Biochemistry and Biophysics Reports最新文献

Exploring the impact of hAMSCs secretome on Panc1 cells via TNF-α/NF-κB (p50/p65)/Caspase 3 signaling pathways: An in vitro study 探讨hAMSCs分泌组通过TNF-α/NF-κB (p50/p65)/Caspase 3信号通路对Panc1细胞的影响：体外研究

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports

Pub Date : 2025-12-12 DOI: 10.1016/j.bbrep.2025.102411

Aynaz Khalafi, Fatemeh Safari

The second most prevalent cause of mortality worldwide is cancer. Pancreatic cancer, known as the “king of cancers” due to its unfavorable prognosis and absence of symptoms, is among the fatal types of cancer. Despite the availability of various cancer therapy options, current strategies are often ineffective. Therefore, there is a constant need to explore novel platforms with low side effects and high efficacy. The application of stem cells or their derivatives in treating diseases including cancer, has become well-established. This study, focuses on investigating the effects of the secretome of human mesenchymal stem cells (hAMSCs) on Panc1 pancreatic cancer cells through the tumor necrosis factor-alpha (TNF-α)/nuclear factor-κB (NF-κB)/Caspase 3 signaling pathways. A co-culture system using 6-well plates transwell was utilized for this purpose. After 72 h, cell death in hAMSCs-treated Panc1 cells was analyzed through the TNF-α/NF-κB (p50/p65)/Caspase 3 signaling pathways using Western blot and enzyme-linked immunosorbent assay (ELISA). DAPI staining was used to visualize cell death in hAMSCs-treated Panc1 cells. The results showed an up-regulation of TNF-α, IL-1β, IL-8, p-IKK, p-IKKα, p-IKKβ, p-IκB, p53, PUMA, Caspase 3, and NF-κB (p50/p65) as well as a down-regulation of IKβ. These findings suggest that the secretome of hAMSCs promotes both inflammation and apoptosis in Panc1 pancreatic cancer cells simultaneously.

全球第二大死因是癌症。胰腺癌因预后不良、无症状而被称为“癌症之王”，是致命的癌症之一。尽管有各种各样的癌症治疗选择，目前的策略往往是无效的。因此，不断需要探索副作用低、疗效高的新型平台。干细胞或其衍生物在治疗包括癌症在内的疾病方面的应用已经得到证实。本研究主要探讨人间充质干细胞（hAMSCs）分泌组通过肿瘤坏死因子-α (TNF-α)/核因子-κB (NF-κB)/Caspase 3信号通路对胰腺胰腺胰腺细胞Panc1的影响。为此，采用transwell 6孔板共培养系统。72 h后，采用Western blot和酶联免疫吸附试验（ELISA），通过TNF-α/NF-κB (p50/p65)/Caspase 3信号通路分析hamscs处理的Panc1细胞的细胞死亡情况。DAPI染色显示hamscs处理的Panc1细胞的细胞死亡情况。结果显示TNF-α、IL-1β、IL-8、p-IKK、p-IKKα、p-IKKβ、p- i -κB、p53、PUMA、Caspase 3、NF-κB （p50/p65）表达上调，IKβ表达下调。这些发现表明hAMSCs分泌组同时促进Panc1胰腺癌细胞的炎症和凋亡。

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引用次数: 0

MicroRNA-181a in acute lymphoblastic leukemia: expression pattern, target genes and its potential as a biomarker MicroRNA-181a在急性淋巴细胞白血病中的表达模式、靶基因及其作为生物标志物的潜力

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports

Pub Date : 2025-12-11 DOI: 10.1016/j.bbrep.2025.102405

Mohammad Jahanpanah, Abtin Ghasempour, Diana Mokhtari, Shirin Eshghi, Mahsa Mousakhan Bakhtiari, Mohammad Taha Salmanifard Ardestani, Leila Jafari, Maryam Behfar, Amir Ali Hamidieh

