共查询到20条相似文献,搜索用时 31 毫秒
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PIWI‐interacting RNAs (piRNAs) guide PIWI proteins to silence transposable elements and safeguard fertility in germ cells. Many protein factors required for piRNA biogenesis localize to perinuclear ribonucleoprotein (RNP) condensates named nuage, where target silencing and piRNA amplification are thought to occur. In mice, some of the piRNA factors are found in discrete cytoplasmic foci called processing bodies (P‐bodies). However, the dynamics and biological significance of such compartmentalization of the piRNA pathway remain unclear. Here, by analyzing the subcellular localization of functional mutants of piRNA factors, we show that piRNA factors are actively compartmentalized into nuage and P‐bodies in silkworm cells. Proper demixing of nuage and P‐bodies requires target cleavage by the PIWI protein Siwi and ATP hydrolysis by the DEAD‐box helicase BmVasa, disruption of which leads to promiscuous overproduction of piRNAs deriving from non‐transposable elements. Our study highlights a role of dynamic subcellular compartmentalization in ensuring the fidelity of piRNA biogenesis. 相似文献
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Marie Sofie Mller Sita Vaag Olesen Ingemar Andr 《Protein science : a publication of the Protein Society》2021,30(6):1144
Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra‐high affinity interactions (K d <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra‐high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra‐high affinity complex (K d of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra‐high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra‐high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra‐high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild‐type, could occur without loss of affinity. This demonstrates that ultra‐high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface. 相似文献
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Sarah L Caddy Marina Vaysburd Guido Papa Mark Wing Kevin OConnell Diana Stoycheva Stian Foss Jan Terje Andersen Annette Oxenius Leo C James 《The EMBO journal》2021,40(5)
Nucleoprotein (N) is an immunodominant antigen in many enveloped virus infections. While the diagnostic value of anti‐N antibodies is clear, their role in immunity is not. This is because while they are non‐neutralising, they somehow clear infection by coronavirus, influenza and LCMV in vivo. Here, we show that anti‐N immune protection is mediated by the cytosolic Fc receptor and E3 ubiquitin ligase TRIM21. Exploiting LCMV as a model system, we demonstrate that TRIM21 uses anti‐N antibodies to target N for cytosolic degradation and generate cytotoxic T cells (CTLs) against N peptide. These CTLs rapidly eliminate N‐peptide‐displaying cells and drive efficient viral clearance. These results reveal a new mechanism of immune synergy between antibodies and T cells and highlights N as an important vaccine target. 相似文献
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Vu Thuy Khanh LeTrilling Sofia Banchenko Darius Paydar Pia Madeleine Leipe Lukas Binting Simon Lauer Andrea Graziadei Robin Klingen Christine Gotthold Jrg Bürger Thilo Bracht Barbara Sitek Robert Jan Lebbink Anna Malyshkina Thorsten Mielke Juri Rappsilber Christian MT Spahn Sebastian Voigt Mirko Trilling David Schwefel 《The EMBO journal》2023,42(5)
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Integrated requirement of non‐specific and sequence‐specific DNA binding in Myc‐driven transcription
Paola Pellanda Mattia Dalsass Marco Filipuzzi Alessia Loffreda Alessandro Verrecchia Virginia Castillo Cano Hugo Thabussot Mirko Doni Marco J Morelli Laura Soucek Theresia Kress Davide Mazza Marina Mapelli MarieEve Beaulieu Bruno Amati Arianna Sab 《The EMBO journal》2021,40(10)
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Bo Zhang Youyou Yan Yangqin Gu Fei Teng Xu Lin Xinglu Zhou Jinxin Che Xiaowu Dong Lixin Zhou Nengming Lin 《Journal of cellular and molecular medicine》2022,26(2):515
Pancreatic cancer is one of the most notorious diseases for being asymptomatic at early stage and high mortality rate thereafter. However, either chemotherapy or targeted therapy has rarely achieved success in recent clinical trials for pancreatic cancer. Novel therapeutic regimens or agents are urgently in need. Ibr‐7 is a novel derivative of ibrutinib, displaying superior antitumour activity in pancreatic cancer cells than ibrutinib. In vitro studies showed that ibr‐7 greatly inhibited the proliferation of BxPC‐3, SW1990, CFPAC‐1 and AsPC‐1 cells via the induction of mitochondrial‐mediated apoptosis and substantial suppression of mTOR/p70S6K pathway. Moreover, ibr‐7 was able to sensitize pancreatic cancer cells to gemcitabine through the efficient repression of TRIM32, which was positively correlated with the proliferation and invasiveness of pancreatic cancer cells. Additionally, knockdown of TRIM32 diminished mTOR/p70S6K activity in pancreatic cancer cells, indicating a positive feedback loop between TRIM32 and mTOR/p70S6K pathway. To conclude, this work preliminarily explored the role of TRIM32 in the malignant properties of pancreatic cancer cells and evaluated the possibility of targeting TRIM32 to enhance effectiveness of gemcitabine, thereby providing a novel therapeutic target for pancreatic cancer. 相似文献
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Lucas R. Struble Audrey L. Smith William E. Lutz Gabrielle Grubbs Satish Sagar Kenneth W. Bayles Prakash Radhakrishnan Surender Khurana Dalia ElGamal Gloria E. O. Borgstahl 《Protein science : a publication of the Protein Society》2022,31(5)
The COVID‐19 pandemic caused by SARS‐CoV‐2 infection has led to socio‐economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID‐19. SARS‐CoV‐2 spike is a target of interest for the development of therapeutic targets. We developed a robust SARS‐CoV‐2 S spike expression and purification protocol from insect cells and studied four recombinant SARS‐CoV‐2 spike protein constructs based on the original SARS‐CoV‐2 sequence using a baculovirus expression system: a spike protein receptor‐binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S‐RBD‐eGFP), spike ectodomain coupled to a fluorescent tag (S‐Ecto‐eGFP), spike ectodomain with six proline mutations and a foldon domain (S‐Ecto‐HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S‐Ecto‐HexaPro(‐F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S‐Ecto‐eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation). 相似文献
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David Hoffmann Stefan Mereiter Yoo Jin Oh Vanessa Monteil Elizabeth Elder Rong Zhu Daniel Canena Lisa Hain Elisabeth Laurent Clemens GrünwaldGruber Miriam Klausberger Gustav Jonsson Max J Kellner Maria Novatchkova Melita Ticevic Antoine Chabloz Gerald Wirnsberger Astrid Hagelkruys Friedrich Altmann Lukas Mach Johannes Stadlmann Chris Oostenbrink Ali Mirazimi Peter Hinterdorfer Josef M Penninger 《The EMBO journal》2021,40(19)
New SARS‐CoV‐2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N‐glycan sites of Spike remain highly conserved among SARS‐CoV‐2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate‐binding proteins (lectins) to probe critical sugar residues on the full‐length trimeric Spike and the receptor binding domain (RBD) of SARS‐CoV‐2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single‐molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD‐ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS‐CoV‐2 infections. These data report the first extensive map and 3D structural modelling of lectin‐Spike interactions and uncovers candidate receptors involved in Spike binding and SARS‐CoV‐2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS‐CoV‐2 viral entry holds promise for pan‐variant therapeutic interventions. 相似文献
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