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The impact of cell states on heterochromatin dynamics. 细胞状态对异染色质动态的影响
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-06 DOI: 10.1042/bcj20240139
Abby Trouth,Giovana M B Veronezi,Srinivas Ramachandran
Establishing, maintaining, and removing histone post-translational modifications associated with heterochromatin is critical for shaping genomic structure and function as a cell navigates different stages of development, activity, and disease. Dynamic regulation of the repressive chromatin landscape has been documented in several key cell states - germline cells, activated immune cells, actively replicating, and quiescent cells - with notable variations in underlying mechanisms. Here, we discuss the role of cell states of these diverse contexts in directing and maintaining observed chromatin landscapes. These investigations reveal heterochromatin architectures that are highly responsive to the functional context of a cell's existence and, in turn, their contribution to the cell's stable identity.
当细胞处于发育、活动和疾病的不同阶段时,建立、维持和去除与异染色质相关的组蛋白翻译后修饰对于塑造基因组结构和功能至关重要。抑制性染色质景观的动态调控已在几种关键细胞状态(生殖细胞、活化的免疫细胞、活跃的复制细胞和静止细胞)中得到证实,其基本机制存在显著差异。在这里,我们将讨论这些不同背景下的细胞状态在引导和维持所观察到的染色质景观中的作用。这些研究揭示了异染色质结构对细胞存在的功能环境的高度响应,反过来,它们对细胞的稳定特性也有贡献。
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引用次数: 0
Pyroptotic executioner pore-forming protein GSDMD forms oligomeric assembly and exhibits amyloid-like attributes that could contribute for its pore-forming function. 嗜火刽子手孔道形成蛋白 GSDMD 形成寡聚体集合体,并表现出淀粉样特性,这可能有助于其孔道形成功能。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-06 DOI: 10.1042/BCJ20240416
Shamaita Chatterjee, Tarang Gupta, Gurvinder Kaur, Kausik Chattopadhyay

Gasdermin D (GSDMD) is the chief executioner of inflammatory cell death or pyroptosis. During pyroptosis, proteolytic processing of GSDMD releases its N-terminal domain (NTD), which then forms large oligomeric pores in the plasma membranes. Membrane pore-formation by NTD allows the release of inflammatory cytokines and causes membrane damage to induce cell death. Structural mechanisms of GSDMD-mediated membrane pore-formation have been extensively studied. However, less effort has been made to understand the physicochemical properties of GSDMD and their functional implications. Here, we explore detailed characterization of the physicochemical properties of mouse GSDMD (mGSDMD), and their implications in regulating the pore-forming function. Our study reveals that mGSDMD shows some of the hallmark features of amyloids, and forms oligomeric assemblies in solution that are critically dependent on the disulphide bond-forming ability of the protein. mGSDMD oligomeric assemblies do not resemble typical amyloid fibrils/aggregates, and do not show resistance to proteolytic degradation that is otherwise observed with the conventional amyloids. Our results further elucidate the essential role of an amyloid-prone region (APR) in the oligomerization and amyloid-like features of mGSDMD. Furthermore, alteration of this APR leads to compromised pore-forming ability and cell-killing activity of NTD released from mGSDMD. Taken together, our study for the first time provides crucial new insights regarding implications of the amyloid-like property of mGSDMD in regulating its pore-forming function, which is an essential requirement for this pyroptotic executioner. To the best of our knowledge, such mode of regulation of mGSDMD-function has not been appreciated so far.

