Biochimie最新文献_第2页

Human anti-apoptotic Bcl-2 and Bcl-xL proteins protect yeast cells from aging induced oxidative stress 人类抗凋亡蛋白 Bcl-2 和 Bcl-xL 保护酵母细胞免受老化诱导的氧化应激。

IF 3.3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2025-02-01 DOI: 10.1016/j.biochi.2024.10.009

Ayşenur Güler , Berna Kavakcıoğlu Yardımcı , Nihal Şimşek Özek

Aging is a degenerative, biological, and time-dependent process that affects all organisms. Yeast aging is a physiological phenomenon characterized by the progressive transformation of yeast cells, resulting in modifications to their viability and vitality. Aging in yeast cells is comparable to that in higher organisms in some respects; however, due to their straightforward and well-characterized genetic makeup, these cells present unique advantages when it comes to researching the aging process. Here, we assessed the impact of human anti-apoptotic Bcl-2 and Bcl-xL proteins on aging using a yeast model. The findings clearly showed that these proteins exhibited remarkable anti-aging properties in yeast cells. Our data indicate that the presence of both proteins enhanced the reproductive survival of aging cells, likely by effecting the components functioning as both pro- and anti-oxidants, depending on the stage of yeast cell lifespan. Both proteins partially protected yeast cells from aging-related morphological deformations and cellular damage during the aging period. In particular, Bcl-xL expressing yeast cells reached the maximum activity levels for almost all of the major antioxidant enzymes and the total antioxidant status on the 8th day of lifespan and could provide effective protection at the latest stage of the investigated aging period. The chemometric data analysis of IR spectra confirmed the findings of the morphological and biochemical analyses. In this regard, specifically, understanding the mechanism of action on the cellular redox state of Bcl-xL in yeast may facilitate comprehension of its indirect antioxidant function in higher eukaryotes.

衰老是一种影响所有生物的退化性、生物性和时间依赖性过程。酵母衰老是一种生理现象，其特点是酵母细胞逐渐发生转变，导致其活力和生命力发生改变。酵母细胞的衰老在某些方面可与高等生物的衰老相媲美；然而，由于酵母细胞的基因构成简单明了、特征清晰，因此在研究衰老过程方面具有独特的优势。在这里，我们利用酵母模型评估了人类抗凋亡蛋白 Bcl-2 和 Bcl-xL 对衰老的影响。研究结果清楚地表明，这些蛋白在酵母细胞中表现出显著的抗衰老特性。我们的数据表明，这两种蛋白质的存在提高了衰老细胞的繁殖存活率，这可能是通过影响作为促氧化剂和抗氧化剂的成分（取决于酵母细胞的寿命阶段）来实现的。在衰老过程中，两种蛋白都能部分保护酵母细胞免受与衰老相关的形态畸变和细胞损伤。其中，表达 Bcl-xL 的酵母细胞在寿命的第 8 天几乎所有主要抗氧化酶和总抗氧化状态的活性都达到了最高水平，并能在所研究的衰老期的最后期提供有效的保护。红外光谱的化学计量数据分析证实了形态和生化分析的结果。在这方面，了解 Bcl-xL 在酵母中对细胞氧化还原状态的作用机制可能有助于理解其在高等真核生物中的间接抗氧化功能。

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引用次数: 0

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IF 3.3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2025-02-01 DOI: 10.1016/S0300-9084(25)00003-3

引用次数: 0

Site-directed mutagenesis of nattokinase: Unveiling structure-function relationship for enhanced functionality 纳豆激酶的定点突变：揭示结构-功能关系以增强功能

IF 3.3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2025-02-01 DOI: 10.1016/j.biochi.2024.09.014

Ankush Jain, Pradeep Kumar Anand, Jagdeep Kaur

Site-directed mutagenesis was employed to investigate the structure-function relationship of nattokinase (NK) and its effect on the enzymatic activity, thermostability, pH tolerance, and fibrinolytic properties of NK. Specific mutations (T270S, V271I, E262D, and A259T) were introduced within the nk gene, targeting regions predicted to be involved in substrate binding. The NK(E262D) mutant exhibited a significant increase in enzymatic activity (2-fold) and catalytic efficiency (2.2-fold) as assessed by N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA) hydrolysis, compared to the wild type. In silico analysis supported these findings, demonstrating lower binding energy for the NK(E262D) mutant, suggesting stronger fibrin affinity. Thermostability assays revealed that NK(E262D) and NK(A259T) displayed exceptional stability, retaining enzyme activity at 60 °C. All mutants exhibited a broader pH tolerance range (pH 5.0–10.0) compared to the wild-type NK. The fibrinolytic activity assay revealed that the E262D mutant possessed the highest fibrinolytic activity (2414 U/mg), surpassing the wild-type. This study reported an NK variant with improved enzymatic activity, thermostability, and fibrinolytic properties.