The exact etiology of Acute Lymphoblastic Leukemia (ALL) is not yet fully understood. Inherited genetic abnormalities and exposure to environmental risk factors are implicated in pathogenesis of ALL patients. Over the past two decades, epigenetic factors like microRNAs (miRs) have been in the spotlight and shown to have a pivotal role in the pathogenicity of ALL. MiRs are a group of small non-coding single-stranded RNAs (∼22 nucleotides) involved in post-transcriptional regulation of gene expression. MiR-181a has been shown to have a dual behavior in different types of cancers. This dual behavior of miR-181a shows that it should be investigated in a disease-specific manner. In this study, 24 studies were reviewed in different sections including the expression pattern of miR-181a, its target genes, and its potential as a biomarker in ALL from January 2000 to March 2025. Results of the reviewed studies conclusively indicate that miR-181a-5p is upregulated in pediatric ALL patients compared to normal control groups, and miR-181a-5p acts as an oncomir in pediatric ALL patients. Nevertheless, there are only a few studies in adults with inconclusive results. Furthermore, miR-181a-5p might be the upstream activator of the leukemic T-cell proliferation by having direct and indirect effects on several genes. Recent studies indicate that this miR can be implemented in diagnosing and classifying central nervous system (CNS) involvement in ALL patients, which is a devastating challenge in the field of hematology-oncology.

急性淋巴细胞白血病（ALL）的确切病因尚不完全清楚。遗传异常和暴露于环境危险因素与ALL患者的发病机制有关。在过去的二十年里，表观遗传因子如microRNAs （miRs）已经成为人们关注的焦点，并被证明在ALL的致病性中起着关键作用。MiRs是一组小的非编码单链rna（约22个核苷酸），参与基因表达的转录后调控。MiR-181a已被证明在不同类型的癌症中具有双重行为。miR-181a的这种双重行为表明它应该以疾病特异性的方式进行研究。在这项研究中，从2000年1月到2025年3月，我们回顾了24项研究，包括miR-181a的表达模式、它的靶基因，以及它作为ALL生物标志物的潜力。综述的研究结果最终表明，与正常对照组相比，miR-181a-5p在儿科ALL患者中表达上调，miR-181a-5p在儿科ALL患者中起肿瘤作用。然而，只有少数针对成人的研究没有确定的结果。此外，miR-181a-5p可能是白血病t细胞增殖的上游激活因子，对几个基因有直接和间接的影响。最近的研究表明，该miR可用于ALL患者中枢神经系统（CNS）病变的诊断和分类，这是血液肿瘤学领域的一个毁灭性挑战。

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引用次数: 0

TMEM207-mediated the impairment of skin regeneration through YAP sequestration in an allergic contact dermatitis model 在过敏性接触性皮炎模型中，tmem207通过YAP隔离介导皮肤再生损伤

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports

Pub Date : 2025-12-11 DOI: 10.1016/j.bbrep.2025.102409

Shusuke Nomura, Yusuke Kito, Chiemi Saigo, Tamotsu Takeuchi

Aim

The Yes-associated protein (YAP) family of transcriptional coactivators has emerged as a potent promoter of cell proliferation in many types of stem/progenitor cells and cancers. Skin is a squamous epithelium that is continuously regenerated by stem/progenitor cells in the basal layer and is capable of wound healing, and YAP also plays an important role in maintaining skin homeostasis and cellular proliferation. Therefore, we focused on YAP and investigated the importance of YAP regulation in allergic contact dermatitis from the perspective of wound healing and regeneration.

Methods

We investigated the expression and pathological characteristics of Transmembrane protein 207 (TMEM207), focusing on YAP-mediated regulation in atopic models, as abnormal TMEM207 expression may cause various functional abnormalities or regulate the function of YAP.

Results

TMEM207 was detected not only in the stomach and large intestine but also in the bulge region of the sebaceous glands and hair roots in mice expressing TMEM207. In addition, ATP binding cassette subfamily B member 1 (ABCB1) expression decreased in the sebaceous glands, and MIB E3 Ubiquitin Protein Ligase 1 (MIB-1) expression diminished in the epidermis. Furthermore, although we were unable to confirm the binding of TMEM207 to NEDD4, we confirmed the binding of TMEM207 to the Yes1-associated transcription factor (YAP) by immunoprecipitation, and a decrease in the nuclear localization of YAP was observed by immunohistochemical staining.