Gasdermin D(GSDMD)是炎性细胞死亡或化脓作用的主要执行者。在化脓过程中,GSDMD 的蛋白水解过程会释放出其 N 端结构域(NTD),然后在质膜上形成大的寡聚孔。NTD 形成的膜孔可释放炎性细胞因子,并造成膜损伤,从而诱导细胞死亡。人们对 GSDMD 介导的膜孔形成的结构机制进行了广泛研究。然而,人们对 GSDMD 的理化性质及其功能影响的了解却较少。在此,我们探讨了小鼠 GSDMD(mGSDMD)理化性质的详细特征及其在调节孔形成功能方面的意义。我们的研究发现,mGSDMD 显示出淀粉样蛋白的一些标志性特征,并在溶液中形成寡聚体集合体,而这种集合体的形成主要依赖于蛋白质的二硫键形成能力。mGSDMD 寡聚体集合体与典型的淀粉样纤维/聚集体并不相似,也不像传统的淀粉样蛋白那样表现出抗蛋白水解降解的能力。我们的研究结果进一步阐明了淀粉样蛋白易形成区(APR)在 mGSDMD 的寡聚化和淀粉样特征中的重要作用。此外,APR的改变导致mGSDMD释放的NTD的孔形成能力和细胞杀伤活性受损。综上所述,我们的研究首次就 mGSDMD 的淀粉样特性在调节其孔隙形成功能方面的影响提供了至关重要的新见解,而孔隙形成功能是这种火性刽子手的基本要求。据我们所知,迄今为止还没有人了解过 mGSDMD 功能的这种调控模式。
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引用次数: 0
Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain. 组蛋白去乙酰化酶 7 通过一种与酶无关的机制激活 6-磷酸葡萄糖酸脱氢酶,该机制涉及 N 端蛋白-蛋白相互作用结构域。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-06 DOI: 10.1042/BCJ20240380
Yizhuo Wang, James E B Curson, Divya Ramnath, Kaustav Das Gupta, Robert C Reid, Denuja Karunakaran, David P Fairlie, Matthew J Sweet

Histone deacetylase 7 (HDAC7) is a member of the class IIa family of classical HDACs with important roles in cell development, differentiation, and activation, including in macrophages and other innate immune cells. HDAC7 and other class IIa HDACs act as transcriptional repressors in the nucleus but, in some cell types, they can also act in the cytoplasm to modify non-nuclear proteins and/or scaffold signalling complexes. In macrophages, HDAC7 is a cytoplasmic protein with both pro- and anti-inflammatory functions, with the latter activity involving activation of the pentose phosphate pathway (PPP) enzyme 6-phosphogluconate dehydrogenase (6PGD) and the generation of anti-inflammatory metabolite ribulose-5-phosphate. Here, we used ectopic expression systems and biochemical approaches to investigate the mechanism by which HDAC7 promotes 6PGD enzyme activity. We reveal that HDAC7 enzyme activity is not required for its activation of 6PGD and that the N-terminal protein-protein interaction domain of HDAC7 is sufficient to initiate this response. Mechanistically, the N-terminus of HDAC7 increases the affinity of 6PGD for NADP+, promotes the generation of a shorter form of 6PGD, and enhances the formation of higher order protein complexes, implicating its scaffolding function in engagement of the PPP. This contrasts with the pro-inflammatory function of HDAC7 in macrophages, in which it promotes deacetylation of the glycolytic enzyme pyruvate kinase M2 for inflammatory cytokine production.

组蛋白去乙酰化酶 7(HDAC7)是经典 HDAC IIa 类家族的成员,在细胞发育、分化和活化(包括巨噬细胞和其他先天性免疫细胞)中发挥着重要作用。HDAC7 和其他 IIa 类 HDAC 在细胞核中充当转录抑制因子,但在某些细胞类型中,它们也能在细胞质中改变非核蛋白和/或支架信号复合物。在巨噬细胞中,HDAC7是一种细胞质蛋白,具有促炎和抗炎两种功能,后者的活性涉及激活磷酸戊糖途径(PPP)酶6-磷酸葡萄糖酸脱氢酶(6PGD)和生成抗炎代谢产物核酮糖-5-磷酸。在这里,我们利用异位表达系统和生化方法研究了 HDAC7 促进 6PGD 酶活性的机制。我们发现,HDAC7 激活 6PGD 并不需要 HDAC7 的酶活性,HDAC7 的 N 端蛋白-蛋白相互作用结构域足以启动这一反应。从机理上讲,HDAC7 的 N 端增加了 6PGD 对 NADP+ 的亲和力,促进了较短形式 6PGD 的生成,并增强了高阶蛋白复合物的形成,这意味着它在参与 PPP 过程中的支架功能。这与 HDAC7 在巨噬细胞中的促炎功能形成鲜明对比,在巨噬细胞中,HDAC7 促进糖酵解酶丙酮酸激酶 M2 的去乙酰化,从而产生炎性细胞因子。
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引用次数: 0
Epigenetics and alternative splicing in cancer: old enemies, new perspectives. 癌症中的表观遗传学和替代剪接:老对手,新视角。
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-06 DOI: 10.1042/bcj20240221
Madhura R Pandkar,Sanjeev Shukla
In recent years, significant strides in both conceptual understanding and technological capabilities have bolstered our comprehension of the factors underpinning cancer initiation and progression. While substantial insights have unraveled the molecular mechanisms driving carcinogenesis, there has been an overshadowing of the critical contribution made by epigenetic pathways, which works in concert with genetics. Mounting evidence demonstrates cancer as a complex interplay between genetics and epigenetics. Notably, epigenetic elements play a pivotal role in governing alternative pre-mRNA splicing, a primary contributor to protein diversity. In this review, we have provided detailed insights into the bidirectional communication between epigenetic modifiers and alternative splicing, providing examples of specific genes and isoforms affected. Notably, succinct discussion on targeting epigenetic regulators and the potential of the emerging field of epigenome editing to modulate splicing patterns is also presented. In summary, this review offers valuable insights into the intricate interplay between epigenetics and alternative splicing in cancer, paving the way for novel approaches to understanding and targeting this critical process.