为了研究纳豆激酶（NK）的结构-功能关系及其对 NK 的酶活性、热稳定性、pH 耐受性和纤维蛋白溶解特性的影响，我们采用了定点诱变技术。研究人员在 nk 基因中引入了特定的突变（T270S、V271I、E262D 和 A259T），这些突变针对的是预测与底物结合有关的区域。与野生型相比，NK(E262D)突变体的酶活性（2倍）和催化效率（2.2倍）均有显著提高。硅学分析证实了这些发现，证明 NK(E262D) 突变体的结合能更低，表明其纤维蛋白亲和力更强。热稳定性测定显示，NK(E262D)和NK(A259T)表现出了极高的稳定性，在60°C时仍能保持酶活性。与野生型 NK 相比，所有突变体的 pH 值耐受范围更广（pH 值为 5.0-10.0）。纤溶活性测定显示，E262D 突变体具有最高的纤溶活性（2414 U/mg ），超过了野生型。该研究报告了一种酶活性、热稳定性和纤维蛋白溶解特性均得到改善的 NK 变体。

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引用次数: 0

Robust approach for production of the human oncology target Aurora kinase B in complex with its binding partner INCENP 生产人类肿瘤靶标极光激酶 b 与其结合伙伴 incenp 复合物的稳健方法。

IF 3.3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2025-02-01 DOI: 10.1016/j.biochi.2024.10.011

Jonna Mattsson , Per Rogne , Maréne Landström , Magnus Wolf-Watz

Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers. The human Aurora kinase B has in particular proven challenging to study with structural biology approaches, and this is mainly due to difficulties in producing the large quantities of active enzyme required for such studies. Here, we present a novel and E. coli-based production system that allows for production of milligram quantities of well-folded and active human Aurora B in complex with its binding partner INCENP. The complex is produced as a continuous polypeptide chain and the resulting fusion protein is cleaved with TEV protease to generate a stable and native heterodimer of the Aurora B:INCENP complex. The activity, stability and degree of phosphorylation of the protein complex was quantified by using a coupled ATPase assay, ³¹P NMR spectroscopy and mass spectrometry. The developed production system enables isotope labeling and we here report the first ¹H–¹⁵N-HSQC of the human Aurora B:INCENP complex. Our developed production strategy paves the way for future structural and functional studies of Aurora B and can as such assist the development of novel anticancer drugs targeting this important mitotic protein kinase.

蛋白激酶是许多真核生物信号转导级联中的关键角色，因此常常与人类疾病相关。在人类中，有丝分裂蛋白激酶家族中的极光激酶由三个成员组成：所有这三个成员都参与细胞分裂，并对各种人类癌症产生影响。事实证明，用结构生物学方法研究人类极光激酶 B 尤其具有挑战性，这主要是由于难以生产此类研究所需的大量活性酶。在这里，我们展示了一种基于大肠杆菌的新型生产系统，该系统可以生产折叠良好、具有活性的人极光激酶 B 与其结合伙伴 INCENP 复合物的毫克级数量。该复合物以连续多肽链的形式产生，产生的融合蛋白经 TEV 蛋白酶裂解后生成稳定的 Aurora B:INCENP 复合物异源二聚体。利用耦合 ATPase 分析法、31P NMR 光谱法和质谱法对蛋白复合物的活性、稳定性和磷酸化程度进行了量化。开发的生产系统可以进行同位素标记，我们在此首次报告了人类 Aurora B:INCENP 复合物的 1H-15N-HSQC 结果。我们开发的生产策略为今后 Aurora B 的结构和功能研究铺平了道路，从而有助于开发针对这一重要有丝分裂蛋白激酶的新型抗癌药物。

