PaperPlayer biorxiv molecular biology

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.223594v1?rss=1

Authors: Lorenzo, J. P., Molla, L., Ibarra, I. L., Ruf, S., Subramani, P. G., Boulais, J., Harjanto, D., Vonica, A., Di Noia, J. M., Dieterich, C., Zaugg, J. B., Papavasiliou, F. N.

Abstract:

APOBEC2 is a member of the prolific activation induced cytidine deaminase/ apolipoprotein B editing complex (AID/APOBEC) family of DNA or RNA editors. This family of nucleic acid editors has diverse molecular roles ranging from antibody diversification to RNA transcript editing. However, even though APOBEC2 is an evolutionarily conserved zinc-dependent cytidine deaminase, it neither has an established molecular substrate nor function. In this work, we use the C2C12 skeletal myoblast differentiation model to confirm that APOBEC2 is upregulated during differentiation and is critical to proper differentiation. Furthermore, we show that APOBEC2 has none of the attributed molecular functions of the AID/APOBEC family, such as mRNA editing, DNA demethylation, and DNA mutation. Unexpectedly, we reveal that APOBEC2 binds chromatin at promoter regions of actively transcribed genes, and binding correlates with transcriptional repression of non-myogenesis related gene pathways. APOBEC2 occupied regions co-occur with sequence motifs for several transcription factors such as Specificity Protein/Kruppel-like Factor (SP/KLF), and we demonstrate in vitro that APOBEC2 binds directly and co-operatively to double stranded DNA containing a SP1 binding site. Finally, protein-proximity data show that APOBEC2 directly interacts with histone deacetylase (HDAC) transcriptional co-repressor complexes. Taken together, these data suggest a role for APOBEC2 as a transcriptional repressor for muscle differentiation, a novel role that is unique among AID/APOBEC family members.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.226803v1?rss=1

Authors: Esposito, C. L., Autiero, I., Basal, M. A., Sandomenico, A., Ummarino, S., Borchiellini, M., Ruvo, M., Catuogno, S., Ebralidze, A. K., de Franciscis, V., Di Ruscio, A.

Abstract:

DNA methylation is a fundamental epigenetic modification regulating gene expression. Aberrant DNA methylation is the most common molecular lesion in cancer cells. However, medical intervention has been limited to the use of toxic, unspecific demethylating drugs. Aptamers are novel high affinity targeting ligand molecules. By conjugating the inherent DNMT1 inhibiting capabilities of RNA to an aptamer platform, we generated a first-of-its kind aptamer approach that can target and neutralize DNMT1 function-the aptaDiR. Molecular modelling of RNA-DNMT1 complexes coupled with biochemical and cellular assays enabled the identification and characterization of aptaDiR. This novel RNA bio-drug blocks DNA methylation and impairs cancer cell viability. Collectively, we present an innovative RNA-based approach to modulate DNMT1 activity in cancer or diseases characterized by aberrant DNA methylation and suggest the first alternative strategy to overcome the limitations of currently approved hypomethylating protocols, which will greatly improve clinical intervention on DNA methylation.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.226944v1?rss=1

Authors: Freier, R., Aragon, E., Baginski, B., Pluta, R., Martin-Malpartida, P., Ruiz, L., Condeminas, M., Gonzalez, C., Macias, M. J.

Abstract:

Thioredoxins (Trxs) are ubiquitous enzymes that regulate the redox state in cells. In Drosophila, there are two germline-specific Trxs, Deadhead (Dhd) and TrxT. Both proteins belong to the L(3)mbt malignant brain tumor signature and to the MMS survival network of genes that mediate the cellular response to DNA damage. Dhd is a maternal protein required for early embryogenesis that promotes protamine-histone exchange in fertilized eggs and midblastula transition. TrxT is testis-specific and associates with the lampbrush loops of the Y chromosome. Here we present the first structures of Dhd and TrxT that unveil new features of these Thioredoxins. Dhd is highly positively charged, unusual in canonical Trxs. This positively charged surface can facilitate its approximation to DNA and to protamine oligomers, to promote chromatin remodeling. On the other hand, TrxT contains a C-terminal extension, mostly unstructured and highly flexible, which wraps the conserved core through a closed conformation. This extension partially covers the catalytic site and modulates the redox activity of the protein. The information provided by these structures can guide future work aimed at understanding how redox inputs modulate the initial steps of embryo development in Drosophila and may help in the design of molecular inhibitors through a structure-based approach.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.227223v1?rss=1

Authors: Haque, S., Vaiselbuh, S.