近年来,我们在概念理解和技术能力方面都取得了长足进步,从而提高了对癌症发生和发展的基本因素的认识。虽然我们对致癌的分子机制有了深入的了解,但却忽略了与遗传学协同作用的表观遗传学途径所做出的重要贡献。越来越多的证据表明,癌症是遗传学和表观遗传学之间复杂的相互作用。值得注意的是,表观遗传因子在调控替代性前核糖核酸剪接方面起着关键作用,而这种剪接是导致蛋白质多样性的主要因素。在这篇综述中,我们详细介绍了表观遗传修饰因子与替代剪接之间的双向交流,并举例说明了受影响的特定基因和同工酶。值得注意的是,本综述还简明扼要地讨论了以表观遗传调节因子为靶点以及表观遗传组编辑这一新兴领域在调节剪接模式方面的潜力。总之,这篇综述对癌症中表观遗传学和替代剪接之间错综复杂的相互作用提供了宝贵的见解,为了解和靶向这一关键过程的新方法铺平了道路。
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引用次数: 0
ATP-competitive inhibitors of PI3K enzymes demonstrate an isoform selective dual action by controlling membrane binding. PI3K 酶的 ATP 竞争性抑制剂通过控制膜结合,显示出同工酶选择性的双重作用。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-01 DOI: 10.1042/BCJ20240479
Grace Q Gong, Glenn Robert Masson, Woo-Jeong Jeong Lee, James Mj Dickson, Jackie D Kendall, Manoj K Rathinaswamy, Christina M Buchanan, Martin Middleditch, Brady Owen, Julie A Spicer, Gordon W Rewcastle, William A Denny, John E Burke, Peter R Shepherd, Roger L Williams, Jack U Flanagan

PI3Kα, consisting of the p110α isoform of the catalytic subunit of PI 3-kinase (encoded by PIK3CA) and the p85α regulatory subunit (encoded by PI3KR1) is activated by growth factor receptors. The identification of common oncogenic mutations in PIK3CA has driven the development of many inhibitors that bind to the ATP-binding site in the p110α subunit. Upon activation, PI3Kα undergoes conformational changes that promote its membrane interaction and catalytic activity, yet the effects of ATP-site directed inhibitors on the PI3Kα membrane interaction are unknown. Using FRET and Biolayer Interferometry assays, we show that a class of ATP-site directed inhibitors represented by GSK2126458 block the growth factor activated PI3KαWT membrane interaction, an activity dependent on the ligand forming specific ATP-site interactions. The membrane interaction for hot spot oncogenic mutations that bypass normal p85α regulatory mechanisms was insensitive to GSK2126458, while GSK2126458 could regulate mutations found outside of these hot spot regions. Our data show that the effect of GSK126458 on the membrane interaction requires the enzyme to revert from its growth factor activated state to a basal state. We find that an ATP substrate analogue can increase the wild type PI3Kα membrane interaction, uncovering a substrate based regulatory event that can be mimicked by different inhibitor chemotypes. Our findings, together with the discovery of small molecule allosteric activators of PI3Kα illustrate that PI3Kα membrane interactions can be modulated by factors related to ligand binding both within the ATP site and at allosteric sites.

