Journal of Molecular Biology最新文献

Assembly of the human multi-tRNA synthetase complex through leucine zipper motifs. 通过亮氨酸拉链图案组装人类多 tRNA 合成酶复合物。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-11-12 DOI: 10.1016/j.jmb.2024.168865

Dong Kyu Kim, Kayoung Lee, Beom Sik Kang

Aminoacyl-tRNA synthetases (ARSs) are responsible for the ligation of amino acids to their cognate tRNAs. In human, nine ARSs form a multi-tRNA synthetase complex (MSC) with three ARS-interacting multifunctional proteins (AIMPs). Among the components of MSC, arginyl-tRNA synthetase 1 (RARS1) and two AIMPs (AIMP1 and AIMP2) have leucine zipper (LZ) motifs, which they utilize for their assembly in an MSC. RARS1 and AIMP1 have two LZ motifs (LZ1 and LZ2) in their N-terminus, respectively, while AIMP2 has one LZ motif between its lysyl-tRNA synthetase 1 (KARS1)-binding motif and glutathione transferase-homology domain, which links aspartyl-tRNA synthetase 1 (DARS1). Although the interaction mode between AIMP1 and RARS1, which also binds glutaminyl-tRNA synthetase 1 (QARS1), has been revealed, the mode in the presence of AIMP2 is still ambiguous since AIMP2 is known to not only bind to AIMP1 but also form a homodimer through its LZ. Here, we determined a crystal structure of the LZ complex of AIMP1 and AIMP2 and revealed the interaction mode of a heterotrimeric complex of RARS1, AIMP1, and AIMP2. The complex is established by a three-stranded coiled-coil structure with RARS1 LZ1, AIMP1 LZ1, and AIMP2 LZ and is completed with a two-stranded coiled-coil structure of RARS1 LZ2 and AIMP1 LZ2. In the human MSC, this heterotrimeric complex of RARS1, AIMP1, and AIMP2 allows for a subcomplex of fourteen protein molecules, in which two QARS1-RARS1-AIMP1-AIMP2-2×KARS1 complexes are linked separately to a dimeric DARS1.

氨基酰-tRNA 合成酶（ARSs）负责将氨基酸连接到它们的同源 tRNA 上。在人类体内，九个 ARS 与三个 ARS 相互作用的多功能蛋白（AIMPs）组成了一个多 tRNA 合成酶复合物（MSC）。在 MSC 的组成成分中，精氨酰-tRNA 合成酶 1（RARS1）和两个 AIMPs（AIMP1 和 AIMP2）具有亮氨酸拉链（LZ）基序，它们利用这些基序组装成 MSC。RARS1 和 AIMP1 的 N 端分别有两个 LZ 基序（LZ1 和 LZ2），而 AIMP2 的赖氨酰-tRNA 合成酶 1（KARS1）结合基序和谷胱甘肽转移酶同源结构域之间有一个 LZ 基序，该结构域连接天冬氨酰-tRNA 合成酶 1（DARS1）。虽然 AIMP1 与 RARS1（RARS1 也能结合谷氨酰胺酰-tRNA 合成酶 1（QARS1））之间的相互作用模式已被揭示，但由于已知 AIMP2 不仅能与 AIMP1 结合，还能通过其 LZ 形成同源二聚体，因此在 AIMP2 存在时的相互作用模式仍不明确。在这里，我们测定了 AIMP1 和 AIMP2 的 LZ 复合物的晶体结构，并揭示了 RARS1、AIMP1 和 AIMP2 的异源三聚体复合物的相互作用模式。该复合物由 RARS1 LZ1、AIMP1 LZ1 和 AIMP2 LZ 的三股线圈结构构成，并由 RARS1 LZ2 和 AIMP1 LZ2 的两股线圈结构完成。在人类间充质干细胞中，这种由 RARS1、AIMP1 和 AIMP2 组成的异三聚体复合物可形成由 14 个蛋白质分子组成的亚复合物，其中两个 QARS1-RARS1-AIMP1-AIMP2-2×KARS1 复合物分别与二聚体 DARS1 相连。

