Keratin 19 binds and regulates cytoplasmic HNRNPK mRNA targets in triple-negative breast cancerBMC Molecular and Cell Biology - Tập 24 - Trang 1-13 - 2023
Arwa Fallatah, Dimitrios G. Anastasakis, Amirhossein Manzourolajdad, Pooja Sharma, Xiantao Wang, Alexis Jacob, Sarah Alsharif, Ahmed Elgerbi, Pierre A. Coulombe, Markus Hafner, Byung Min Chung
Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long non-coding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes cell proliferation and cancer metastasis. However, the mechanism of HNRNPK cytoplasmic localization, its cytoplasmic RNA ligands, and impact on post-transcriptional gene regulation remain uncharacterized. Here we show that the intermediate filament protein Keratin 19 (K19) directly interacts with HNRNPK and sequesters it in the cytoplasm. Correspondingly, in K19 knockout breast cancer cells, HNRNPK does not localize in the cytoplasm, resulting in reduced cell proliferation. We comprehensively mapped HNRNPK binding sites on mRNAs and showed that, in the cytoplasm, K19-mediated HNRNPK-retention increases the abundance of target mRNAs bound to the 3’ untranslated region (3’UTR) at the expected cytidine-rich (C-rich) sequence elements. Furthermore, these mRNAs protected by HNRNPK in the cytoplasm are typically involved in cancer progression and include the p53 signaling pathway that is dysregulated upon HNRNPK knockdown (HNRNPK KD) or K19 knockout (KRT19 KO). This study identifies how a cytoskeletal protein can directly regulate gene expression by controlling the subcellular localization of RNA-binding proteins to support pathways involved in cancer progression.
Identification of universal and cell-type specific p53 DNA bindingBMC Molecular and Cell Biology - Tập 21 - Trang 1-12 - 2020
Antonina Hafner, Lyubov Kublo, Michael Tsabar, Galit Lahav, Jacob Stewart-Ornstein
The tumor suppressor p53 is a major regulator of the DNA damage response and has been suggested to selectively bind and activate cell-type specific gene expression programs. However recent studies and meta-analyses of genomic data propose largely uniform, and condition independent p53 binding and thus question the selective and cell-type dependent function of p53. To systematically assess the cell-type specificity of p53, we measured its association with DNA in 12 p53 wild-type cancer cell lines, from a range of epithelial linages, in response to ionizing radiation. We found that the majority of bound sites were occupied across all cell lines, however we also identified a subset of binding sites that were specific to one or a few cell lines. Unlike the shared p53-bound genome, which was not dependent on chromatin accessibility, the association of p53 with these atypical binding sites was well explained by chromatin accessibility and could be modulated by forcing cell state changes such as the epithelial-to-mesenchymal transition. Our study reconciles previous conflicting views in the p53 field, by demonstrating that although the majority of p53 DNA binding is conserved across cell types, there is a small set of cell line specific binding sites that depend on cell state.
Recognizing ion ligand binding sites by SMO algorithmBMC Molecular and Cell Biology - Tập 20 - Trang 1-9 - 2019
Shan Wang, Xiuzhen Hu, Zhenxing Feng, Xiaojin Zhang, Liu Liu, Kai Sun, Shuang Xu
In many important life activities, the execution of protein function depends on the interaction between proteins and ligands. As an important protein binding ligand, the identification of the binding site of the ion ligands plays an important role in the study of the protein function. In this study, four acid radical ion ligands (NO2−,CO32−,SO42−,PO43−) and ten metal ion ligands (Zn2+,Cu2+,Fe2+,Fe3+,Ca2+,Mg2+,Mn2+,Na+,K+,Co2+) are selected as the research object, and the Sequential minimal optimization (SMO) algorithm based on sequence information was proposed, better prediction results were obtained by 5-fold cross validation. An efficient method for predicting ion ligand binding sites was presented.
Dự đoán vị trí liên kết ion gốc axit bằng bộ phân loại K-lân cận gần nhất Dịch bởi AI BMC Molecular and Cell Biology - - 2019
Liu Liu, Xiuzhen Hu, Zhenxing Feng, Xiaojin Zhang, Shan Wang, Shuang Xu, Kai Sun
Tóm tắtĐặt vấn đềCác protein thực hiện chức năng của chúng bằng cách tương tác với các ion gốc axit. Gần đây, việc dự đoán chính xác các vị trí liên kết của các ligand ion gốc axit đã trở thành một thách thức trong lĩnh vực thiết kế thuốc phân tử.