Conclusion

Our findings indicate that abnormal expression of TMEM207 is involved in the decline of skin regeneration capacity through YAP, leading to the aggravation of Allergic contact dermatitis.

yes相关蛋白（YAP）转录共激活因子家族已成为许多类型的干细胞/祖细胞和癌症中细胞增殖的有效促进子。皮肤是一种鳞状上皮，由基底层的干细胞/祖细胞不断再生，具有伤口愈合的能力，YAP在维持皮肤稳态和细胞增殖方面也起着重要作用。因此，我们以YAP为研究对象，从伤口愈合和再生的角度探讨YAP调控在变应性接触性皮炎中的重要性。方法研究跨膜蛋白207 （Transmembrane protein 207, TMEM207）的表达及病理特征，重点研究YAP在特应性模型中的调控作用，因为TMEM207的异常表达可能导致各种功能异常或调控YAP的功能。结果表达TMEM207的小鼠不仅在胃和大肠中检测到stmem207，而且在皮脂腺凸起区和毛根中也检测到stmem207。此外，ATP结合盒B亚家族成员1 （ABCB1）在皮脂腺中的表达降低，MIB E3泛素蛋白连接酶1 （MIB-1）在表皮中的表达降低。此外，虽然我们无法证实TMEM207与NEDD4的结合，但我们通过免疫沉淀证实了TMEM207与yes1相关转录因子（YAP）的结合，并且通过免疫组织化学染色观察到YAP的核定位减少。结论TMEM207的异常表达通过YAP参与皮肤再生能力下降，导致变应性接触性皮炎加重。

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引用次数: 0

Methodological validation of a cryo-TEM-based detection method for empty capsid ratio in recombinant adeno-associated virus 重组腺相关病毒空衣壳比冷冻tem检测方法的方法学验证

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports

Pub Date : 2025-12-10 DOI: 10.1016/j.bbrep.2025.102397

Lingyi Xu , Jinhuan Chen , Wei Zhu , Yaokun Zhao , Fangfang Zheng , Rong Du , Yuwei Jiang , Yidan Yang , Yuanyuan Chen , Yuanshu Dong , Zhengxi Zhang , Yong Tong

Recombinant adeno-associated virus (rAAV) is one of the most promising vectors for gene therapy. It consists of a protein capsid that encapsulates the genetic material. However, during the production process, except for the full particles containing the genome, a variety of types of particles are also produced concomitantly, such as empty particles (no DNA encapsulated in the capsid), viral fragments, damaged viruses, and viral aggregates. Empty particles have been reported to induce unnecessary immune response and reduce transduction efficiency. Therefore, a quantitative method that objectively assesses the content of rAAV particles containing the genome is crucial for quality measurement. In this study, we developed a technique to detect the proportion of empty capsids in rAAV samples by using cryogenic transmission electron microscopy (cryo-TEM). This method not only accurately quantifies the percentage of empty capsids, but also allows for the characterization and analysis of other different particle types within the sample. Our comprehensive evaluation of specificity, precision, accuracy, linearity, and limit of quantitation (LOQ) demonstrates the advantages of cryo-TEM technology for the quantitative analysis of rAAV empty capsid ratio. Moreover, this research offers a thorough, multidimensional approach to enhance the understanding and implementation of quality control for rAAV.