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引用次数: 0

Corrigendum to “The Role of ATG9 Vesicles in Autophagosome Biogenesis” [J. Mol. Biol. 436(15) (2024) 168489] ATG9 小泡在自噬体生物生成中的作用》[J. Mol. Biol. 436(15) (2024) 168489]的更正。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-11-12 DOI: 10.1016/j.jmb.2024.168849

Elisabeth Holzer , Sascha Martens , Susanna Tulli

引用次数: 0

Structural studies on Mycobacterial NudC reveal a class of zinc independent NADH pyrophosphatase. 对分枝杆菌 NudC 的结构研究揭示了一类独立于锌的 NADH 焦磷酸酶。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-11-07 DOI: 10.1016/j.jmb.2024.168864

Lingyu Meng, Zhaojian Sun, Yulong Zhang, Yan Dong, Xiaoan Du, Yujian Wu, Yuan Yuan, Yirong Sun, Yong Xu, Huaiwei Ding, Jinsong Liu, Jinxin Xu

Non-tuberculous mycobacteria (NTM) have emerged as an increasing threat to public health, due to the extreme antibiotic resistance. NADH pyrophosphatase (NudC) was proposed involving in mycobacterial resistance to the first line anti-tubercular drug isoniazid (INH) or its analog ethionamide (ETH), by hydrolyzing their NAD modified active forms (NAD-INH and NAD-ETH). In this study, we performed enzymatic and structural studies on NudC from M. abscessus (NudC_Mab), which is highly resistant to isoniazid and emerging as the most worrisome NTM. We determined the crystal structures of NudC_Mab in apo form, substrate NAD-bound form and product AMP-bound form. We observed the mode for the Nudix motif of NudC_Mab capturing the pyrophosphate group of NAD mediated by three divalent cation ions, which provides details for understanding the mechanism on NudC hydrolyzing NAD(H) or NAD-capped substrate. Interestingly, our structures revealed a novel subclass NudC from mycobacteria characterized by a unique arginine residue on the conserved QPWPFPxS motif, as well as a unique tower domain that replaces a well-defined zinc-binding motif in E.coli NudC and catalytic domain of mammalian Nudt12. Thus, our structural studies on NudC_Mab not only present a class of zinc independent NADH pyrophosphatase in mycobacteria, but also may facilitate the design of NudC inhibitors for the treatment of mycobacteria infections in combination with INH or ETH.

非结核分枝杆菌（NTM）对抗生素具有极强的耐药性，对公共卫生的威胁日益严重。有人提出，NADH焦磷酸酶（NudC）通过水解NAD修饰的活性形式（NAD-INH和NAD-ETH），参与分枝杆菌对一线抗结核药物异烟肼（INH）或其类似物乙硫酰胺（ETH）的耐药性。在本研究中，我们对来自脓肿霉菌的 NudC（NudCMab）进行了酶学和结构研究，脓肿霉菌对异烟肼高度耐药，正在成为最令人担忧的非典型肺炎霉菌。我们测定了 NudCMab 的蛋白酶、底物 NAD 结合型和产物 AMP 结合型晶体结构。我们观察到 NudCMab 的 Nudix 基序在三个二价阳离子的介导下捕获 NAD 的焦磷酸基团的模式，这为了解 NudC 水解 NAD(H) 或 NAD 封闭底物的机制提供了细节。有趣的是，我们的结构发现了一种来自分枝杆菌的新型亚类 NudC，其特点是在保守的 QPWPFPxS 基序上有一个独特的精氨酸残基，还有一个独特的塔状结构域，取代了大肠杆菌 NudC 中定义明确的锌结合基序和哺乳动物 Nudt12 的催化结构域。因此，我们对 NudCMab 的结构研究不仅展示了分枝杆菌中一类锌独立的 NADH 焦磷酸酶，还有助于设计 NudC 抑制剂，与 INH 或 ETH 联用治疗分枝杆菌感染。

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引用次数: 0

Revealing Missing Protein–Ligand Interactions Using AlphaFold Predictions 利用 AlphaFold 预测揭示缺失的蛋白质配体相互作用。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-11-06 DOI: 10.1016/j.jmb.2024.168852