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引用次数: 0

The effect of extremolytes ectoine and hydroxyectoine on the heat-induced protein aggregation: The case of growth hormone 极性溶解物埃克托因和羟基埃克托因对热诱导蛋白质聚集的影响：以生长激素为例

IF 3.3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2025-02-01 DOI: 10.1016/j.biochi.2024.10.006

Rūta Gruškienė, Jolanta Sereikaitė

The extremolytes ectoine and hydroxyectoine are osmolytes found in extremophilic microorganisms. They are stabilisers of proteins and other macromolecules, including DNA and lipids. The aim of the study was to investigate the effect of the additives on the heat-induced aggregation of mink growth hormone as a model protein. The first-order rate constants of protein aggregation were determined at 60 °C depending on the additive concentration and pH of the solution. The onset temperature of aggregation was also recorded using a circular dichroism spectropolarimeter. The study showed that the effect of the additives depended on the pH of the solution. The first-order rate constants of aggregation were lower when the protein molecule had a negative charge. The effect also depended on the structure of the extremolyte itself. When the protein molecule was positively charged, hydroxyectoine destabilised the mink growth hormone molecule and promoted the aggregation. The different effects of the additives were determined by the different interactions with the protein molecules, as shown by circular dichroism measurements and previously by fluorescence spectroscopy. Therefore, when using ectoine or hydroxyectoine for protein formulation, the effect of the additive should be carefully analysed for each protein individually.

嗜极微生物中的极性溶解物ectoine 和 hydroxyectoine 是渗透溶解物。它们是蛋白质和其他大分子（包括 DNA 和脂质）的稳定剂。本研究旨在探讨添加剂对作为模型蛋白质的水貂生长激素受热诱导聚集的影响。根据添加剂的浓度和溶液的 pH 值，测定了 60 °C 时蛋白质聚集的一阶速率常数。此外，还使用圆二色性分光光度计记录了聚集的起始温度。研究表明，添加剂的效果取决于溶液的 pH 值。当蛋白质分子带负电荷时，聚集的一阶速率常数较低。这种影响还取决于极溶解物本身的结构。当蛋白质分子带正电荷时，羟基环氧乙烷会破坏水貂生长激素分子的稳定性并促进聚集。添加剂的不同作用是由其与蛋白质分子的不同相互作用决定的，圆二色性测量和之前的荧光光谱法都证明了这一点。因此，在使用埃克托因或羟基埃克托因配制蛋白质时，应仔细分析添加剂对每种蛋白质的影响。

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引用次数: 0

Luminescence-based complementation assay to assess target engagement and cell permeability of glycolate oxidase (HAO1) inhibitors 基于荧光的互补测定法，用于评估乙醇酸氧化酶（HAO1）抑制剂的靶参与性和细胞渗透性。

IF 3.3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2025-01-01 DOI: 10.1016/j.biochi.2024.08.011

Sabrina R. Mackinnon , Tryfon Zarganes-Tzitzikas , Cassandra J. Adams , Paul E. Brennan , Wyatt W. Yue

Glycolate oxidase (HAO1) catalyses the synthesis of glyoxylate, a common metabolic intermediate that causes renal failure if accumulated. HAO1 inhibition is an emerging treatment for primary hyperoxaluria, a rare disorder of glyoxylate metabolism. Here we report the first cell-based measurement of inhibitor uptake and engagement with HAO1, by adapting the cellular thermal shift assay (CETSA) based on Nano luciferase complementation and luminescence readout. By profiling the interaction between HAO1 and four well-characterised inhibitors in intact and lysed HEK293T cells, we showed that our CETSA method differentiates between low-permeability/high-engagement and high-permeability/low-engagement ligands and is able to rank HAO1 inhibitors in line with both recombinant protein methods and previously reported indirect cellular assays. Our methodology addresses the unmet need for a robust, sensitive, and scalable cellular assay to guide HAO1 inhibitor development and, in broader terms, can be rapidly adapted for other targets to simultaneously monitor compound affinity and cellular permeability.