重组腺相关病毒（rAAV）是最有前途的基因治疗载体之一。它由包裹遗传物质的蛋白质衣壳组成。然而，在生产过程中，除了含有基因组的完整颗粒外，还会同时产生各种类型的颗粒，如空颗粒（衣壳中没有封装DNA）、病毒片段、受损病毒和病毒聚集体。据报道，空颗粒可诱导不必要的免疫反应并降低转导效率。因此，一种客观评估含有基因组的rAAV颗粒含量的定量方法对于质量测量至关重要。在这项研究中，我们开发了一种利用低温透射电子显微镜（cro - tem）检测rAAV样品中空衣壳比例的技术。该方法不仅可以准确地定量空衣壳的百分比，还可以对样品中其他不同颗粒类型进行表征和分析。我们对特异度、精密度、准确度、线性度和定量限（LOQ）进行了综合评价，证明了冷冻透射电镜技术在rAAV空衣壳比定量分析中的优势。此外，本研究还提供了一种全面、多维的方法来增强对rAAV质量控制的理解和实施。

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引用次数: 0

Comparative analysis of activation of macrophages/microglia in diabetic retinopathy and Familial Exudative Vitreoretinopathy 糖尿病视网膜病变与家族性渗出性玻璃体视网膜病变中巨噬细胞/小胶质细胞活化的比较分析

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports

Pub Date : 2025-12-09 DOI: 10.1016/j.bbrep.2025.102396

Jiakai Li , Ping Fei , Xiang Zhang , Yuqing Rao , Jing Li , Peiquan Zhao

Familial Exudative Vitreoretinopathy (FEVR) and Diabetic Retinopathy (DR) are two prominent retinal diseases. The role of macrophages/microglia in the vascular dynamics of FEVR and DR is unknown and thus addressed in this study. FZD4 knockout mouse, a model for FEVR in human characterized by genetic mutations affecting angiogenesis, exhibited reduced b-wave amplitudes and decreased vascular density, replicating human FEVR symptoms. Conversely, STZ-treated C57/BL6 mouse developed heightened fasting glucose levels, reduced insulin content, and increased retinal vasculature, aligning with DR features. Further analysis revealed significant differences in macrophage/microglia populations between the two diseases. In DR, a marked increase in both number and M2-like polarization of retinal macrophages/microglia was observed, contrasting with FEVR. Moreover, DR induced substantial proinflammatory differentiation of macrophages/microglia, evidenced by elevated cytokines such as IL-1β, TNF-α, and IFNɣ. Both conditions significantly upregulated Ang-1 and IL-10, with a more pronounced IL-10 increase in DR, suggesting a more active role in tissue and vessel remodeling. Notably, DR induced higher levels of anti-inflammatory factors like bFGF, TIMP-1, TGFβ1, and VEGF-A compared to FEVR, suggesting a balance of inflammation initiation, progression and resolution. These findings highlight the distinct roles of macrophages/microglia in FEVR and DR, providing insights into their contributions to disease pathogenesis and potential therapeutic strategies through reprogramming macrophages/microglia.

家族性渗出性玻璃体视网膜病变（FEVR）和糖尿病性视网膜病变（DR）是两种突出的视网膜疾病。巨噬细胞/小胶质细胞在FEVR和DR的血管动力学中的作用尚不清楚，因此在本研究中进行了研究。FZD4基因敲除小鼠是一种以影响血管生成的基因突变为特征的人类FEVR模型，其表现出b波振幅降低和血管密度降低，复制了人类FEVR症状。相反，stz处理的C57/BL6小鼠空腹血糖水平升高，胰岛素含量降低，视网膜血管增加，与DR特征一致。进一步分析显示，两种疾病之间巨噬细胞/小胶质细胞群存在显著差异。在DR中，与FEVR相比，视网膜巨噬细胞/小胶质细胞的数量和m2样极化均明显增加。此外，DR诱导巨噬细胞/小胶质细胞大量促炎分化，IL-1β、TNF-α和IFN α等细胞因子升高证明了这一点。两种情况下均显著上调Ang-1和IL-10，其中IL-10在DR中升高更为明显，表明其在组织和血管重塑中发挥更积极的作用。值得注意的是，与FEVR相比，DR诱导了更高水平的抗炎因子，如bFGF、TIMP-1、tgf - β1和VEGF-A，这表明炎症的发生、进展和消退是平衡的。这些发现突出了巨噬细胞/小胶质细胞在FEVR和DR中的独特作用，为巨噬细胞/小胶质细胞重编程对疾病发病机制和潜在治疗策略的贡献提供了见解。