Nahuel Escobedo , Tadeo Saldaño , Juan Mac Donagh , Luciana Rodriguez Sawicki , Nicolas Palopoli , Sebastian Fernandez Alberti , Maria Silvina Fornasari , Gustavo Parisi

Protein–ligand interactions represent an essential step to understand the bases of molecular recognition, an intense field of research in many scientific areas. Structural biology has played a central role in unveiling protein–ligand interactions, but current techniques are still not able to reliably describe the interactions of ligands with highly flexible regions. In this work, we explored the capacity of AlphaFold2 (AF2) to estimate the presence of interactions between ligands and residues belonging to disordered regions. As these interactions are missing in the crystallographic-derived structures, we called them “ghost interactions”. Using a set of protein structures experimentally obtained after AF2 was trained, we found that the obtained models are good predictors of regions associated with order–disorder transitions. Additionally, we found that AF2 predicts residues making ghost interactions with ligands, which are mostly buried and show differential evolutionary conservation with the rest of the residues located in the flexible region. Our findings could fuel current areas of research that consider, given their biological relevance and their involvement in diseases, intrinsically disordered proteins as potentially valuable targets for drug development.

蛋白质与配体的相互作用是了解分子识别基础的重要一步，也是许多科学领域的热门研究领域。结构生物学在揭示蛋白质-配体相互作用方面发挥了核心作用，但目前的技术仍无法可靠地描述配体与高柔性区域的相互作用。在这项工作中，我们探索了 AlphaFold2（AF2）估计配体与属于无序区域的残基之间是否存在相互作用的能力。由于这些相互作用在晶体学结构中缺失，我们称之为 "幽灵相互作用"。通过使用一组经过 AF2 训练后通过实验获得的蛋白质结构，我们发现所获得的模型可以很好地预测与有序-无序转换相关的区域。此外，我们还发现 AF2 可以预测与配体发生鬼影作用的残基，这些残基大多被埋藏，并与位于柔性区域的其他残基显示出不同的进化守恒性。鉴于内在无序蛋白质的生物学相关性及其与疾病的关系，我们的研究结果可以推动当前的研究领域，使其成为有价值的潜在药物开发目标。

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引用次数: 0

Pim1 is Critical in T-cell-independent B-cell Response and MAPK Activation in B Cells Pim1 在 B 细胞的独立于 T 细胞的 B 细胞反应和 MAPK 激活中至关重要。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-11-04 DOI: 10.1016/j.jmb.2024.168824

Dongya Cui , Yongguang Zhang , Baijiao Zheng , Liling Chen , Jianhui Wei , Danfeng Lin , Miaohui Huang , Hekang Du , Qi Chen

The Pim family consists of three members that encode a distinct class of highly conserved serine/threonine kinases. In this study, we generated and examined mice with hematopoiesis-specific deletion of Pim1 and bone marrow (BM) chimeric mice with B-cell-specific targeted deletion of Pim1. Pim1 was expressed at all stages of B-cell development and hematopoietic-specific deletion of Pim1 altered B-cell development in BM, spleen and peritoneal. However, Pim1 deficiency did not affect T-cell development. Studies of BM chimeric mice showed that Pim1 is required in a cell-intrinsic manner to maintain normal B-cell development. Pim1 deficiency led to significant changes in B cell antibody responses. Additionally, Pim1 deficiency resulted in reduced B cell receptor (BCR)-induced cell proliferation and cell cycle progression. Examination of the various BCR-activated signaling pathways in Pim1-deficient B cells reveals defective activation of mitogen-activated protein kinases (MAPKs), which are known to regulate genes involved in cell proliferation and survival. qRT-PCR analysis of BCR-engaged B cells from Pim1-deficient B cells revealed reduced expression of cyclin-dependent kinase (CDK) and cyclin genes, including CDK2, CCNB1 and CCNE1. In conclusion, Pim1 plays a crucial role in B-cell development and B cell activation.