Kết quảTrong nghiên cứu này, chúng tôi đã đề xuất một phương pháp cải tiến để dự đoán các vị trí liên kết ion gốc axit bằng cách sử dụng bộ phân loại K-lân cận gần nhất. Trong khi đó, chúng tôi đã xây dựng các tập dữ liệu của bốn ligand ion gốc axit (NO2−, CO32−, SO42−, PO43−) từ cơ sở dữ liệu BioLip. Sau đó, dựa trên độ dài cửa sổ tối ưu cho từng ligand ion gốc axit, chúng tôi đã tinh chỉnh thông tin thành phần và thông tin bảo tồn vị trí và trích xuất chúng làm các tham số đặc trưng cho bộ phân loại K-lân cận gần nhất. Trong kết quả của quá trình kiểm tra chéo 5 lần, hệ số tương quan Matthew cao hơn 0.45, các giá trị độ chính xác, độ nhạy và độ đặc hiệu đều cao hơn 69.2%, và tỷ lệ dương tính giả thấp hơn 30.8%. Hơn nữa, chúng tôi cũng đã thực hiện một bài kiểm tra độc lập để kiểm tra tính khả thi của phương pháp được đề xuất. Trong các kết quả thu được, độ nhạy cao hơn 40.9%, các giá trị độ chính xác và độ đặc hiệu đều cao hơn 84.2%, hệ số tương quan Matthew cao hơn 0.116, và tỷ lệ dương tính giả thấp hơn 15.4%. Cuối cùng, chúng tôi đã xác định các vị trí liên kết của sáu ligand ion kim loại. Trong các kết quả được dự đoán, các giá trị độ chính xác, độ nhạy và độ đặc hiệu đều cao hơn 77.6%, hệ số tương quan Matthew cao hơn 0.6, và tỷ lệ dương tính giả thấp hơn 19.6%.
Kết luậnTóm lại, các kết quả tốt từ phương pháp dự đoán của chúng tôi đã bổ sung những hiểu biết mới trong việc dự đoán các vị trí liên kết của các ligand ion gốc axit.
Identification of a druggable binding pocket in the spike protein reveals a key site for existing drugs potentially capable of combating Covid-19 infectivityBMC Molecular and Cell Biology - Tập 21 - Trang 1-13 - 2020
Elliot D. Drew, Robert W. Janes
Following the recent outbreak of the new coronavirus pandemic (Covid-19), the rapid determination of the structure of the homo-trimeric spike glycoprotein has prompted the study reported here. The aims were to identify potential “druggable” binding pockets in the protein and, if located, to virtual screen pharmaceutical agents currently in use for predicted affinity to these pockets which might be useful to restrict, reduce, or inhibit the infectivity of the virion. Our analyses of this structure have revealed a key potentially druggable pocket where it might be viable to bind pharmaceutical agents to inhibit its ability to infect human cells. This pocket is found at the inter-chain interface that exists between two domains prior to the virion binding to human Angiotensin Converting Enzyme 2 (ACE2) protein. One of these domains is the highly mobile receptor binding domain, which must move into position to interact with ACE2, which is an essential feature for viral entry to the host cell. Virtual screening with a library of purchasable drug molecules has identified pharmaceuticals currently in use as prescription and over the counter medications that, in silico, readily bind into this pocket. This study highlights possible drugs already in use as pharmaceuticals that may act as agents to interfere with the movements of the domains within this protein essential for the infectivity processes and hence might slow, or even halt, the infection of host cells by this new coronavirus. As these are existing pharmaceuticals already approved for use in humans, this knowledge could accelerate their roll-out, through repurposing, for affected individuals and help guide the efforts of other researchers in finding effective treatments for the disease.