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引用次数: 0

Signalling pathways in hepatocellular carcinoma (HCC) metastasis and invasion: Molecular mechanisms and therapeutic implications 肝细胞癌（HCC）转移和侵袭的信号通路：分子机制和治疗意义

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports

Pub Date : 2025-12-09 DOI: 10.1016/j.bbrep.2025.102403

Jayanta Das , Bhupen Barman , Phulen Sarma , Bipul Kumar Das , Rajiv Chetia , Partha Pratim Kalita

HCC is one of the deadliest malignancies with a rising global occurrence and poor prognosis. Metastasis and invasion are essential processes in the HCC progression, and have a profound bearing on clinical outcome. This review explores the key signalling pathways involved in HCC metastasis and invasion, focusing on their molecular mechanisms, crosstalk, and therapeutic implications. Alongside the discussion of the Wnt/β-catenin, TGF-β, PI3K/AKT/mTOR, MAPK/ERK, HGF/c-MET, Notch and Hippo-YAP/TAZ pathways, are known to contribute to promoting aggressive HCC behaviour. Stromal interactions, extracellular matrix remodelling, hypoxia and angiogenesis as well as the tumour microenvironment are also highlighted. These pathways are subject to current therapeutic treatments in the form of tyrosine kinase inhibitors and monoclonal antibodies, and research prospective of the Wnt/β-catenin blocker, TGF-β inhibitors, etc. The variations in tumours and resistance patterns to treatment and their existing problems in treating HCC are addressed. The review evaluates new therapeutic targets offering a foundation for further research and clinical advancements in this challenging field.

HCC是最致命的恶性肿瘤之一，全球发病率不断上升，预后不良。转移和侵袭是HCC发展的重要过程，对临床预后有着深远的影响。本文综述了参与HCC转移和侵袭的关键信号通路，重点讨论了它们的分子机制、串扰和治疗意义。除了讨论Wnt/β-catenin外，TGF-β、PI3K/AKT/mTOR、MAPK/ERK、HGF/c-MET、Notch和Hippo-YAP/TAZ通路也有助于促进HCC的侵袭性行为。间质相互作用，细胞外基质重塑，缺氧和血管生成以及肿瘤微环境也被强调。这些途径受到当前以酪氨酸激酶抑制剂和单克隆抗体形式的治疗，以及Wnt/β-catenin阻断剂、TGF-β抑制剂等的研究前景。讨论了肝癌治疗中肿瘤和耐药模式的变化及其存在的问题。该综述评估了新的治疗靶点，为这一具有挑战性的领域的进一步研究和临床进展提供了基础。

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引用次数: 0

Heme as activator and target for artemisinin: Towards multiple pharmacological bioactivity 血红素作为青蒿素的激活剂和靶点：走向多重药理生物活性

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports

Pub Date : 2025-12-09 DOI: 10.1016/j.bbrep.2025.102398

Pan Zhu , Xinyi Sun , Yuting Fang , Yufei Li , Liang Guo

Artemisinin, a sesquiterpene trioxane derived from the plant Artemisia annua, possesses a distinctive peroxide bridge structure that endows it with remarkable biological activity, primarily through its interaction with heme. Given artemisinin's well-established clinical safety profile, researchers are increasingly investigating its potential applications in antifungal, anticancer, and antiviral treatments. This review employs heme as a foundational element to dissect the mechanisms underlying the diverse pharmacological actions of artemisinin, thereby expanding its application prospects. It also offers valuable insights for the development of novel pharmaceuticals and innovative therapeutic strategies targeting various human diseases.