Pim 家族由三个成员组成，它们编码一类不同的高度保守的丝氨酸/苏氨酸激酶。在这项研究中，我们产生并研究了造血特异性缺失 Pim1 的小鼠和骨髓（BM）嵌合体小鼠。Pim1 在 B 细胞发育的各个阶段都有表达，造血特异性缺失 Pim1 会改变 BM、脾脏和腹膜中 B 细胞的发育。然而，Pim1 的缺失并不影响 T 细胞的发育。对BM嵌合小鼠的研究表明，Pim1需要以细胞内在方式维持正常的B细胞发育。Pim1 缺乏会导致 B 细胞抗体反应发生显著变化。此外，Pim1 缺乏还会导致 B 细胞受体（BCR）诱导的细胞增殖和细胞周期进展减少。对 Pim1 缺乏的 B 细胞中 BCR 激活的各种信号通路进行的研究发现，丝裂原活化蛋白激酶（MAPKs）的激活存在缺陷，而众所周知，MAPKs 可调节参与细胞增殖和存活的基因；对 Pim1 缺乏的 B 细胞中 BCR 激活的 B 细胞进行的 qRT-PCR 分析显示，细胞周期蛋白依赖性激酶（CDK）和细胞周期蛋白基因（包括 CDK2、CCNB1 和 CCNE1）的表达减少。总之，Pim1 在 B 细胞发育和 B 细胞活化中起着至关重要的作用。

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引用次数: 0

A Nanobody Toolbox for Recognizing Distinct Epitopes on Cas9 用于识别 Cas9 上不同表位的纳米抗体工具箱。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-10-30 DOI: 10.1016/j.jmb.2024.168836

Jack Boylan , Rebecca A Shrem , Isabel C. Vallecillo-Viejo , Craig L. Duvall , Brian E. Wadzinski , Benjamin W. Spiller

Cas9s and fusions of Cas9s have emerged as powerful tools for genetic manipulations. Fusions of Cas9 with other DNA editing enzymes have led to variants capable of single base editing and catalytically dead Cas9s have emerged as tools to specifically target desired regions of a genome. Here we describe the generation of a panel of nanobodies directed against three unique epitopes on Streptococcus pyogenes Cas9. The nanobodies were identified from a nanobody library derived from an alpaca that had been immunized with Cas9. The most potent binders recognize Cas9 and RNA bound Cas9 equally well and do not inhibit Cas9 cleavage of target DNA. These nanobodies bind non-overlapping epitopes as determined by ELISA based epitope binning experiments and mass photometry. We present the sequences of these clones and supporting biochemical data so the broader scientific community can access these reagents.

Cas9s 和 Cas9s 融合体已成为基因操作的强大工具。Cas9与其他DNA编辑酶的融合产生了能够进行单碱基编辑的变体，而催化死亡的Cas9则成为了特异性靶向基因组所需区域的工具。在这里，我们描述了针对化脓性链球菌 Cas9 上的三个独特表位生成纳米抗体的过程。这些纳米抗体是从用 Cas9 免疫的羊驼身上提取的纳米抗体库中鉴定出来的。最有效的结合体同样能识别Cas9和RNA结合的Cas9，而且不会抑制Cas9裂解靶DNA。根据基于酶联免疫吸附法的表位分选实验和质量光度测定法确定，这些纳米抗体结合的表位并不重叠。我们介绍了这些克隆的序列和支持性生化数据，以便更广泛的科学界能够获得这些试剂。

引用次数: 0

Translation Complex Profile Sequencing Allows Discrimination of Leaky Scanning and Reinitiation in Upstream Open Reading Frame-controlled Translation 通过翻译复合体轮廓测序，可以分辨上游开放阅读框控制翻译中的漏扫描和再启动。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-10-30 DOI: 10.1016/j.jmb.2024.168850

