in a large part, by post-translational modifications, such as phosphorylation, ubiquitylation, and modification with ubiquitin-like proteins (UBLs). The importance of ubiquitylation and UBLylation with SUMO in DNA repair is well established, with the modified targets and downstream signalling consequences relatively well characterised. However, the role of dedicated erasers for ubiquitin and UBLs, known as deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) respectively, in genome stability is less well established, particularly for emerging UBLs such as ISG15 and UFM1. In this review, we provide an overview of the known regulatory roles and mechanisms of DUBs and ULPs involved in genome stability pathways. Expanding our understanding of the molecular agents and mechanisms underlying the removal of ubiquitin and UBL modifications will be fundamental for progressing our knowledge of the DDR and likely provide new therapeutic avenues for relevant human diseases, such as cancer.
保持基因组的稳定性需要专门的 DNA 修复和信号传递过程,这对染色体的忠实复制和繁殖至关重要。这些 DNA 损伤应答(DDR)机制可抵消日常外源性和内源性基因毒性压力可能造成的诱变影响。这些 DNA 修复途径的内在要素是蛋白质因子的活性,它们在 DNA 损伤发生时启动修复过程。这些 DDR 因子的调控、协调和安排在很大程度上是通过翻译后修饰进行的,如磷酸化、泛素化和泛素样蛋白(UBLs)修饰。泛素化和与 SUMO 的 UBL 化在 DNA 修复中的重要性已得到公认,被修饰的目标和下游信号后果也相对明确。然而,泛素和 UBLs 的专用清除剂(分别称为去泛素化酶(DUBs)和泛素样蛋白酶(ULPs))在基因组稳定性中的作用还不太明确,尤其是对于 ISG15 和 UFM1 等新出现的 UBLs。在这篇综述中,我们概述了参与基因组稳定性途径的 DUBs 和 ULPs 的已知调控作用和机制。扩大我们对消除泛素和 UBL 修饰的分子介质和机制的认识,将是促进我们对 DDR 的了解的基础,并有可能为癌症等相关人类疾病提供新的治疗途径。
{"title":"DoUBLing up: ubiquitin and ubiquitin-like proteases in genome stability","authors":"Foster, Benjamin M., Wang, Zijuan, Schmidt, Christine K.","doi":"10.1042/bcj20230284","DOIUrl":"https://doi.org/10.1042/bcj20230284","url":null,"abstract":"Maintaining stability of the genome requires dedicated DNA repair and signalling processes that are essential for the faithful duplication and propagation of chromosomes. These DNA damage response (DDR) mechanisms counteract the potentially mutagenic impact of daily genotoxic stresses from both exogenous and endogenous sources. Inherent to these DNA repair pathways is the activity of protein factors that instigate repair processes in response to DNA lesions. The regulation, coordination, and orchestration of these DDR factors is carried out, in a large part, by post-translational modifications, such as phosphorylation, ubiquitylation, and modification with ubiquitin-like proteins (UBLs). The importance of ubiquitylation and UBLylation with SUMO in DNA repair is well established, with the modified targets and downstream signalling consequences relatively well characterised. However, the role of dedicated erasers for ubiquitin and UBLs, known as deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) respectively, in genome stability is less well established, particularly for emerging UBLs such as ISG15 and UFM1. In this review, we provide an overview of the known regulatory roles and mechanisms of DUBs and ULPs involved in genome stability pathways. Expanding our understanding of the molecular agents and mechanisms underlying the removal of ubiquitin and UBL modifications will be fundamental for progressing our knowledge of the DDR and likely provide new therapeutic avenues for relevant human diseases, such as cancer.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140346240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anastasiya Potapenko, Jennilee M Davidson, Albert Lee, Angela S Laird
Machado-Joseph disease (MJD) is a devastating and incurable neurodegenerative disease characterised by progressive ataxia, difficulty speaking and swallowing. Consequently, affected individuals ultimately become wheelchair dependent, require constant care, and face a shortened life expectancy. The monogenic cause of MJD is expansion of a trinucleotide (CAG) repeat region within the ATXN3 gene, which results in polyglutamine (polyQ) expansion within the resultant ataxin-3 protein. While it is well established that the ataxin-3 protein functions as a deubiquitinating (DUB) enzyme and is therefore critically involved in proteostasis, several unanswered questions remain regarding the impact of polyQ expansion in ataxin-3 on its DUB function. Here we review the current literature surrounding ataxin-3's DUB function, its DUB targets, and what is known regarding the impact of polyQ expansion on ataxin-3's DUB function. We also consider the potential neuroprotective effects of ataxin-3's DUB function, and the intersection of ataxin-3's role as a DUB enzyme and regulator of gene transcription. Ataxin-3 is the principal pathogenic protein in MJD and also appears to be involved in cancer. As aberrant deubiquitination has been linked to both neurodegeneration and cancer, a comprehensive understanding of ataxin-3's DUB function is important for elucidating potential therapeutic targets in these complex conditions. In this review, we aim to consolidate knowledge of ataxin-3 as a DUB and unveil areas for future research to aid therapeutic targeting of ataxin-3's DUB function for the treatment of MJD and other diseases.
