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Journal articleJalan A, Sammon D, Hartgerink J, et al., 2020,
Chain alignment of collagen I deciphered using computationally designed heterotrimers
, Nature Chemical Biology, Vol: 16, Pages: 423-429, ISSN: 1552-4450The most abundant member of the collagen protein family, collagen I (COL1), is composed of two similar (chain A) and one unique (chain B) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contained a putative Von Willebrand Factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register.
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Journal articleBrezovjakova H, Tomlinson C, Mohd Naim N, et al., 2019,
Junction Mapper is a novel computer vision tool to decipher cell-cell contact phenotypes.
, Elife, Vol: 8Stable cell-cell contacts underpin tissue architecture and organization. Quantification of junctions of mammalian epithelia requires laborious manual measurements that are a major roadblock for mechanistic studies. We designed Junction Mapper as an open access, semi-automated software that defines the status of adhesiveness via the simultaneous measurement of pre-defined parameters at cell-cell contacts. It identifies contacting interfaces and corners with minimal user input and quantifies length, area and intensity of junction markers. Its ability to measure fragmented junctions is unique. Importantly, junctions that considerably deviate from the contiguous staining and straight contact phenotype seen in epithelia are also successfully quantified (i.e. cardiomyocytes or endothelia). Distinct phenotypes of junction disruption can be clearly differentiated among various oncogenes, depletion of actin regulators or stimulation with other agents. Junction Mapper is thus a powerful, unbiased and highly applicable software for profiling cell-cell adhesion phenotypes and facilitate studies on junction dynamics in health and disease.
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Journal articleKemp P, Griffiths M, Polkey M, 2019,
Muscle wasting in the presence of disease, why is it so variable?
, Biological Reviews, Vol: 94, Pages: 1038-105, ISSN: 1464-7931Skeletal muscle wasting is a common clinical feature of many chronic diseases and also occurs in response to single acute events. The accompanying loss of strength can lead to significant disability, increased care needs and have profound negative effects on quality of life. As muscle is the most abundant source of amino acids in the body, it appears to function as a buffer for fuel and substrates that can be used to repair damage elsewhere and to feed the immune system. In essence, the fundamentals of muscle wasting are simple: less muscle is made than is broken down. However, although well‐described mechanisms modulate muscle protein turnover, significant individual differences in the amount of muscle lost in the presence of a given severity of disease complicate the understanding of underlying mechanisms and suggest that individuals have different sensitivities to signals for muscle loss. Furthermore, the rate at which muscle protein is turned over under normal conditions means that clinically significant muscle loss can occur with changes in the rate of protein synthesis and/or breakdown that are too small to be measurable. Consequently, the changes in expression of factors regulating muscle turnover required to cause a decline in muscle mass are small and, except in cases of rapid wasting, there is no consistent pattern of change in the expression of factors that regulate muscle mass. MicroRNAs are fine tuners of cell phenotype and are therefore ideally suited to cause the subtle changes in proteome required to tilt the balance between synthesis and degradation in a way that causes clinically significant wasting. Herein we present a model in which muscle loss as a consequence of disease in non‐muscle tissue is modulated by a set of microRNAs, the muscle expression of which is associated with severity of disease in the non‐muscle tissue. These microRNAs alter fundamental biological processes including the synthesis of ribosomes and mitochondria leading to reduce
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Journal articleAkram K, Yates L, Mongey R, et al., 2019,
Live imaging of alveologenesis in precision-cut lung slices reveals dynamic epithelial cell behaviour
, Nature Communications, Vol: 10, Pages: 1-16, ISSN: 2041-1723Damage to alveoli, the gas-exchanging region of the lungs, is a component of many chronic and acute lung diseases. In addition, insufficient generation of alveoli results in bronchopulmonary dysplasia, a disease of prematurity. Therefore visualising the process of alveolar development (alveologenesis) is critical for our understanding of lung homeostasis and for the development of treatments to repair and regenerate lung tissue. Using long-term, time-lapse imaging of precision-cut lung slices, we show alveologenesis for the first time. We reveal that during this process, epithelial cells are highly mobile and we identify specific cell behaviours that contribute to alveologenesis: cell clustering, hollowing and cell extension. Using the cytoskeleton inhibitors blebbistatin and cytochalasin D, we showed that cell migration is a key driver of alveologenesis. This study reveals important novel information about lung biology and provides a new system in which to manipulate alveologenesis genetically and pharmacologically.
