Biology subjects

Sendino, M.

Publications and source records attributed to Sendino, M..

3 recordsLinked to original sources

bioRxiv2024

Collablots: Quantification of collagen VI levels and its structural disorganisation in cell cultures from patients with collagen VI-related dystrophies

AimsThis study aims to develop a quantitative method for assessing collagen VI expression in cell cultures, which is crucial for the diagnosis and treatment of collagen VI-related dystrophies. MethodsWe developed a combined in-cell western (ICW) and on-cell western (OCW) assay, that we have called collablot to quantify collagen VI and its organisation in the extracellular matrix of cell cultures from patients and healthy controls. To optimise it, we optimised cell density and the protocols to induce collagen expression in cultures, as well as the cell fixation and permeabilisation methods. This was completed with a thorough selection of collagen antibodies and a collagen hybridising peptide (CHP). We then used collablots to compare cultures from patients and controls and evaluate therapeutic interventions in the cultures. ResultsCollablots enabled the quantification of collagen VI expression in both control and patient cells, aligning with immunocytochemistry findings and detecting variations in collagen VI expression following treatment of the cultures. Additionally, CHP analysis revealed a marked increase in collagen network disruption in patients compared to the controls. ConclusionsThe collablot assay represents an optimal method for quantifying collagen VI expression and its organisation in culture and assessing the effect of therapies. Key Points- Evaluating therapies for collagen VI-related dystrophies (COL6-RD) requires the quantification of collagen VI levels. - Collablot assays are a novel method for quantifying collagen VI expression and its structural organisation in cell culture. - Due to the significant role of phenotype heterogeneity in this complex disease, quantifying collagen alone might not be adequate for diagnosing COL6-RD, but the addition of a peptide to quantify collagen disorganisation could help in the characterisation of patient cultures.

neuroscience↗

bioRxiv2020

The strength of a NES motif in the nucleocapsid protein of human coronaviruses is related to genus, but not to pathogenic capacity

Seven members of the Coronaviridae family infect humans, but only three (SARS-CoV, SARS-CoV-2 and MERS-CoV) cause severe disease with a high case fatality rate. Using in silico analyses (machine learning techniques and comparative genomics), several features associated to coronavirus pathogenicity have been recently proposed, including a potential increase in the strength of a predicted novel nuclear export signal (NES) motif in the nucleocapsid protein. Here, we have used a well-established nuclear export assay to experimentally establish whether the recently proposed nucleocapsid NESs are capable of mediating nuclear export, and to evaluate if their activity correlates with coronavirus pathogenicity. The six NES motifs tested were functional in our assay, but displayed wide differences in export activity. Importantly, these differences in NES strength were not related to strain pathogenicity. Rather, we found that the NESs of the strains belonging to the genus Alphacoronavirus were markedly stronger than the NESs of the strains belonging to the genus Betacoronavirus. We conclude that, while some of the genomic features recently identified in silico could be crucial contributors to coronavirus pathogenicity, this does not appear to be the case of nucleocapsid NES activity, as it is more closely related to coronavirus genus than to pathogenic capacity.

molecular biology↗

bioRxiv2020

Using a simple cellular assay to map NES motifs in cancer-related proteins, gain insight into CRM1-mediated NES export, and search for NES-harbouring micropeptides

The nuclear export receptor CRM1 (XPO1) recognizes and binds specific sequence motifs termed nuclear export signals (NESs) in cargo proteins. About 200 NES motifs have been identified, but over a thousand human proteins are potential CRM1 cargos, and most of their NESs remain to be identified. On the other hand, the interaction of NES peptides with the "NES-binding groove" of CRM1 has been studied in detail using structural and biochemical analyses, but a better understanding of CRM1 function requires further investigation of how the results from these in vitro studies translate into actual NES export in a cellular context. Here we show that a simple cellular assay, based on a recently described reporter (SRVB/A), can be applied to identify novel potential NESs motifs, and to obtain relevant information on different aspects of CRM1-mediated NES export. Using cellular assays, we first map 19 new sequence motifs with nuclear export activity in 14 cancer-related proteins that are potential CRM1 cargos. Next, we investigate the effect of mutations in individual NES-binding groove residues, providing further insight into CRM1-mediated NES export. Finally, we extend the search for CRM1-dependent NESs to a recently uncovered, but potentially vast, set of small proteins called micropeptides. By doing so, we report the first NES-harbouring human micropeptides.

molecular biology↗