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Now showing 1 - 10 of 27
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    DeepsmirUD: Prediction of Regulatory Effects on microRNA Expression Mediated by Small Molecules Using Deep Learning
    (Basel : Molecular Diversity Preservation International, 2023) Sun, Jianfeng; Ru, Jinlong; Ramos-Mucci, Lorenzo; Qi, Fei; Chen, Zihao; Chen, Suyuan; Cribbs, Adam P.; Deng, Li; Wang, Xia
    Aberrant miRNA expression has been associated with a large number of human diseases. Therefore, targeting miRNAs to regulate their expression levels has become an important therapy against diseases that stem from the dysfunction of pathways regulated by miRNAs. In recent years, small molecules have demonstrated enormous potential as drugs to regulate miRNA expression (i.e., SM-miR). A clear understanding of the mechanism of action of small molecules on the upregulation and downregulation of miRNA expression allows precise diagnosis and treatment of oncogenic pathways. However, outside of a slow and costly process of experimental determination, computational strategies to assist this on an ad hoc basis have yet to be formulated. In this work, we developed, to the best of our knowledge, the first cross-platform prediction tool, DeepsmirUD, to infer small-molecule-mediated regulatory effects on miRNA expression (i.e., upregulation or downregulation). This method is powered by 12 cutting-edge deep-learning frameworks and achieved AUC values of 0.843/0.984 and AUCPR values of 0.866/0.992 on two independent test datasets. With a complementarily constructed network inference approach based on similarity, we report a significantly improved accuracy of 0.813 in determining the regulatory effects of nearly 650 associated SM-miR relations, each formed with either novel small molecule or novel miRNA. By further integrating miRNA–cancer relationships, we established a database of potential pharmaceutical drugs from 1343 small molecules for 107 cancer diseases to understand the drug mechanisms of action and offer novel insight into drug repositioning. Furthermore, we have employed DeepsmirUD to predict the regulatory effects of a large number of high-confidence associated SM-miR relations. Taken together, our method shows promise to accelerate the development of potential miRNA targets and small molecule drugs.
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    Recent developments in peptide-based nucleic acid delivery
    (Basel : MDPI, 2008) Veldhoen, Sandra; Laufer, Sandra D.; Restle, Tobias
    Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cellpenetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10-30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisenseoligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls.
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    Identification of cleavage sites and substrate proteins for two mitochondrial intermediate peptidases in Arabidopsis thaliana
    (Oxford : Oxford University Press, 2015) Carrie, Chris; Venne, A. Saskia; Zahedi, René P.; Soll, Jürgen
    Most mitochondrial proteins contain an N-terminal targeting signal that is removed by specific proteases following import. In plant mitochondria, only mitochondrial processing peptidase (MPP) has been characterized to date. Therefore, we sought to determine the substrates and cleavage sites of the Arabidopsis thaliana homologues to the yeast Icp55 and Oct1 proteins, using the newly developed ChaFRADIC method for N-terminal protein sequencing. We identified 88 and seven putative substrates for Arabidopsis ICP55 and OCT1, respectively. It was determined that the Arabidopsis ICP55 contains an almost identical cleavage site to that of Icp55 from yeast. However, it can also remove a far greater range of amino acids. The OCT1 substrates from Arabidopsis displayed no consensus cleavage motif, and do not contain the classical –10R motif identified in other eukaryotes. Arabidopsis OCT1 can also cleave presequences independently, without the prior cleavage of MPP. It was concluded that while both OCT1 and ICP55 were probably acquired early on in the evolution of mitochondria, their substrate profiles and cleavage sites have either remained very similar or diverged completely.
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    The bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles
    (London : Nature Publishing Group, 2013) Matkovic, Tanja; Siebert, Matthias; Knoche, Elena; Depner, Harald; Mertel, Sara; Owald, David; Schmidt, Manuela; Thomas, Ulrich; Sickmann, Albert; Kamin, Dirk; Hell, Stefan W.; Bürger, Jörg; Hollmann, Christina; Mielke, Thorsten; Wichmann, Carolin; Sigrist, Stephan J.
    Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca2+ channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca2+ channel-coupled SV release slots available per AZ and thereby sets the size of the RRP.
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    LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus
    (Hoboken, NJ [u.a.] : Wiley, 2022) Wu, Zhixiao; Berlemann, Lena A.; Bader, Verian; Sehr, Dominik A.; Dawin, Eva; Covallero, Alberto; Meschede, Jens; Angersbach, Lena; Showkat, Cathrin; Michaelis, Jonas B.; Münch, Christian; Rieger, Bettina; Namgaladze, Dmitry; Herrera, Maria Georgina; Fiesel, Fabienne C.; Springer, Wolfdieter; Mendes, Marta; Stepien, Jennifer; Barkovits, Katalin; Marcus, Katrin; Sickmann, Albert; Dittmar, Gunnar; Busch, Karin B.; Riedel, Dietmar; Brini, Marisa; Tatzelt, Jörg; Cali, Tito; Winklhofer, Konstanze F.
    Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.