Abstract:

Exosomes are nanovesicles (~30-150 nm diameters) released via an endocytic pathway in almost all mammalian cell types. Exosomes are composed of a lipid bilayer membrane that encloses RNA, miRNA, proteins and DNA. This manuscript unravels how exosome cargo is collected by a highly precise process delineating two separate mRNA transcript entities encoding cytoplasmic and nuclear proteins separately. Ultracentrifuge isolated exosomes were directly converted into cDNA (Exo-cDNA), by a method developed in our laboratory. Cellular RNA was extracted from each cell line and cDNA was prepared (Cell-cDNA). We amplified mRNA transcripts translating cytoplasmic proteins CD10 and CXCR4 and mRNA transcripts translating nuclear proteins such as proliferating cell nuclear antigen (PCNA), CREB-BP, activation induced cytidine deaminase (AID), and terminal deoxynucleotidyl transferase (TdT). We amplified all four different mRNA transcripts (PCNA, CREB-BP, AID, and TdT) from cellular cDNA but none from exosomal cDNA (Exo-cDNA). These findings suggest that exosomes carry mRNA transcripts encoding cytoplasmic proteins only but mRNA transcripts encoding nuclear proteins could not be detected. This important observation could prove to be crucial for the exosome research community since it sheds light on one of the limitations relating to the use of exosomes as biomarkers in cancer biology and other diseases.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.27.221622v1?rss=1

Authors: Mukhopadhya, A., Santoro, J., Moran, B., Useckaite, Z., O'Driscoll, L.

Abstract:

This study aimed to separate and characterise extracellular vesicles (EV) from infant milk formula (IMF) and skim milk (SM), to determine how representative the EV content of IMF is to SM. Contaminant casein micelles, due to their abundance and overlapping size, were removed followed by either differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). Characterisation included BCA, SDS-PAGE, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). NTA reported significantly reduced concentrations of EVs/particles in IMF versus SM; TEM showed intact SM-derived EVs to sparse and disrupted EV-like structures in IMF. Compared to IMF, noticeably stronger bands for EV biomarkers were observed by immunoblotting in SM, indicating compromised EV proteins in IMF; also supported by IFCM. Altogether, we established that EVs are substantially compromised during IMF processing. Furthermore, an optimised method combining acid pre-treatment and GUC for EV separation from milk products has been established.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.225201v1?rss=1

Authors: Probst, V., Pacheco, F., Nielsen, F. C., Bagger, F. O.

Abstract:

Single cell mRNA sequencing technologies have transformed our understanding of cellular heterogeneity and identity. For sensitive discovery or clinical marker estimation where high transcript capture per cell is needed only plate-based techniques currently offer sufficient resolution. Here, we present a performance evaluation of three different plate-based scRNA-seq protocols. Our evaluation is aimed towards applications requiring high gene detection sensitivity, reproducibility between samples, and minimum hands-on time, as is required, for example, in clinical use. We included two commercial kits, NEBNext(R) Single Cell/ Low Input RNA Library Prep Kit (NEB(R)), SMART-seq(R) HT kit (Takara(R)), and the non-commercial protocol Genome & Transcriptome sequencing (G&T). G&T delivered the highest detection of genes per single cell, at the absolute lowest price. Takara(R) kit presented similar high gene detection per single cell, and high reproducibility between sample, but at the absolute highest price. NEBNext(R) delivered a lower detection of genes but remain an alternative to more expensive commercial kits.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.28.224832v1?rss=1

Authors: Robinson, E. L., Drawnel, F. M., Mehdi, S., Archer, C. R., Liu, W., Okkenhaug, H., Alkass, K., Aronsen, J. M., Nagaraju, C. K., Sjaastad, I., Sipido, K. R., Bergmann, O., Arthur, J. S. C., Wang, X., Roderick, H. L.