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Journal articleKemp P, Connolly, Paul R, et al., 2017,
miR-424-5p reduces ribosomal RNA and protein synthesis in muscle wasting
, Journal of Cachexia, Sarcopenia and Muscle, Vol: 9, Pages: 400-416, ISSN: 2190-6009Background: A loss of muscle mass occurs as a consequence of a range of chronic and acute diseases as well as in older age. This wasting results from an imbalance of protein synthesis and degradation with a reduction in synthesis and resistance to anabolic stimulation often reported features. Ribosomes are required for protein synthesis so changes in the control of ribosome synthesis is a potential contributor to muscle wasting. MicroRNAs (miRNAs) are known regulators of muscle phenotype and have been shown to modulate components of the protein synthetic pathway. One miRNA that is predicted to target a number of components of protein synthetic pathway is miR-424-5p, which is elevated in the quadriceps of patients with chronic obstructive pulmonary disease (COPD).Methods: Targets of miR-424-5p were identified by Ago2 pull-down and the effects of the miRNA on RNA and protein expression were determined by qPCR and western blotting in muscle cells in vitro. Protein synthesis was determined by puromycin incorporation in vitro. The miRNA was over-expressed in the tibialis anterior muscle of mice by electroporation and the effects quantified. Finally, quadriceps expression of the miRNA was determined by qPCR in patients with COPD, intensive care unit acquired weakness (ICUAW), and in patients undergoing aortic surgery as well as in individuals from the Hertfordshire Sarcopenia Study.Results: Pull-down assays showed that miR-424-5p bound to mRNAs encoding proteins associated with muscle protein synthesis. The most highly enriched mRNAs encoded proteins required for the Pol I RNA pre-initiation complex (PIC) required for rRNA transcription, (PolR1A and Upstream binding transcription factor, UBTF). In vitro, miR-424-5p reduced expression of these RNAs, reduced rRNA levels and inhibited protein synthesis. In mice, over-expression of miR-322 (rodent miR-424 orthologue) caused fibre atrophy and reduced UBTF expression and rRNA levels. In humans elevated miR-424-5p as
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Journal articlefarre garros, paul R, connolly M, et al., 2017,
miR-542 promotes mitochondrial dysfunction and SMAD activity and is raised in ICU Acquired Weakness
, American Journal of Respiratory and Critical Care Medicine, Vol: 196, Pages: 1-12, ISSN: 1073-449XRationale: Loss of skeletal muscle mass and function is a common consequence of critical illness and a range of chronic diseases but the mechanisms by which this occurs are unclear. Objectives: We aimed to identify miRNAs that were increased in the quadriceps of patients with muscle wasting and to determine the molecular pathways by which they contributed to muscle dysfunction. Methods: miR-542-3p/-5p were quantified in the quadriceps of patients with COPD and intensive care unit acquired weakness (ICUAW). The effect of miR-542-3p/5p was determined on mitochondrial function and TGF-β signaling in vitro and in vivo. Measurements and main results: miR-542-3p/5p were elevated in patients with COPD but more markedly in patients with ICUAW. In vitro, miR-542-3p suppressed the expression of the mitochondrial ribosomal protein MRPS10, and reduced 12S rRNA expression suggesting mitochondrial ribosomal stress. miR-542-5p increased nuclear phospho-SMAD2/3 and suppressed expression of SMAD7, SMURF1 and PPP2CA, proteins that inhibit or reduce SMAD2/3 phosphorylation suggesting that miR-542-5p increased TGF-β signaling. In mice, miR-542 over-expression caused muscle wasting, reduced mitochondrial function, 12S rRNA expression and SMAD7 expression, consistent with the effects of the miRNAs in vitro. Similarly, in patients with ICUAW, the expression of 12S rRNA and of the inhibitors of SMAD2/3 phosphorylation were reduced, indicative of mitochondrial ribosomal stress and increased TGF-β signaling. In patients undergoing aortic surgery, pre-operative levels of miR-542-3p/5p were positively correlated with muscle loss following surgery. Conclusion; Elevated miR-542-3p/5p may cause muscle atrophy in ICU patients through the promotion of mitochondrial dysfunction and activation of SMAD2/3 phosphorylation.
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Journal articleJuskaite V, Corcoran DS, Leitinger B, 2017,
Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers
, Elife, Vol: 6, Pages: 1-27, ISSN: 2050-084XThe collagen-binding receptor tyrosine kinase DDR1 (discoidin domain receptor 1) is a drug target for a wide range of human diseases, but the molecular mechanism of DDR1 activation is poorly defined. Here we co-expressed different types of signalling-incompetent DDR1 mutants ('receiver') with functional DDR1 ('donor') and demonstrate phosphorylation of receiver DDR1 by donor DDR1 in response to collagen. Making use of enforced covalent DDR1 dimerisation, which does not affect receptor function, we show that receiver dimers are phosphorylated in trans by the donor; this process requires the kinase activity of the donor but not that of the receiver. The receiver ectodomain is not required, but phosphorylation in trans is abolished by mutation of the transmembrane domain. Finally, we show that mutant DDR1 that cannot bind collagen is recruited into DDR1 signalling clusters. Our results support an activation mechanism whereby collagen induces lateral association of DDR1 dimers and phosphorylation between dimers.
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Journal articleErasmus JC, Bruche S, Pizarro L, et al., 2017,
Corrigendum: Defining functional interactions during biogenesis of epithelial junctions
, Nature Communications, Vol: 8, Pages: 14195-14195, ISSN: 2041-1723The original version of this Article (https://doi.org/10.1038/ncomms13542) contained an error in the spelling of the author Tommaso Poggioli, which was incorrectly given as Tommaso Pogglioli. This has now been corrected in both the PDF and HTML versions of the Article.
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