Dmitri E. Andreev , Jack A.S. Tierney , Pavel V. Baranov

Upstream open reading frames (uORFs) are a class of translated regions (translons) in mRNA 5′ leaders. uORFs are believed to be pervasive regulators of the translation of mammalian mRNAs. Some uORFs are highly repressive but others have little or no impact on downstream mRNA translation either due to inefficient recognition of their start codon(s) or/and due to efficient reinitiation after uORF translation. While experiments with uORF reporter constructs proved to be instrumental in the investigation of uORF-mediated mechanisms of translation control, they can have serious limitations as manipulations with uORF sequences can yield various artefacts. Here we propose a general approach for using translation complex profiling (TCP-seq) data for exploring uORF regulatory characteristics. Using several examples, we show how TCP-seq could be used to estimate both repressiveness and modes of action of individual uORFs. We demonstrate how this approach could be used to assess the mechanisms of uORF-mediated translation control in the mRNA of several human genes, including EIF5, IFRD1, MDM2, MIEF1, PPP1R15B, TAF7, and UCP2.

上游开放阅读框（uORFs）是 mRNA 5' 头部的一类翻译区域（转译子）。据信，uORFs 是哺乳动物 mRNA 翻译的普遍调节因子。有些 uORF 具有高度抑制作用，但有些 uORF 对下游 mRNA 翻译影响很小或没有影响，原因可能是对其起始密码子的识别效率不高，或者/和由于 uORF 翻译后的有效再启动。尽管使用 uORF 报告构建体进行的实验被证明有助于研究 uORF 介导的翻译控制机制，但它们也有严重的局限性，因为对 uORF 序列的操作可能会产生各种假象。在这里，我们提出了一种使用翻译复合剖析（TCP-seq）数据探索 uORF 调控特性的通用方法。通过几个例子，我们展示了如何利用 TCP-seq 估算单个 uORF 的抑制性和作用模式。我们展示了这种方法如何用于评估 uORF 介导的几个人类基因 mRNA 翻译控制机制，包括 EIF5、IFRD1、MDM2、MIEF1、PPP1R15B、TAF7 和 UCP2。

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引用次数: 0

HulaCCR1, a pump-like cation channelrhodopsin discovered in a lake microbiome 在湖泊微生物群中发现的泵状阳离子通道罗多素的扩展 N 端区和 ETD 基团的作用。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-10-29 DOI: 10.1016/j.jmb.2024.168844

Shunki Takaramoto , Shai Fainsod , Takashi Nagata , Andrey Rozenberg , Oded Béjà , Keiichi Inoue

Channelrhodopsins are light-gated ion channels consisting of seven transmembrane helices and a retinal chromophore, which are used as popular optogenetic tools for modulating neuronal activity. Cation channelrhodopsins (CCRs), first recognized as the photoreceptors in the chlorophyte Chlamydomonas reinhardtii, have since been identified in diverse species of green algae, as well in other unicellular eukaryotes. The CCRs from non-chlorophyte species are commonly referred to as bacteriorhodopsin-like cation channelrhodopsins, or BCCRs, as most of them feature the three characteristic amino acid residues of the “DTD motif” in the third transmembrane helix (TM3 or helix C) matching the canonical DTD motif of the well-studied archaeal light-driven proton pump bacteriorhodopsin. Here, we report characterization of HulaCCR1, a novel BCCR identified through metatranscriptomic analysis of a unicellular eukaryotic community in Lake Hula, Israel. Interestingly, HulaCCR1 has an ETD motif in which the first residue of the canonical motif is substituted for glutamate. Electrophysiological measurements of the wild-type and a mutant with a DTD motif of HulaCCR1 suggest the critical role of the first glutamate in spectral tuning and channel gating. Additionally, HulaCCR1 exhibits long extensions at the N- and C-termini. Photocurrents recorded from a truncated variant without the signal peptide predicted at the N-terminus were diminished, and membrane localization of the truncated variant significantly decreased, indicating that the signal peptide is important for membrane trafficking of HulaCCR1. These characteristics of HulaCCR1 would be related to a new biological significance in the original unidentified species, distinct from those known for other BCCRs.