Abstract:

Heart failure is a leading cause of death, developing subsequent to deleterious hypertrophic cardiac remodelling associated with pathologies including hypertension and myocardial infarction. MAPK signalling pathways, acting via transcription factors, play a key role in coordinating the induction of gene expression during hypertrophy. Induction of the immediate early gene (IEG) response, characterised by increased expression of AP-1 factors, is a necessary and early event in this process. How MAPK signalling and IEG expression are coupled during cardiac hypertrophy is not yet resolved. Here, in vitro, in rodent models and in human samples, we demonstrate that the MAPK-stimulated IEG response depends on the mitogen and stress activated protein kinase (MSK) and its phosphorylation of histone H3 at serine 28 (H3S28). Brg1, a BAF60 ATP-dependent chromatin remodelling complex component, is recruited to IEG promoters harbouring phosphorylated H3S28, initiating their expression. In the absence of MSK activity and IEG induction, the hypertrophic response is suppressed. Together, these studies provide new mechanistic insights into hypertrophic gene regulation and highlight the role of signalling to the epigenome in cardiac hypertrophy.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.223859v1?rss=1

Authors: Litov, L., Petkov, P., Rangelov, M., Ilieva, N., Lilkova, E., Todorova, N., Krachmarova, E., Malinova, K., Gospodinov, A., Hristova, R., Ivanov, I., Nacheva, G.

Abstract:

Timely control of the cytokine release syndrome (CRS) at the severe stage of COVID-19 is key to improving the treatment success rate and reducing the mortality rate. The inhibition of the activity of the two key cytokines, IFN{gamma} and IL-6, can significantly reduce or even reverse the development of the cytokine storm. The objective of our investigations is to reveal the anti-inflammatory potential of heparin for prevention and suppression of the development of CRS in acute COVID-19 patients. The effect of low-molecular-weight heparin (LMWH) on IFN{gamma} signalling inside the stimulated WISH cells was investigated by measuring its antiproliferative activity and the translocation of phosphorylated STAT1 in the nucleus. The mechanism of heparin binding to IFN{gamma} and IL-6 and therefore inhibition of their activity was studied by means of extensive molecular-dynamics simulations. We find that LMWH binds with high affinity to IFN{gamma} and is able to inhibit fully the interaction with its cellular receptor. It also influences the biological activity of IL-6 by binding to either IL-6 or IL-6/IL-6Ralpha thus preventing the formation of the IL-6/IL-6R/gp130 signalling complex. Our conclusion is that heparin is a potent anti-inflammatory agent that can be used in acute inflammatory conditions, due to its potential to inhibit both IFN{gamma} and IL-6 signalling pathways. Based on our results and available clinical observations, we suggest the administration of LMWH to COVID-19 patients in the initial stages of the acute phase. The beginning of the treatment and the dosage should be based on a careful follow-up of the platelet count and the D-dimer, IL-6, IFN, T-cells, and B-cells levels.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.28.225359v1?rss=1

Authors: Zeng, J., Santos, A. F., Mukadam, A., Osswald, M., Luptak, J., Jacques, D., Dickson, C., Renner, N., Johnson, C., Vaysburd, M., McEwan, W. A., Morais-de-Sa, E., Clift, D., James, L. C.

Abstract:

Trim-Away is a powerful new technology that exploits off-the-shelf antibodies and the E3 RING ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically-activated upon substrate engagement during either its normal immune function or when re-purposed for targeted protein degradation is unknown. Here we show that a mechanism of substrate-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce an antiviral response or drive Trim-Away. We harness this mechanism to expand the Trim-Away toolbox with highly-active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has important implications for targeted protein degradation technologies.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.28.222919v1?rss=1

Authors: Ruiz-Lopez, N., Perez-Sancho, J., Esteban del Valle, A., Haslam, R. P., Vanneste, S., Catala, R., Perea-Resa, C., Van Damme, D., Garcia-Hernandez, S., Albert, A., Vallarino, J., Lin, J., Friml, J., Macho, A., Salinas, J., Rosado, A., Napier, J. A., Amorim-Silva, V., Botella, M. A.

Abstract:

Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to WT while the levels of most glycerolipid species remain unchanged. Additionally, SYT1-GFP preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a crucial SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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