青蒿素是一种从植物黄花蒿中提取的倍半萜三氧环，具有独特的过氧化物桥结构，主要通过与血红素的相互作用赋予其显著的生物活性。鉴于青蒿素已确立的临床安全性，研究人员正在越来越多地研究其在抗真菌、抗癌和抗病毒治疗方面的潜在应用。本文以血红素为基础元素，剖析青蒿素多种药理作用的机制，从而拓展其应用前景。它还为开发针对各种人类疾病的新型药物和创新治疗策略提供了宝贵的见解。

引用次数: 0

Development of novel anti-CDH1/E-cadherin monoclonal antibodies for versatile applications 新型多功能抗cdh1 / e -钙粘蛋白单克隆抗体的研制

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports

Pub Date : 2025-12-08 DOI: 10.1016/j.bbrep.2025.102401

Rena Ubukata, Hiroyuki Suzuki, Mika K. Kaneko, Yukinari Kato

Cadherin (CDH)-mediated extracellular homophilic binding is crucial for maintaining tissue homeostasis. The epithelial cell-cell adhesion molecule cadherin 1 (CDH1/E‐cadherin) forms the adherens junctions in epithelial cells, and the loss of CDH1 facilitates the migration and invasion of carcinoma cells. Although several anti-CDH1 monoclonal antibodies (mAbs) are available for western blotting and immunohistochemistry (IHC), a highly sensitive anti-CDH1 mAb suitable for flow cytometry has not been developed. We developed anti-CDH1 mAbs through a flow cytometry-based high-throughput screening. Two anti-CDH1 mAb clones, Ca₁Mab-3 (IgG₁, κ) and Ca₁Mab-5 (IgG₁, κ), reacted with human CDH1-overexpressed Chinese hamster ovary-K1 (CHO/CDH1) cells in flow cytometry. Furthermore, Ca₁Mab-3 and Ca₁Mab-5 recognized endogenous CDH1-expressing human luminal-type breast cancer cells, such as MCF-7, but not triple-negative breast cancer cells, like MDA-MB-231. The dissociation constant values of Ca₁Mab-3 and Ca₁Mab-5 for CHO/CDH1 were determined as 5.9 × 10⁻¹⁰ M and 1.8 × 10⁻⁹ M, respectively. Ca₁Mab-3 and Ca₁Mab-5 can detect endogenous CDH1 in western blotting and IHC using a cell block. Furthermore, Ca₁Mab-5 is available for IHC in formalin-fixed paraffin-embedded tumor tissues. These results indicate that Ca₁Mab-3 and Ca₁Mab-5 are versatile for basic research and are expected to contribute to clinical applications, such as tumor diagnosis and therapy.

钙粘蛋白（CDH）介导的细胞外亲同性结合对维持组织稳态至关重要。上皮细胞-细胞粘附分子cadherin 1 （CDH1/E‐cadherin）在上皮细胞中形成粘附连接，CDH1的缺失促进了癌细胞的迁移和侵袭。虽然有几种抗cdh1单克隆抗体（mAb）可用于western blotting和免疫组织化学（IHC），但尚未开发出适合流式细胞术的高灵敏度抗cdh1 mAb。我们通过基于流式细胞术的高通量筛选开发了抗cdh1单克隆抗体。两种抗CDH1单抗克隆Ca1Mab-3 （IgG1, κ）和Ca1Mab-5 （IgG1, κ）在流式细胞术中与人CDH1过表达的中国仓鼠卵巢k1 （CHO/CDH1）细胞发生反应。此外，Ca1Mab-3和Ca1Mab-5可以识别内源性表达cdh1的人发光型乳腺癌细胞，如MCF-7，但不能识别三阴性乳腺癌细胞，如MDA-MB-231。测定Ca1Mab-3和Ca1Mab-5对CHO/CDH1的解离常数分别为5.9 × 10−10 M和1.8 × 10−9 M。Ca1Mab-3和Ca1Mab-5可以在western blotting和细胞块免疫组化中检测内源性CDH1。此外，Ca1Mab-5可用于福尔马林固定石蜡包埋肿瘤组织的免疫组化。这些结果表明，Ca1Mab-3和Ca1Mab-5可用于基础研究，并有望在肿瘤诊断和治疗等临床应用中发挥作用。