{"title":"The deubiquitinase function of ataxin-3 and its role in the pathogenesis of Machado-Joseph disease and other diseases.","authors":"Anastasiya Potapenko, Jennilee M Davidson, Albert Lee, Angela S Laird","doi":"10.1042/BCJ20240017","DOIUrl":"10.1042/BCJ20240017","url":null,"abstract":"<p><p>Machado-Joseph disease (MJD) is a devastating and incurable neurodegenerative disease characterised by progressive ataxia, difficulty speaking and swallowing. Consequently, affected individuals ultimately become wheelchair dependent, require constant care, and face a shortened life expectancy. The monogenic cause of MJD is expansion of a trinucleotide (CAG) repeat region within the ATXN3 gene, which results in polyglutamine (polyQ) expansion within the resultant ataxin-3 protein. While it is well established that the ataxin-3 protein functions as a deubiquitinating (DUB) enzyme and is therefore critically involved in proteostasis, several unanswered questions remain regarding the impact of polyQ expansion in ataxin-3 on its DUB function. Here we review the current literature surrounding ataxin-3's DUB function, its DUB targets, and what is known regarding the impact of polyQ expansion on ataxin-3's DUB function. We also consider the potential neuroprotective effects of ataxin-3's DUB function, and the intersection of ataxin-3's role as a DUB enzyme and regulator of gene transcription. Ataxin-3 is the principal pathogenic protein in MJD and also appears to be involved in cancer. As aberrant deubiquitination has been linked to both neurodegeneration and cancer, a comprehensive understanding of ataxin-3's DUB function is important for elucidating potential therapeutic targets in these complex conditions. In this review, we aim to consolidate knowledge of ataxin-3 as a DUB and unveil areas for future research to aid therapeutic targeting of ataxin-3's DUB function for the treatment of MJD and other diseases.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11088879/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140142709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Uday Kiran Velagapudi, Élise Rouleau-Turcotte, Ramya Billur, Xuwei Shao, Manisha Patil, Ben E Black, John M Pascal, Tanaji T Talele
Catalytic poly(ADP-ribose) production by PARP1 is allosterically activated through interaction with DNA breaks, and PARP inhibitor compounds have the potential to influence PARP1 allostery in addition to preventing catalytic activity. Using the benzimidazole-4-carboxamide pharmacophore present in the first generation PARP1 inhibitor veliparib, a series of 11 derivatives was designed, synthesized, and evaluated as allosteric PARP1 inhibitors, with the premise that bulky substituents would engage the regulatory helical domain (HD) and thereby promote PARP1 retention on DNA breaks. We found that core scaffold modifications could indeed increase PARP1 affinity for DNA; however, the bulk of the modification alone was insufficient to trigger PARP1 allosteric retention on DNA breaks. Rather, compounds eliciting PARP1 retention on DNA breaks were found to be rigidly held in a position that interferes with a specific region of the HD domain, a region that is not targeted by current clinical PARP inhibitors. Collectively, these compounds highlight a unique way to trigger PARP1 retention on DNA breaks and open a path to unveil the pharmacological benefits of such inhibitors with novel properties.
PARP1 催化聚(ADP-核糖)的产生是通过与 DNA 断裂的相互作用异构激活的,PARP 抑制剂化合物除了阻止催化活性外,还有可能影响 PARP1 的异构性。利用第一代 PARP1 抑制剂 veliparib 中的苯并咪唑-4-甲酰胺药理结构,我们设计、合成并评估了 11 种系列衍生物作为异构 PARP1 抑制剂,前提是大块取代基会与 HD 调控结构域接触,从而促进 PARP1 在 DNA 断裂处的保留。我们发现,核心支架修饰确实可以增加 PARP1 对 DNA 的亲和力;但是,仅靠大块修饰不足以引发 PARP1 在 DNA 断裂处的异构滞留。相反,研究发现,能诱导 PARP1 在 DNA 断裂处滞留的化合物被严格固定在一个位置上,从而干扰了 HD 结构域的特定区域,而目前的临床 PARP 抑制剂并不针对该区域。总之,这些化合物突显了一种独特的方式来触发 PARP1 在 DNA 断裂处的滞留,并为揭示这种具有新特性的抑制剂的药理作用开辟了一条道路。
{"title":"Novel modifications of PARP inhibitor veliparib increase PARP1 binding to DNA breaks.","authors":"Uday Kiran Velagapudi, Élise Rouleau-Turcotte, Ramya Billur, Xuwei Shao, Manisha Patil, Ben E Black, John M Pascal, Tanaji T Talele","doi":"10.1042/BCJ20230406","DOIUrl":"10.1042/BCJ20230406","url":null,"abstract":"<p><p>Catalytic poly(ADP-ribose) production by PARP1 is allosterically activated through interaction with DNA breaks, and PARP inhibitor compounds have the potential to influence PARP1 allostery in addition to preventing catalytic activity. Using the benzimidazole-4-carboxamide pharmacophore present in the first generation PARP1 inhibitor veliparib, a series of 11 derivatives was designed, synthesized, and evaluated as allosteric PARP1 inhibitors, with the premise that bulky substituents would engage the regulatory helical domain (HD) and thereby promote PARP1 retention on DNA breaks. We found that core scaffold modifications could indeed increase PARP1 affinity for DNA; however, the bulk of the modification alone was insufficient to trigger PARP1 allosteric retention on DNA breaks. Rather, compounds eliciting PARP1 retention on DNA breaks were found to be rigidly held in a position that interferes with a specific region of the HD domain, a region that is not targeted by current clinical PARP inhibitors. Collectively, these compounds highlight a unique way to trigger PARP1 retention on DNA breaks and open a path to unveil the pharmacological benefits of such inhibitors with novel properties.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11070930/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139899305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Laura Weatherdon, Kate Stuart, Megan Cassidy, Alberto Moreno de la Gándara, Hanneke Okkenhaug, Markus Muellener, Grahame Mckenzie, Simon J Cook, Rebecca Gilley
The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or protein interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging 'hits' from drug discovery screens.