通道闪烁蛋白（Channelrhodopsins）是一种光门控离子通道，由七个跨膜螺旋和一个视网膜发色团组成，是调节神经元活动的常用光遗传工具。阳离子通道视网膜素（CCRs）最早被认为是叶绿体衣藻的光感受器，后来又在多种绿藻和其他单细胞真核生物中被发现。来自非叶绿体物种的 CCRs 通常被称为 "类细菌视紫红质通道视紫红质"（bacteriorhodopsin-like channelrhodopsins）或 BCCRs，因为它们中的大多数都具有第三个跨膜螺旋（TM3 或螺旋 C）中 "DTD 基序 "的三个特征性氨基酸残基，与已被充分研究的古生光驱动质子泵细菌视紫红质的典型 DTD 基序相匹配。在这里，我们报告了 HulaCCR1 的特征，它是通过对以色列胡拉湖中的单细胞真核生物群落进行元转录本组分析而发现的一种新型 BCCR。有趣的是，HulaCCR1 有一个 ETD 基团，其中标准基团的第一个残基被谷氨酸取代。对 HulaCCR1 的野生型和具有 DTD 基序的突变体进行的电生理测量表明，第一个谷氨酸在光谱调谐和通道门控中起着关键作用。此外，HulaCCR1 在 N 端和 C 端有较长的延伸。一个在 N 端没有信号肽的截短变体记录到的光电流减弱，截短变体的膜定位显著降低，这表明信号肽对 HulaCCR1 的膜运输非常重要。HulaCCR1 的这些特征将与原始未确定物种的新生物学意义有关，有别于已知的其他 BCCRs。

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引用次数: 0

The Hsp90 Molecular Chaperone as a Global Modifier of the Genotype-Phenotype-Fitness Map: An Evolutionary Perspective Hsp90分子伴侣是基因型-表型-适配性图谱的全球修饰因子：进化的视角

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-10-29 DOI: 10.1016/j.jmb.2024.168846

José Aguilar-Rodríguez , Christopher M. Jakobson , Daniel F. Jarosz

Global modifier genes influence the mapping of genotypes onto phenotypes and fitness through their epistatic interactions with genetic variants on a massive scale. The first such factor to be identified, Hsp90, is a highly conserved molecular chaperone that plays a central role in protein homeostasis. Hsp90 is a “hub of hubs” that chaperones proteins engaged in many key cellular and developmental regulatory networks. These clients, which are enriched in kinases, transcription factors, and E3 ubiquitin ligases, drive diverse cellular functions and are themselves highly connected. By contrast to many other hub proteins, the abundance and activity of Hsp90 changes substantially in response to shifting environmental conditions. As a result, Hsp90 modifies the functional impact of many genetic variants simultaneously in a manner that depends on environmental stress. Studies in diverse organisms suggest that this coupling between Hsp90 function and challenging environments exerts a substantial impact on what parts of the genome are visible to natural selection, expanding adaptive opportunities when most needed. In this Perspective, we explore the multifaceted role of Hsp90 as global modifier of the genotype-phenotype-fitness map as well as its implications for evolution in nature and the clinic.

全局修饰基因通过与遗传变异的大规模表观相互作用，影响着基因型与表型的映射和适应性。第一个被发现的此类因子 Hsp90 是一种高度保守的分子伴侣，在蛋白质平衡中发挥着核心作用。Hsp90 是一个 "枢纽中的枢纽"，它为参与许多关键细胞和发育调控网络的蛋白质提供伴侣。这些客户包括激酶、转录因子和 E3 泛素连接酶，它们驱动着多种细胞功能，而且本身也高度关联。与许多其他枢纽蛋白不同的是，Hsp90 的丰度和活性会随着环境条件的变化而发生重大变化。因此，Hsp90 以一种取决于环境压力的方式同时改变了许多基因变异的功能影响。对不同生物的研究表明，Hsp90 功能与挑战性环境之间的这种耦合关系对基因组中哪些部分可以被自然选择发现产生了重大影响，从而在最需要的时候扩大了适应机会。在本《视角》中，我们将探讨Hsp90作为基因型-表型-适配性图谱全球修饰因子的多方面作用及其对自然界和临床进化的影响。

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引用次数: 0

Conformational Differences in the Light Chain Constant Domain of Immunoglobulin G and Free Light Chain May Influence Proteolysis in AL Amyloidosis 免疫球蛋白 G 的轻链恒定域和游离轻链的构象差异可能会影响 AL 淀粉样变性病的蛋白水解。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-10-28 DOI: 10.1016/j.jmb.2024.168837