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引用次数: 0

Influence of PLIN5 and lipid composition on lipid droplet contact sites with other organelles PLIN5和脂质组成对脂滴与其他细胞器接触部位的影响

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports

Pub Date : 2025-12-08 DOI: 10.1016/j.bbrep.2025.102402

Mahsa Mohammadian , Shima Asfia , Ralf Seemann

Lipid droplets (LDs) maintain cellular lipid homeostasis through dynamic interactions with other organelles. Understanding how these contact sites form is crucial for uncovering the mechanisms of lipid exchange and signaling. In this study, we used an in vitro model to investigate how lipid composition and the LD-associated protein perilipin 5 (PLIN5) influence contact formation between an LD monolayer and a bilayer membrane. Artificial LDs consisting of triolein and coated with either a DOPE or DOPC monolayer containing PLIN5 or not were incubated with large unilamellar vesicles (LUVs) that mimic the bilayer membrane of the organelle. Using double fluorescence labeling of the LUV bilayer and the core, we can distinguish between fusion of the LUV bilayer with the LDs and stable attachment of LUVs to the LD’s surface. Our results show that the probability of fusion between LDs and LUVs is greatly increased for DOPE-coated LDs, while PLIN5 promotes the stable attachment of LUVs to the LD’s surface and prevents fusion. These observations illustrate how certain lipid and protein components can modulate contact formation between LDs and membranes in a controlled in vitro system, and provide a basis for future studies on the molecular mechanisms of organelle communication.

脂滴（ld）通过与其他细胞器的动态相互作用维持细胞脂质稳态。了解这些接触位点如何形成对于揭示脂质交换和信号传导机制至关重要。在这项研究中，我们使用了一个体外模型来研究脂质组成和LD相关蛋白perilipin 5 （PLIN5）如何影响LD单层和双层膜之间的接触形成。由三油酸组成的人工ld表面涂有掺杂或不含PLIN5的DOPC单层，与模拟细胞器双层膜的大单层囊泡（LUVs）一起孵育。通过对LUV双层和核心的双重荧光标记，我们可以区分LUV双层与LD的融合和LUV在LD表面的稳定附着。我们的研究结果表明，dope涂层的LD与luv之间的融合概率大大增加，而PLIN5促进luv在LD表面的稳定附着并阻止融合。这些观察结果说明了在受控的体外系统中，某些脂质和蛋白质成分如何调节lld和膜之间的接触形成，并为未来细胞器通讯的分子机制研究提供了基础。

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引用次数: 0

Reconstitution of CFTR ubiquitination identifies lysine-420 as a regulator of cell surface residence and current 重组CFTR泛素化鉴定赖氨酸420作为细胞表面驻留和电流的调节剂

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports

Pub Date : 2025-12-08 DOI: 10.1016/j.bbrep.2025.102393

Jennifer L. Goeckeler-Fried , Xuemei Zeng , Jeong S. Hong , Disha Joshi , Zhengrong Yang , Fan Jiang , John C. Kappes , Eric J. Sorscher , Jeffrey L. Brodsky

Background

The most common loss-of-function mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) is F508del. The misfolded F508del-CFTR protein is targeted for endoplasmic reticulum associated degradation (ERAD), a pathway in which non-native proteins are ubiquitinated and degraded by the proteasome. Because the identification of ubiquitinated residues would highlight how F508del-CFTR is selected for premature degradation, ubiquitination profiles in CFTR- and F508del-CFTR-expressing cells have been examined. Several ubiquitin ligases modify CFTR, however, the relative CFTR-directed activity of each ligase is unknown.

Methods

We reconstituted CFTR ubiquitination using purified CFTR and components of the ubiquitination machinery. Since prior work implicated the Carboxyl terminus of Hsp70-Interacting Protein (CHIP) ubiquitin ligase in both ERAD and plasma membrane turnover, CFTR ubiquitination was examined in the presence of CHIP and a companion ubiquitin conjugating enzyme.