{"title":"Reporter cell lines to screen for inhibitors or regulators of the KRAS-RAF-MEK1/2-ERK1/2 pathway.","authors":"Laura Weatherdon, Kate Stuart, Megan Cassidy, Alberto Moreno de la Gándara, Hanneke Okkenhaug, Markus Muellener, Grahame Mckenzie, Simon J Cook, Rebecca Gilley","doi":"10.1042/BCJ20240015","DOIUrl":"10.1042/BCJ20240015","url":null,"abstract":"<p><p>The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or protein interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging 'hits' from drug discovery screens.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11088904/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139911924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sophia Bustraan, Jane Bennett, Chad Whilding, Betheney R Pennycook, David Smith, Alexis R Barr, Jon Read, David Carling, Alice Pollard
Adipogenesis, defined as the development of mature adipocytes from stem cell precursors, is vital for the expansion, turnover and health of adipose tissue. Loss of adipogenic potential in adipose stem cells, or impairment of adipogenesis is now recognised as an underlying cause of adipose tissue dysfunction and is associated with metabolic disease. In this study, we sought to determine the role of AMP-activated protein kinase (AMPK), an evolutionarily conserved master regulator of energy homeostasis, in adipogenesis. Primary murine adipose-derived stem cells were treated with a small molecule AMPK activator (BI-9774) during key phases of adipogenesis, to determine the effect of AMPK activation on adipocyte commitment, maturation and function. To determine the contribution of the repression of lipogenesis by AMPK in these processes, we compared the effect of pharmacological inhibition of acetyl-CoA carboxylase (ACC). We show that AMPK activation inhibits adipogenesis in a time- and concentration-dependent manner. Transient AMPK activation during adipogenic commitment leads to a significant, ACC-independent, repression of adipogenic transcription factor expression. Furthermore, we identify a striking, previously unexplored inhibition of leptin gene expression in response to both short-term and chronic AMPK activation irrespective of adipogenesis. These findings reveal that in addition to its effect on adipogenesis, AMPK activation switches off leptin gene expression in primary mouse adipocytes independently of adipogenesis. Our results identify leptin expression as a novel target of AMPK through mechanisms yet to be identified.
{"title":"AMP-activated protein kinase activation suppresses leptin expression independently of adipogenesis in primary murine adipocytes.","authors":"Sophia Bustraan, Jane Bennett, Chad Whilding, Betheney R Pennycook, David Smith, Alexis R Barr, Jon Read, David Carling, Alice Pollard","doi":"10.1042/BCJ20240003","DOIUrl":"10.1042/BCJ20240003","url":null,"abstract":"<p><p>Adipogenesis, defined as the development of mature adipocytes from stem cell precursors, is vital for the expansion, turnover and health of adipose tissue. Loss of adipogenic potential in adipose stem cells, or impairment of adipogenesis is now recognised as an underlying cause of adipose tissue dysfunction and is associated with metabolic disease. In this study, we sought to determine the role of AMP-activated protein kinase (AMPK), an evolutionarily conserved master regulator of energy homeostasis, in adipogenesis. Primary murine adipose-derived stem cells were treated with a small molecule AMPK activator (BI-9774) during key phases of adipogenesis, to determine the effect of AMPK activation on adipocyte commitment, maturation and function. To determine the contribution of the repression of lipogenesis by AMPK in these processes, we compared the effect of pharmacological inhibition of acetyl-CoA carboxylase (ACC). We show that AMPK activation inhibits adipogenesis in a time- and concentration-dependent manner. Transient AMPK activation during adipogenic commitment leads to a significant, ACC-independent, repression of adipogenic transcription factor expression. Furthermore, we identify a striking, previously unexplored inhibition of leptin gene expression in response to both short-term and chronic AMPK activation irrespective of adipogenesis. These findings reveal that in addition to its effect on adipogenesis, AMPK activation switches off leptin gene expression in primary mouse adipocytes independently of adipogenesis. Our results identify leptin expression as a novel target of AMPK through mechanisms yet to be identified.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11088909/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139680691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}