MazEF-rifampicin interaction suggests a mechanism for rifampicin induced inhibition of persistersBMC Molecular and Cell Biology - - 2020
Cyrus Alexander, Ankeeta Guru, Pinkilata Pradhan, Sunanda Mallick, Nimai Charan Mahanandia, Bharat Bhusan Subudhi, Tushar Kant Beuria
Persistence is a natural phenomenon whereby a subset of a population of isogenic bacteria either grow slow or become dormant conferring them with the ability to withstand various stresses including antibiotics. In a clinical setting bacterial persistence often leads to the recalcitrance of various infections increasing the treatment time and cost. Additionally, some studies also indicate that persistence can also pave way for the emergence of resistant strains. In a laboratory setting this persistent phenotype is enriched in nutritionally deprived environments. Consequently, in a batch culture the late stationary phase is enriched with persistent bacteria. The mechanism of persister cell formation and its regulation is not well understood. Toxin-antitoxin (TA) systems have been implicated to be responsible for bacterial persistence and rifampicin is used to treat highly persistent bacterial strains. The current study tries to explore a possible interaction between rifampicin and the MazEF TA system that furthers the former’s success rate in treating persistent bacteria. In the current study we found that the population of bacteria in the death phase of a batch culture consists of metabolically inactive live cells resembling persisters, which showed higher membrane depolarization as compared to the log phase bacteria. We also observed an increase in the expression of the MazEF TA modules in this phase. Since rifampicin is used to kill the persisters, we assessed the interaction of rifampicin with MazEF complex. We showed that rifampicin moderately interacts with MazEF complex with 1:1 stoichiometry. Our study suggests that the interaction of rifampicin with MazEF complex might play an important role in inhibition of persisters.
Interaction between NSMCE4A and GPS1 links the SMC5/6 complex to the COP9 signalosomeBMC Molecular and Cell Biology - Tập 21 - Trang 1-10 - 2020
András Horváth, Gergely Rona, Michele Pagano, Philip W. Jordan
The SMC5/6 complex, cohesin and condensin are the three mammalian members of the structural maintenance of chromosomes (SMC) family, large ring-like protein complexes that are essential for genome maintenance. The SMC5/6 complex is the least characterized complex in mammals; however, it is known to be involved in homologous recombination repair (HRR) and chromosome segregation. In this study, a yeast two-hybrid screen was used to help elucidate novel interactions of the kleisin subunit of the SMC5/6 complex, NSMCE4A. This approach discovered an interaction between NSMCE4A and GPS1, a COP9 signalosome (CSN) component, and this interaction was further confirmed by co-immunoprecipitation. Additionally, GPS1 and components of SMC5/6 complex colocalize during interphase and mitosis. CSN is a cullin deNEDDylase and is an important factor for HRR. Depletion of GPS1, which has been shown to negatively impact DNA end resection during HRR, caused an increase in SMC5/6 levels at sites of laser-induced DNA damage. Furthermore, inhibition of the dennedylation function of CSN increased SMC5/6 levels at sites of laser-induced DNA damage. Taken together, these data demonstrate for the first time that the SMC5/6 and CSN complexes interact and provides evidence that the CSN complex influences SMC5/6 functions during cell cycle progression and response to DNA damage.
The Flot2 component of the lipid raft changes localization during neural differentiation of P19C6 cellsBMC Molecular and Cell Biology - Tập 20 - Trang 1-9 - 2019
Kei Hanafusa, Nobuhiro Hayashi
Flotillin-2 (Flot2) is a lipid raft scaffold protein that is thought to be related to neural differentiation. Flot2 is phosphorylated by Fyn, a Src kinase, and causes raft-dependent endocytosis; however, the exact role of Flot2 in neural differentiation remains unclear. To reveal the roles of lipid raft-associated proteins during neural differentiation, we tried to analyze the expression and localization. In this study, we found that the expression levels of the Flot2 and Fyn proteins increased in whole-cell lysates of P19C6 cells after neural differentiation. In addition, sucrose density fractionation and immunofluorescence experiments revealed an increase in the localization of Flot2 and Fyn to lipid rafts after neural differentiation. We also found that Fyn partially colocalized with Flot2 lipid rafts in neural cells. The observed distribution of Fyn and level of inactivated Fyn and/or c-Src in detergent–resistant membrane (DRM) fractions suggests that the amount of activated Fyn might increase in DRM fractions after neural differentiation. Overall these findings suggest that Flot2 lipid rafts are associated with Fyn, and that Fyn phosphorylates Flot2 during neural differentiation of P19C6 cells.