Results

Mass spectrometry identified 16 modified lysines, half of which were previously identified after CFTR was isolated from cells. One lysine, K420, which resides in the regulatory insertion, had been implicated in cyclic nucleotide-dependent activation of CFTR. Here, we find that mutation of K420 increases cell surface levels of CFTR, an effect which in turn increases forskolin-dependent short circuit current.

Conclusions

We establish a system in which residue-specific modifications of CFTR by any component of the ubiquitin machinery can now be surveyed.

在编码囊性纤维化跨膜传导调节因子（CFTR）的基因中，最常见的功能缺失突变是F508del。错误折叠的F508del-CFTR蛋白是内质网相关降解（ERAD）的靶标，这是一种非天然蛋白被蛋白酶体泛素化和降解的途径。由于泛素化残基的鉴定将突出F508del-CFTR是如何被选择用于过早降解的，因此我们研究了CFTR-和F508del-CFTR表达细胞中的泛素化谱。几种泛素连接酶修饰CFTR，然而，每种连接酶的相对CFTR定向活性是未知的。方法利用纯化的CFTR和泛素化机制的组成部分重组CFTR泛素化。由于先前的研究表明hsp70相互作用蛋白（CHIP）泛素连接酶的羧基端参与ERAD和质膜转换，因此在CHIP和伴随的泛素结合酶存在的情况下，研究了CFTR泛素化。结果质谱鉴定出16种修饰赖氨酸，其中一半是在CFTR分离后鉴定出来的。一种赖氨酸，K420，位于调控插入中，与环核苷酸依赖性的CFTR激活有关。在这里，我们发现K420的突变增加了细胞表面CFTR的水平，这种效应反过来又增加了福斯克林依赖的短路电流。结论我们建立了一个系统，在这个系统中，任何泛素机制的组成部分对CFTR的残基特异性修饰现在都可以被调查。

{"title":"Reconstitution of CFTR ubiquitination identifies lysine-420 as a regulator of cell surface residence and current","authors":"Jennifer L. Goeckeler-Fried , Xuemei Zeng , Jeong S. Hong , Disha Joshi , Zhengrong Yang , Fan Jiang , John C. Kappes , Eric J. Sorscher , Jeffrey L. Brodsky","doi":"10.1016/j.bbrep.2025.102393","DOIUrl":"10.1016/j.bbrep.2025.102393","url":null,"abstract":"<div><h3>Background</h3><div>The most common loss-of-function mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) is F508del. The misfolded F508del-CFTR protein is targeted for endoplasmic reticulum associated degradation (ERAD), a pathway in which non-native proteins are ubiquitinated and degraded by the proteasome. Because the identification of ubiquitinated residues would highlight how F508del-CFTR is selected for premature degradation, ubiquitination profiles in CFTR- and F508del-CFTR-expressing cells have been examined. Several ubiquitin ligases modify CFTR, however, the relative CFTR-directed activity of each ligase is unknown.</div></div><div><h3>Methods</h3><div>We reconstituted CFTR ubiquitination using purified CFTR and components of the ubiquitination machinery. Since prior work implicated the Carboxyl terminus of Hsp70-Interacting Protein (CHIP) ubiquitin ligase in both ERAD and plasma membrane turnover, CFTR ubiquitination was examined in the presence of CHIP and a companion ubiquitin conjugating enzyme.</div></div><div><h3>Results</h3><div>Mass spectrometry identified 16 modified lysines, half of which were previously identified after CFTR was isolated from cells. One lysine, K420, which resides in the regulatory insertion, had been implicated in cyclic nucleotide-dependent activation of CFTR. Here, we find that mutation of K420 increases cell surface levels of CFTR, an effect which in turn increases forskolin-dependent short circuit current.</div></div><div><h3>Conclusions</h3><div>We establish a system in which residue-specific modifications of CFTR by any component of the ubiquitin machinery can now be surveyed.</div></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":"45 ","pages":"Article 102393"},"PeriodicalIF":2.2,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145748902","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

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