乙醛酸氧化酶（HAO1）能催化乙醛酸盐的合成，乙醛酸盐是一种常见的代谢中间产物，一旦积累就会导致肾功能衰竭。抑制 HAO1 是治疗原发性高草酸尿症（一种罕见的乙醛酸代谢紊乱）的新兴疗法。在此，我们报告了基于纳米荧光素酶互补和发光读数的细胞热转移分析法（CETSA），首次以细胞为基础测量了抑制剂的吸收和与 HAO1 的相互作用。通过分析 HAO1 与四种特性明确的抑制剂在完整和裂解的 HEK293T 细胞中的相互作用，我们发现我们的 CETSA 方法能区分低渗透性/高参与性和高渗透性/低参与性配体，并能对 HAO1 抑制剂进行分级，这与重组蛋白方法和之前报道的间接细胞检测方法一致。我们的方法满足了对一种稳健、灵敏、可扩展的细胞检测方法的未满足需求，以指导 HAO1 抑制剂的开发。

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引用次数: 0

Enzymatic activity of HIV-1 protease defines migration of tumor cells in vitro and enhances their metastatic activity in vivo hiv-1 蛋白酶的酶活性决定了肿瘤细胞在体外的迁移并增强其在体内的转移活性。

IF 3.3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2025-01-01 DOI: 10.1016/j.biochi.2024.08.009

M. Isaguliants , A. Zhitkevich , S. Petkov , T. Gorodnicheva , D. Mezale , I. Fridrihsone , Y. Kuzmenko , D. Kostyushev , A. Kostyusheva , I. Gordeychuk , E. Bayurova

Overexpression of aspartic proteases, as cathepsin D, is an independent marker of poor prognosis in breast cancer, correlated with the incidence of clinical metastasis. We aimed to find if HIV-1 aspartic protease (PR) can play a similar role. Murine adenocarcinoma 4T1luc2 cells were transduced with lentivirus encoding inactivated drug-resistant PR, generating subclones PR20.1 and PR20.2. Subclones were assessed for production of reactive oxygen species (ROS), expression of epithelial-mesenchymal transition (EMT) factors, and in vitro migratory activity in the presence or absence of antioxidant N-acetyl cysteine and protease inhibitors. Tumorigenic activity was evaluated by implanting cells into BALB/c mice and following tumor growth by calipering and bioluminescence imaging in vivo, and metastases, by organ imaging ex vivo. Both subclones expressed PR mRNA, and PR20.2, also the protein detected by Western blotting. PR did not induce production of ROS, and had no direct effect on cell migration rate, however, treatment with inhibitors of drug-resistant PR suppressed the migratory activity of both subclones. Furthermore, expression of N-cadherin and Vimentin in PR20.2 cells and their migration were enhanced by antioxidant treatment. Sensitivity of in vitro migration to protease inhibitors and to antioxidant, known to restore PR activity, related the effects to the enzymatic activity of PR. In vivo, PR20.2 cells demonstrated higher tumorigenic and metastatic activity than PR20.1 or parental cells. Thus, HIV-1 protease expressed in breast cancer cells determines their migration in vitro and metastatic activity in vivo. This effect may aggravate clinical course of cancers in people living with HIV-1.

天冬氨酸蛋白酶（如 cathepsin D）的过表达是乳腺癌预后不良的独立标志，与临床转移的发生率相关。我们的目的是研究 HIV-1 天冬氨酸蛋白酶（PR）是否也能发挥类似的作用。用编码失活耐药 PR 的慢病毒转导小鼠腺癌 4T1luc2 细胞，产生 PR20.1 和 PR20.2 亚克隆。在抗氧化剂 N-乙酰半胱氨酸和蛋白酶抑制剂存在或不存在的情况下，对子克隆的活性氧（ROS）产生、上皮-间质转化（EMT）因子的表达以及体外迁移活性进行了评估。通过将细胞植入 BALB/c 小鼠体内，并在体内通过量热和生物发光成像跟踪肿瘤生长情况，以及在体外通过器官成像跟踪转移情况，评估了致瘤活性。两个亚克隆都表达 PR mRNA 和 PR20.2，蛋白也通过 Western 印迹检测到。PR不会诱导ROS的产生，对细胞迁移率也没有直接影响，但耐药PR抑制剂会抑制两个亚克隆的迁移活性。此外，PR20.2细胞中N-cadherin和Vimentin的表达及其迁移在抗氧化剂处理后得到增强。体外迁移对蛋白酶抑制剂和抗氧化剂（已知可恢复 PR 活性）的敏感性与 PR 的酶活性有关。在体内，PR20.2 细胞比 PR20.1 或亲代细胞表现出更高的致瘤和转移活性。因此，乳腺癌细胞中表达的 HIV-1 蛋白酶决定了它们在体外的迁移和体内的转移活性。这种效应可能会加重 HIV-1 感染者癌症的临床病程。