PI3Kα 由 PI 3-kinase 催化亚基的 p110α 异构体(由 PIK3CA 编码)和 p85α 调节亚基(由 PI3KR1 编码)组成,由生长因子受体激活。PIK3CA 常见致癌突变的发现推动了许多与 p110α 亚基中的 ATP 结合位点结合的抑制剂的开发。激活后,PI3Kα会发生构象变化,从而促进其膜相互作用和催化活性,但ATP位点定向抑制剂对PI3Kα膜相互作用的影响尚不清楚。我们利用 FRET 和生物层干涉测量法检测表明,以 GSK2126458 为代表的一类 ATP 位点定向抑制剂会阻断生长因子激活的 PI3KαWT 膜相互作用,这种活性依赖于配体形成特定的 ATP 位点相互作用。绕过正常 p85α 调节机制的热点致癌突变的膜相互作用对 GSK2126458 不敏感,而 GSK2126458 能调节这些热点区域之外的突变。我们的数据显示,GSK126458 对膜相互作用的影响需要酶从生长因子激活状态恢复到基础状态。我们发现 ATP 底物类似物能增加野生型 PI3Kα 的膜相互作用,从而揭示了一种基于底物的调控事件,不同的抑制剂化学类型可以模拟这种调控事件。我们的发现以及 PI3Kα 的小分子异构激活剂的发现说明,PI3Kα 膜相互作用可受 ATP 位点和异构位点内配体结合相关因素的调节。
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引用次数: 0
Exopolysaccharide is detrimental for the symbiotic performance of Sinorhizobium fredii HH103 mutants with a truncated lipopolysaccharide core. 外多糖不利于具有截短脂多糖核心的裂殖单胞菌 HH103 突变体的共生性能。
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-25 DOI: 10.1042/bcj20240599
Francisco Fuentes-Romero,Marcello Mercogliano,Stefania De Chiara,Cynthia Alías-Villegas,Pilar Navarro-Gómez,Sebastián Acosta-Jurado,Alba Silipo,Carlos Medina,Miguel-Ángel Rodríguez-Carvajal,Marta S Dardanelli,José-Enrique Ruiz-Sainz,Francisco-Javier López-Baena,Antonio Molinaro,José-María Vinardell,Flaviana Di Lorenzo
The nitrogen-fixing rhizobia-legume symbiosis relies on a complex interchange of molecular signals between the two partners during the whole interaction. On the bacterial side, different surface polysaccharides, such as lipopolysaccharide (LPS) and exopolysaccharide (EPS), might play important roles for the success of the interaction. In a previous work we studied two Sinorhizobium fredii HH103 mutants affected in the rkpK and lpsL genes, which are responsible for the production of glucuronic acid and galacturonic acid, respectively. Both mutants produced an altered LPS, and the rkpK mutant, in addition, lacked EPS. These mutants were differently affected in symbiosis with Glycine max and Vigna unguiculata, with the lpsL mutant showing a stronger impairment than the rkpK mutant. In the present work we have further investigated the LPS structure and the symbiotic abilities of the HH103 lpsL and rkpK mutants. We demonstrate that both strains produce the same LPS, with a truncated core oligosaccharide devoid of uronic acids. We show that the symbiotic performance of the lpsL mutant with Macroptilium atropurpureum and Glycyrrhiza uralensis is worse than that of the rkpK mutant. Introduction of an exoA mutation (which avoids EPS production) in HH103 lpsL improved its symbiotic performance with G. max, M. atropurpureum, and G. uralensis to the level exhibited by HH103 rkpK, suggesting that the presence of EPS might hide the truncated LPS produced by the former mutant.