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    Cardiolipin supports respiratory enzymes in plants in different ways
    (Lausanne : Frontiers Media, 2017) Petereit, Jakob; Katayama, Kenta; Lorenz, Christin; Ewert, Linda; Schertl, Peter; Kitsche, Andreas; Wada, Hajime; Frentzen, Margrit; Braun, Hans-Peter; Eubel, Holger
    In eukaryotes the presence of the dimeric phospholipid cardiolipin (CL) is limited to the mitochondrial membranes. It resides predominantly in the inner membrane where it interacts with components of the mitochondrial electron transfer chain. CL deficiency has previously been shown to affect abundances of the plant NADHdehydrogenase complex and its association with dimeric cyctochrome c reductase. Using an Arabidopsis thaliana knock-out mutant for the final enzyme of CL biosynthesis we here extend current knowledge on the dependence of plant respiration on CL. By correlating respiratory enzyme abundances with enzymatic capacities in mitochondria isolated from wild type, CL deficient and CL complemented heterotrophic cell culture lines a new picture of the participation of CL in plant respiration is emerging. Data indicate a loss of a general reduction of respiratory capacity in CL deficient mitochondria which cannot solely be attributed to decreased abundances or capacities of mitochondrial electron transfer protein complexes and supercomplexes. Instead, it most likely is the result of a loss of the mobile electron carrier cytochrome c. Furthermore, enzymes of the tricarboxylic acid cycle are found to have lower maximum activities in the mutant, including the succinate dehydrogenase complex. Interestingly, abundance of the latter is not altered, indicative of a direct impact of CL deficiency on the enzymatic capacity of this electron transfer chain protein complex.
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    The Caveolin-3 G56S sequence variant of unknown significance: Muscle biopsy findings and functional cell biological analysis
    (Hoboken, NJ : Wiley, 2016) Brauers, Eva; Roos, Andreas; Kollipara, Laxmikanth; Zahedi, René P.; Beckmann, Alf; Mohanadas, Nilane; Bauer, Hartmut; Häusler, Martin; Thoma, Stéphanie; Kress, Wolfram; Senderek, Jan; Weis, Joachim
    Purpose: In the era of next-generation sequencing, we are increasingly confronted with se- quence variants of unknown significance. This phenomenon is also known for variations in Caveolin-3 and can complicate the molecular diagnosis of the disease. Here, we aimed to study the ambiguous character of the G56S Caveolin-3 variant. Experimental design: A comprehensive approach combining genetic and morphological stud- ies of muscle derived from carriers of the G56S Caveolin-3 variant were carried out and linked to biochemical assays (including phosphoblot studies and proteome profiling) and morphological investigations of cultured myoblasts. Results: Muscles showed moderate chronic myopathic changes in all carriers of the variant. Myogenic RCMH cells expressing the G56S Caveolin-3 protein presented irregular Caveolin-3 deposits within the Golgi in addition to a regular localization of the protein to the plasma mem- brane. This result was associated with abnormal findings on the ultra-structural level. Phos- phoblot studies revealed that G56S affects EGFR-signaling. Proteomic profiling demonstrated alterations in levels of physiologically relevant proteins which are indicative for antagonization of G56S Caveolin-3 expression. Remarkably, some proteomic alterations were enhanced by osmotic/mechanical stress. Conclusions and clinical relevance: Our studies suggest that G56S might influence the mani- festation of myopathic changes upon the presence of additional cellular stress burden. Results of our studies moreover improve the current understanding of (genetic) causes of myopathic disorders classified as caveolinopathies.
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    A Homozygous PPP1R21 Splice Variant Associated with Severe Developmental Delay, Absence of Speech, and Muscle Weakness Leads to Activated Proteasome Function
    (Totowa, NJ : Humana Press, 2023) Hentschel, Andreas; Meyer, Nancy; Kohlschmidt, Nicolai; Groß, Claudia; Sickmann, Albert; Schara-Schmidt, Ulrike; Förster, Fabian; Töpf, Ana; Christiansen, Jon; Horvath, Rita; Vorgerd, Matthias; Thompson, Rachel; Polaparapu, Kiran; Lochmüller, Hanns; Preusse, Corinna; Hannappel, Luis; Schänzer, Anne; Grüneboom, Anika; Gangfuß, Andrea; Roos, Andreas
    PPP1R21 acts as a co-factor for protein phosphatase 1 (PP1), an important serine/threonine phosphatase known to be essential for cell division, control of glycogen metabolism, protein synthesis, and muscle contractility. Bi-allelic pathogenic variants in PPP1R21 were linked to a neurodevelopmental disorder with hypotonia, facial dysmorphism, and brain abnormalities (NEDHFBA) with pediatric onset. Functional studies unraveled impaired vesicular transport as being part of PPP1R21-related pathomechanism. To decipher further the pathophysiological processes leading to the clinical manifestation of NEDHFBA, we investigated the proteomic signature of fibroblasts derived from the first NEDHFBA patient harboring a splice-site mutation in PPP1R21 and presenting with a milder phenotype. Proteomic findings and further functional studies demonstrate a profound activation of the ubiquitin–proteasome system with presence of protein aggregates and impact on cellular fitness and moreover suggest a cross-link between activation of the proteolytic system and cytoskeletal architecture (including filopodia) as exemplified on paradigmatic proteins including actin, thus extending the pathophysiological spectrum of the disease. In addition, the proteomic signature of PPP1R21-mutant fibroblasts displayed a dysregulation of a variety of proteins of neurological relevance. This includes increase proteins which might act toward antagonization of cellular stress burden in terms of pro-survival, a molecular finding which might accord with the presentation of a milder phenotype of our NEDHFBA patient.
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    β-adrenoceptor-mediated relaxation of urinary bladder muscle in β2-adrenoceptor knockout mice
    (Lausanne : Frontiers Media, 2016) Propping, Stefan; Lorenz, Kristina; Michel, Martin C.; Wirth, Manfred P.; Ravens, Ursula
    Background and Objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice. Materials and Methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs). Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug’s affinity for β3-AR. L743,337 shifted the CRCs to the right. Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological conditions. Nevertheless, upon removal of this reserve, β3-AR can also mediate murine detrusor relaxation.
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