Elena S. Klimtchuk , Tatiana Prokaeva , Brian H. Spencer , Sherry Wong , Shreya Ghosh , Angela Urdaneta , Gareth Morgan , Thomas E. Wales , Olga Gursky

Immunoglobulin light chain amyloidosis (AL) is a life-threatening disease caused by the deposition of light chain (LC) and its fragments containing variable (V_L) and portions of constant (C_L) domains. AL patients feature either monoclonal free LCs (FLCs) circulating as covalent and noncovalent homodimers, or monoclonal immunoglobulin (Ig) wherein the LC and heavy chain (HC) form disulfide-linked heterodimers, or both. The role of full-length Ig in AL amyloidosis is unclear as prior studies focused on FLC or V_L domain. We used a mammalian cell-based expression system to generate four AL patient-derived full-length IgGs, two non-AL IgG controls, and six corresponding FLC proteins derived from an IGLV6-57 germline precursor. Comparison of proteins’ secondary structure, thermal stability, proteolytic susceptibility, and disulfide link reduction suggested the importance of local vs. global conformational stability. Analysis of IgGs vs. corresponding FLCs using hydrogen–deuterium exchange mass spectrometry revealed major differences in the local conformation/dynamics of the C_L domain. In all IgGs vs. FLCs, segments containing β-strand and α-helix βA_C-αA_CB_C were more dynamic/exposed while segment βD_C-βE_C was less dynamic/exposed. Notably, these segments overlapped proteolysis-prone regions whose in vivo cleavage generates LC fragments found in AL deposits. Altogether, the results suggest that preferential cleavage in segments βA_C-αA_CB_C of FLC or βD_C-βE_C of LC in IgG helps generate amyloid protein precursors. We propose that protecting these segments using small-molecule stabilizers, which bind to the interfacial cavities C_L-C_L in FLC and/or C_L-C_H1 in IgG, is a potential therapeutic strategy to complement current approaches targeting V_L-V_L or V_L-C_L stabilization of LC dimer.

免疫球蛋白轻链淀粉样变性（AL）是一种威胁生命的疾病，由轻链（LC）及其包含可变（VL）和部分恒定（CL）结构域的片段沉积引起。AL 患者的特征是单克隆游离轻链（FLC）以共价和非共价同二聚体形式循环，或单克隆免疫球蛋白（Ig），其中轻链和重链（HC）形成二硫键连接的异二聚体，或两者兼而有之。全长 Ig 在 AL 淀粉样变性中的作用尚不清楚，因为之前的研究主要集中在 FLC 或 VL 结构域。我们使用基于哺乳动物细胞的表达系统生成了四种 AL 患者来源的全长 IgG、两种非 AL IgG 对照以及六种相应的 FLC 蛋白，这些蛋白来源于 IGLV6-57 胚系前体。蛋白质二级结构、热稳定性、蛋白水解敏感性和二硫键还原性的比较表明了局部构象稳定性与整体构象稳定性的重要性。利用氢氘交换质谱分析 IgG 与相应的 FLC，发现 CL 结构域的局部构象/动力学存在重大差异。在所有 IgG 与 FLCs 的对比中，含有 β-链和α-螺旋 βAC-αACBC 的区段的动态/暴露程度较高，而 βDC-βEC 区段的动态/暴露程度较低。值得注意的是，这些片段与蛋白水解易发区重叠，蛋白水解易发区在体内裂解产生 AL 沉积物中的 LC 片段。总之，这些结果表明，IgG 中 FLC 的 βAC-αACBC 段或 LC 的 βDC-βEC 段的优先裂解有助于产生淀粉样蛋白前体。我们建议使用小分子稳定剂来保护这些区段，这种稳定剂可与 FLC 中的 CL-CL 和/或 IgG 中的 CL-CH1 接口空腔结合，是一种潜在的治疗策略，可补充目前以 VL-VL 或 VL-CL 稳定 LC 二聚体为目标的方法。

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引用次数: 0