固氮根瘤菌与豆科植物的共生依赖于双方在整个相互作用过程中分子信号的复杂交换。在细菌方面,不同的表面多糖,如脂多糖(LPS)和外多糖(EPS),可能对相互作用的成功起着重要作用。在之前的工作中,我们研究了两个受 rkpK 和 lpsL 基因影响的 HH103 突变体,这两个基因分别负责产生葡萄糖醛酸和半乳糖醛酸。这两个突变体产生的 LPS 都发生了改变,此外,rkpK 突变体还缺乏 EPS。这些突变体在与 Glycine max 和 Vigna unguiculata 共生时受到的影响不同,lpsL 突变体比 rkpK 突变体受到的影响更大。在本研究中,我们进一步研究了 HH103 lpsL 和 rkpK 突变体的 LPS 结构和共生能力。结果表明,这两种菌株产生的 LPS 结构相同,都有一个不含尿酸的截短核心寡糖。我们发现,lpsL突变体与大肠杆菌(Macroptilium atropurpureum)和甘草(Glycyrrhiza uralensis)的共生性能比rkpK突变体差。在 HH103 lpsL 中引入 exoA 突变(可避免产生 EPS)后,它与 G. max、M. atropurpureum 和 G. uralensis 的共生性能提高到了 HH103 rkpK 的水平,这表明 EPS 的存在可能掩盖了前一突变体产生的截短 LPS。
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引用次数: 0
Comparative Analysis of Canine and Human HtrA2 to Delineate Its Role in Apoptosis and Cancer. 比较分析犬和人的 HtrA2 以确定其在细胞凋亡和癌症中的作用
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-17 DOI: 10.1042/bcj20240295
Snehal Pandav Mudrale,Shubhankar Dutta,Kalyani Natu,Pradip Chaudhari,Kakoli Bose
Therapeutically, targeting the pro- and anti-apoptotic proteins has been one of the major approaches behind devising strategies to combat associated diseases. Human high-temperature requirement serine protease A2 (hHtrA2), which induces apoptosis through both caspase-dependent and independent pathways is implicated in several diseases including cancer, ischemic heart diseases, and neurodegeneration, thus making it a promising target molecule. In the recent past, the canine model has gained prominence in the understanding of human pathophysiology that was otherwise limited to the rodent system. Moreover, canine models in cancer research provide an opportunity to study spontaneous tumors as their size, lifespan, and environmental exposure are significantly closer to that of humans compared to laboratory rodents. Therefore, using HtrA2 as a model protein, comparative analysis has been done to revisit the hypothesis that canines might be excellent models for cancer research. We have performed evolutionary phylogenetic analyses that confirm a close relationship between canine and human HtrA2s. Molecular modeling demonstrates structural similarities including orientation of the catalytic triad residues, followed by in silico docking and molecular dynamics simulation studies that identify the potential interacting partners for canine HtrA2 (cHtrA2). In vitro biophysical and protease studies depict similarities in interaction with their respective substrates as well as transient transfection of cHtrA2 in mammalian cell culture shows induction of apoptosis. This work, therefore, promises to open a new avenue in cancer research through the study of spontaneous cancer model systems in canines.
在治疗方面,靶向促凋亡蛋白和抗凋亡蛋白一直是制定相关疾病防治策略的主要方法之一。人类高温所需的丝氨酸蛋白酶 A2(hHtrA2)可通过依赖和独立于 Caspase 的途径诱导细胞凋亡,与癌症、缺血性心脏病和神经变性等多种疾病有关,因此是一种很有前景的靶分子。近来,犬类模型在了解人类病理生理学方面的作用日益突出,而这些研究原本仅限于啮齿动物系统。此外,癌症研究中的犬类模型为研究自发性肿瘤提供了机会,因为与实验室啮齿类动物相比,犬类的体型、寿命和环境暴露明显更接近人类。因此,我们以 HtrA2 为模型蛋白进行了比较分析,以重新审视犬类可能是癌症研究的绝佳模型这一假设。我们进行了进化系统发育分析,结果证实犬类和人类的 HtrA2 关系密切。分子建模显示了结构上的相似性,包括催化三体残基的取向,随后进行的硅对接和分子动力学模拟研究确定了犬 HtrA2(ctrA2)的潜在相互作用伙伴。体外生物物理和蛋白酶研究表明,犬 HtrA2 与各自底物的相互作用具有相似性,在哺乳动物细胞培养中瞬时转染犬 HtrA2 会诱导细胞凋亡。因此,这项工作有望通过研究犬类自发性癌症模型系统为癌症研究开辟一条新途径。
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引用次数: 0
Yop1 stability and membrane curvature generation propensity are controlled by its oligomerisation interface. Yop1 的稳定性和膜曲率生成倾向受其寡聚界面控制。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-16 DOI: 10.1042/BCJ20240190
Anu V Chandran, Daniel Álvarez, Stefano Vanni, Jason R Schnell

The DP1 family of integral membrane proteins stabilize high membrane curvature in the endoplasmic reticulum and phagophores. Mutations in the human DP1 gene REEP1 are associated with Hereditary Spastic Paraplegia type 31 and distal hereditary motor neuropathy. Four missense mutations map to a putative dimerization interface but the impact of these mutations on DP1 structure and tubule formation are unknown. Combining biophysical measurements, functional assays, and computational modeling in the context of the model protein Yop1, we found that missense mutations have variable effects on DP1 dimer structure and in vitro tubulation activity, and provide mechanistic insights into the role of DP1 oligomerisation on membrane curvature stabilization. Whereas the mutations P71L and S75F decreased dimer homogeneity and led to polydisperse oligomerization and impaired membrane curving activity, A72E introduced new polar interactions between subunits that stabilized the Yop1 dimer and allowed robust tubule formation but prevented formation of more highly-curved lipoprotein particles (LPP). The introduction of a BRIL domain to the cytoplasmic loop of A72E rescued LPP formation, consistent with a requirement for dimer splaying in highly curved membranes. These results suggest that the membrane curving activity of DP1 proteins requires both dimer stability and conformational plasticity at the intermolecular interface.