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引用次数: 0

Using targeted proteomics-based detection of collagen propeptides to quantify fibrillar collagen biogenesis in vitro 利用基于蛋白质组学的胶原蛋白肽靶向检测，量化体外纤维胶原蛋白的生物生成。

IF 3.3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2025-01-01 DOI: 10.1016/j.biochi.2024.09.004

Matthias Kühle, Joachim Kuhn, Thanh-Diep Ly, Cornelius Knabbe, Bastian Fischer

The collagen superfamily, as the major structural component of the extracellular matrix, encompasses 28 distinct subtypes, with type-I and –III forming fibrils crucial for the matrix scaffold. During collagen biogenesis, trimers of type-I and –III procollagen are secreted into the extracellular matrix. The N- and C-terminal propeptides of these trimers are proteolytically cleaved from procollagen during secretion, initiating collagen fibril formation. The propeptides are released into extracellular space and, therefore, have been used to quantify collagen biogenesis. But high-throughput methods for the quantification of these biomarkers are still lacking. This study presents a state-of-the-art multiplexed approach for the simultaneous quantification of PINP, PICP, PIIINP and PIIICP from cell culture supernatants. The ability of targeted proteomics to quantify these propeptides from cell culture samples was assessed in this study. Using tryptic digestion and solid phase extraction, we were able to accurately quantify precollagen propeptides in a range of 3–1000 ng/mL. The assay showed an average inter-assay variance of 6.86 % with an overall recovery ranging from 92 to 98 %. The assay was validated using recombinant protein standards diluted in surrogate matrix and tested using transforming growth factor β1 mediated induction of normal human dermal fibroblasts. In summary, the assay presented in this paper offers a novel, robust, and precise high-throughput method for measuring human collagen propeptides in cell culture supernatants, empowering researchers to assess collagen biogenesis effectively in in vitro experiments.

胶原蛋白超家族是细胞外基质的主要结构成分，包括 28 个不同的亚型，其中 I 型和 -III 型胶原蛋白形成的纤维对基质支架至关重要。在胶原蛋白的生物生成过程中，I 型和 -III 型胶原蛋白的三聚体被分泌到细胞外基质中。在分泌过程中，这些三聚体的 N- 端和 C- 端丙肽会从胶原蛋白中被蛋白水解，从而启动胶原纤维的形成。丙肽释放到细胞外空间，因此被用于量化胶原蛋白的生物生成。但目前仍缺乏高通量的方法来量化这些生物标记物。本研究提出了一种最先进的多重方法，用于同时定量细胞培养上清液中的 PINP、PICP、PIIINP 和 PIIICP。本研究评估了靶向蛋白质组学从细胞培养样本中量化这些肽的能力。通过胰蛋白酶消化和固相萃取，我们能够准确定量 3 至 1000 ng/mL 范围内的前胶原蛋白肽。该测定的平均测定间差异为 6.86%，总体回收率为 92% 至 98%。该测定使用在替代基质中稀释的重组蛋白标准品进行了验证，并使用转化生长因子 β1 介导的正常人真皮成纤维细胞诱导进行了测试。总之，本文介绍的检测方法为测量细胞培养上清中的人胶原蛋白肽提供了一种新颖、稳健、精确的高通量方法，使研究人员能够在体外实验中有效评估胶原蛋白的生物生成。

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引用次数: 0

Extracellular vesicles as a potential source of biomarkers for endocrine disruptors in MASLD: A short review on the case of DEHP 细胞外囊泡作为 MASLD 中内分泌干扰物生物标志物的潜在来源：DEHP 案例简评。

IF 3.3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2025-01-01 DOI: 10.1016/j.biochi.2024.09.009

Pierre-Etienne Merret, Lydie Sparfel, Catherine Lavau, Dominique Lagadic-Gossmann, Corinne Martin-Chouly

Metabolic dysfunction–Associated Steatotic Liver Disease (MASLD) is a chronic disease with increasing prevalence and for which non-invasive biomarkers are needed. Environmental endocrine disruptors (EDs) are known to be involved in the onset and progression of MASLD and assays to monitor their impact on the liver are being developed. Extracellular vesicles (EVs) mediate cell communication and their content reflects the pathophysiological state of the cells from which they are released. They can thus serve as biomarkers of the pathological state of the liver and of exposure to EDs. In this review, we present the relationships between DEHP (Di(2-ethylhexyl) phthalate) and MASLD and highlight the potential of EVs as biomarkers of DEHP exposure and the resulting progression of MASLD.