BACE2 degradation is mediated by both the proteasome and lysosome pathwaysBMC Molecular and Cell Biology - Tập 21 - Trang 1-7 - 2020
Kaixin Qiu, Wenping Liang, Shuai Wang, Tingting Kong, Xin Wang, Chunyan Li, Zhe Wang, Yili Wu
Alzheimer’s disease is the most common neurodegenerative disease in the elderly. Amyloid-β protein (Aβ) is the major component of neuritic plaques which are the hallmark of AD pathology. β-site APP cleaving enzyme 1 (BACE1) is the major β-secretase contributing to Aβ generation. β-site APP-cleaving enzyme 2 (BACE2), the homolog of BACE1, might play a complex role in the pathogenesis of Alzheimer’s disease as it is not only a θ-secretase but also a conditional β-secretase. Dysregulation of BACE2 is observed in Alzheimer’s disease. However, the regulation of BACE2 is less studied compared with BACE1, including its degradation pathways. In this study, we investigated the turnover rates and degradation pathways of BACE2 in both neuronal cells and non-neuronal cells. Both lysosomal inhibition and proteasomal inhibition cause a time- and dose-dependent increase of transiently overexpressed BACE2 in HEK293 cells. The half-life of transiently overexpressed BACE2 protein is approximately 6 h. Moreover, the half-life of endogenous BACE2 protein is approximately 4 h in both HEK293 cells and mouse primary cortical neurons. Furthermore, both lysosomal inhibition and proteasomal inhibition markedly increases endogenous BACE2 in HEK293 cells and mouse primary cortical neurons. This study demonstrates that BACE2 is degraded by both the proteasome and lysosome pathways in both neuronal and non-neuronal cells at endogenous level and in transient overexpression system. It indicates that BACE2 dysregulation might be mediated by the proteasomal and lysosomal impairment in Alzheimer’s disease. This study advances our understanding of the regulation of BACE2 and provides a potential mechanism of its dysregulation in Alzheimer’s disease.
Novel leukocyte-depleted platelet-rich plasma-based skin equivalent as an in vitro model of chronic wounds: a preliminary studyBMC Molecular and Cell Biology - - 2021
Elisa Seria, George Galea, Joseph Borg, Kevin Schembri, Godfrey Grech, Sarah Samut Tagliaferro, Alex E. Felice
Abstract
Background
Chronic leg ulcerations are associated with Haemoglobin disorders, Type2 Diabetes Mellitus, and long-term venous insufficiency, where poor perfusion and altered metabolism develop into a chronic inflammation that impairs wound closure. Skin equivalent organotypic cultures can be engineered in vitro to study skin biology and wound closure by modelling the specific cellular components of the skin. This study aimed to develop a novel bioactive platelet-rich plasma (PRP) leukocyte depleted scaffold to facilitate the study of common clinical skin wounds in patients with poor chronic skin perfusion and low leukocyte infiltration. A scratch assay was performed on the skin model to mimic two skin wound conditions, an untreated condition and a condition treated with recombinant tumour necrotic factor (rTNF) to imitate the stimulation of an inflammatory state. Gene expression of IL8 and TGFA was analysed in both conditions. Statistical analysis was done through ANOVA and paired student t-test. P < 0.05 was considered significant.
Results
A skin model that consisted of a leukocyte-depleted, platelet-rich plasma scaffold was setup with embedded fibroblasts as dermal equivalents and seeded keratinocytes as multi-layered epidermis. Gene expression levels of IL8 and TGFA were significantly different between the control and scratched conditions (p < 0.001 and p < 0.01 respectively), as well as between the control and treated conditions (p < 0.01 and p < 0.001 respectively). The scratch assay induced IL8 upregulation after 3 h (p < 0.05) which continued to increase up to day 1 (p < 0.05). On the other hand, the administration of TNF led to the downregulation of IL8 (p < 0.01), followed by an upregulation on day 2. IL8 gene expression decreased in the scratched condition after day 1 as the natural healing process took place and was lower than in the treated condition on day 8 (p < 0.05).
Both untreated and treated conditions showed a downregulation of TGFA 3 h after scratch when compared with the control condition (p < 0.01). Administration of rTNF showed significant downregulation of TGFA after 24 h when compared with the control (p < 0.01) and treated conditions (p < 0.05).
Conclusion
This study suggests that a leukocyte-depleted PRP-based skin equivalent can be a useful model for the in vitro study of chronic skin wounds related to poor skin perfusion.