DP1 整体膜蛋白家族可稳定内质网和吞噬细胞中的高膜曲率。人类 DP1 基因 REEP1 的突变与遗传性痉挛性截瘫 31 型和远端遗传性运动神经病有关。四个错义突变映射到一个假定的二聚化界面,但这些突变对 DP1 结构和小管形成的影响尚不清楚。结合模型蛋白 Yop1 的生物物理测量、功能测试和计算建模,我们发现错义突变对 DP1 二聚体结构和体外管化活性有不同的影响,并提供了 DP1 寡聚化对膜曲率稳定作用的机理见解。突变 P71L 和 S75F 降低了二聚体的均一性,导致多分散的寡聚化,削弱了膜弯曲活性,而 A72E 则在亚基之间引入了新的极性相互作用,稳定了 Yop1 二聚体,使其能够形成稳健的小管,但却阻止了更高弯曲度的脂蛋白颗粒的形成。在 A72E 的细胞质环上引入 BRIL 结构域可挽救脂蛋白颗粒的形成,这与高弯曲膜对二聚体平展的要求一致。这些结果表明,DP1 蛋白的膜弯曲活性需要二聚体的稳定性和分子间界面的构象可塑性。
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引用次数: 0
Adapting to change: resolving the dynamic and dual roles of NCK1 and NCK2. 适应变化:解决 NCK1 和 NCK2 的动态和双重作用。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-16 DOI: 10.1042/BCJ20230232
Valentine Teyssier, Casey R Williamson, Erka Shata, Stephanie P Rosen, Nina Jones, Nicolas Bisson

Adaptor proteins play central roles in the assembly of molecular complexes and co-ordinated activation of specific pathways. Through their modular domain structure, the NCK family of adaptor proteins (NCK1 and NCK2) link protein targets via their single SRC Homology (SH) 2 and three SH3 domains. Classically, their SH2 domain binds to phosphotyrosine motif-containing receptors (e.g. receptor tyrosine kinases), while their SH3 domains bind polyproline motif-containing cytoplasmic effectors. Due to these functions being established for both NCK1 and NCK2, their roles were inaccurately assumed to be redundant. However, in contrast with this previously held view, NCK1 and NCK2 now have a growing list of paralog-specific functions, which underscores the need to further explore their differences. Here we review current evidence detailing how these two paralogs are unique, including differences in their gene/protein regulation, binding partners and overall contributions to cellular functions. To help explain these contrasting characteristics, we then discuss SH2/SH3 structural features, disordered interdomain linker regions and post-translational modifications. Together, this review seeks to highlight the importance of distinguishing NCK1 and NCK2 in research and to pave the way for investigations into the origins of their interaction specificity.