代谢功能障碍相关性脂肪性肝病（MASLD）是一种慢性疾病，发病率越来越高，需要非侵入性的生物标志物。众所周知，环境内分泌干扰物（EDs）与脂肪肝的发病和进展有关，目前正在开发监测其对肝脏影响的检测方法。细胞外囊泡（EVs）介导细胞间的交流，其内容物反映了释放EVs的细胞的病理生理状态。因此，它们可以作为肝脏病理状态和暴露于 EDs 的生物标志物。在这篇综述中，我们介绍了DEHP（邻苯二甲酸二（2-乙基己酯））与MASLD之间的关系，并强调了EVs作为DEHP暴露及其导致的MASLD进展的生物标志物的潜力。

引用次数: 0

Mutations in the N-domain of aryl hydrocarbon receptor interacting protein affect interactions with heat shock protein 90β and phosphodiesterase 4A5 芳基烃受体相互作用蛋白 N-域的突变会影响与热休克蛋白 90β 和磷酸二酯酶 4A5 的相互作用。

IF 3.3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2025-01-01 DOI: 10.1016/j.biochi.2024.09.005

Marita Vella , Iain W. Manfield , Brandon C. Seychell , Chi H. Trinh , Robert Rambo , G. Nasir Khan , Josanne Vassallo , Thérèse Hunter , Gary J. Hunter

The aryl hydrocarbon receptor interacting protein (AIP) is a cytoplasmic molecular co-chaperone and tumour suppressor that assists in protein stability and complex formation involving the aryl hydrocarbon receptor. Germline mutations in the AIP gene predispose to pituitary tumourigenesis with patients exhibiting an aggressive clinical phenotype. Full length AIP proteins harbouring N-domain mutations (R9Q, R16H, V49 M and K103R) were purified from E.coli utilizing a methodology that maintained structural integrity and monomeric stability. Mutations did not significantly affect the thermal stability of the protein and caused no overall disruptive effect in the protein structure. The mutations studied lowered the binding affinity of AIP towards two of its binding partners; heat shock protein 90β and phosphodiesterase 4A5 (PDE4A5). The inhibition of phosphodiesterase activity by AIP was also greatly reduced by all mutants. While previously published data has mainly concentrated on the tetratricopeptide repeats of the C-domain of AIP, we present clear evidence that AIP N-domain mutations play a significant role in two protein:protein interactions with partner proteins. The complex interactome of AIP suggests that any observable change in one or more of its binding partners cannot be disregarded as it may have repercussions on other biochemical pathways.

芳基烃受体相互作用蛋白（AIP）是一种细胞质分子辅助伴侣和肿瘤抑制因子，有助于蛋白质的稳定性和涉及芳基烃受体的复合物的形成。AIP 基因的种系突变易导致垂体肿瘤发生，患者表现出侵袭性临床表型。利用一种保持结构完整性和单体稳定性的方法，从大肠杆菌中纯化了携带 N-域突变（R9Q、R16H、V49M 和 K103R）的全长 AIP。突变不会对蛋白质的热稳定性产生重大影响，也不会对蛋白质结构造成整体破坏性影响。所研究的突变降低了 AIP 与其两个结合伙伴（热休克蛋白 90β 和磷酸二酯酶 4A5 (PDE4A5)）的结合亲和力。所有突变体对 AIP 磷酸二酯酶活性的抑制作用也大大降低。虽然以前发表的数据主要集中在 AIP C 域的四重肽重复序列上，但我们提出了明确的证据，证明 AIP N 域突变在与伙伴蛋白的两种蛋白：蛋白相互作用中发挥了重要作用。AIP 复杂的相互作用组表明，不能忽视其一个或多个结合伙伴的任何可观察到的变化，因为这可能会对其他生化途径产生影响。

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引用次数: 0