适配蛋白在分子复合物的组装和特定通路的协调激活中发挥着核心作用。通过模块化结构域,NCK 家族的适体蛋白(NCK1 和 NCK2)通过单个 SRC 同源(SH)2 结构域和三个 SH3 结构域连接蛋白质靶标。通常,它们的 SH2 结构域与含磷酸酪氨酸基序的受体(如受体酪氨酸激酶)结合,而它们的 SH3 结构域则与含多脯氨酸基序的细胞质效应物结合。由于 NCK1 和 NCK2 都具有这些功能,因此人们错误地认为它们的作用是多余的。然而,与以前的观点相反,NCK1 和 NCK2 现在具有越来越多的旁系特异性功能,这突出表明有必要进一步探讨它们的差异。在这里,我们回顾了目前的证据,详细说明了这两个旁系亲属的独特性,包括它们在基因/蛋白调控、结合伙伴和对细胞功能的总体贡献方面的差异。为了帮助解释这些截然不同的特征,我们接着讨论了 SH2/SH3 的结构特征、无序的域间连接区和翻译后修饰。本综述旨在强调区分 NCK1 和 NCK2 在研究中的重要性,并为研究它们相互作用特异性的起源铺平道路。
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引用次数: 0
Identification of inhibitors of human ChaC1, a cytoplasmic glutathione degrading enzyme through high throughput screens in yeast. 通过酵母中的高通量筛选鉴定人ChaC1(一种细胞质谷胱甘肽降解酶)的抑制剂。
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-16 DOI: 10.1042/bcj20240447
Shradha Suyal,Chinmayee Choudhury,Deepinder Kaur,Anand K Bachhawat
The cytosolic glutathione-degrading enzyme, ChaC1, is highly up-regulated in several cancers, with the up-regulation correlating to poor prognosis. The ability to inhibit ChaC1 is therefore important in different pathophysiological situations, but is challenging owing to the high substrate Km of the enzyme. As no inhibitors of ChaC1 are known, in this study we have focussed on this goal. We have initially taken a computational approach where a systemic structure-based virtual screening was performed. However, none of the predicted hits proved to be effective inhibitors. Synthetic substrate analogs were also not inhibitory. As both these approaches targeted the active site, we shifted to developing two high-throughput, robust, yeast-based assays that were active site independent. A small molecule compound library was screened using an automated liquid handling system using these screens. The hits were further analyzed using in vitro assays. Among them, juglone, a naturally occurring naphthoquinone, completely inhibited ChaC1 activity with an IC50 of 8.7 µM. It was also effective against the ChaC2 enzyme. Kinetic studies indicated that the inhibition was not competitive with the substrate. Juglone is known to form adducts with glutathione and is also known to selectively inhibit enzymes by covalently binding to active site cysteine residues. However, juglone continued to inhibit a cysteine-free ChaC1 variant, indicating that it was acting through a novel mechanism. We evaluated different inhibitory mechanisms, and also analogues of juglone, and found plumbagin effective as an inhibitor. These compounds are the first inhibitor leads against the ChaC enzymes using a robust yeast screen.
细胞膜谷胱甘肽降解酶 ChaC1 在多种癌症中高度上调,上调与预后不良相关。因此,在不同的病理生理情况下,抑制 ChaC1 的能力非常重要,但由于该酶的底物 Km 很高,因此具有挑战性。由于目前还没有已知的 ChaC1 抑制剂,在这项研究中,我们将重点放在了这一目标上。我们最初采用了一种计算方法,进行了基于系统结构的虚拟筛选。然而,没有一个预测的结果被证明是有效的抑制剂。合成的底物类似物也没有抑制作用。由于这两种方法都针对活性位点,我们转而开发了两种独立于活性位点的高通量、稳健、基于酵母的检测方法。利用这些筛选方法,我们使用自动液体处理系统筛选了一个小分子化合物库。对筛选出的化合物进一步进行了体外分析。其中,juglone(一种天然萘醌)完全抑制了 ChaC1 的活性,IC50 为 8.7 µM。它对 ChaC2 酶也有效。动力学研究表明,这种抑制作用与底物之间不存在竞争关系。众所周知,丁螺环酮可与谷胱甘肽形成加合物,还可通过与活性位点半胱氨酸残基共价结合而选择性地抑制酶。然而,丁螺环酮仍能抑制不含半胱氨酸的 ChaC1 变体,这表明它是通过一种新的机制发挥作用的。我们评估了不同的抑制机制以及朱格隆的类似物,发现plumbagin是一种有效的抑制剂。这些化合物是利用强大的酵母筛选技术找到的首个针对 ChaC 酶的抑制剂线索。
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