Biology subjects

Villar-Briones, A.

Publications and source records attributed to Villar-Briones, A..

4 recordsLinked to original sources

bioRxiv2024

A transphyletic study of metazoan β-catenin protein complexes

{beta}-catenin is essential for various biological processes, such as body axis determination and cell differentiation, during embryonic development in metazoans. {beta}-catenin functions are thought to be exerted through complexes formed with various types of proteins. Although {beta}-catenin complex proteins have been identified in several bilaterians, little is known about the structural and functional properties of {beta}-catenin complexes in early metazoan evolution. In this study, we performed a comparative analysis of {beta}-catenin sequences in nonbilaterian lineages that diverged early in metazoan evolution. We also carried out transphyletic function experiments with {beta}-catenin from nonbilaterian metazoans using developing Xenopus embryos, which included secondary axis induction in embryos and proteomic analysis of {beta}-catenin protein complexes. Comparative functional analysis of nonbilaterian {beta}-catenins demonstrated sequence characteristics important for {beta}-catenin functions, and the deep origin and evolutionary conservation of the cadherin-catenin complex. Proteins co-immunoprecipitated with {beta}-catenin included several proteins conserved among metazoans. These data provide new insights into the conserved repertoire of {beta}-catenin complexes.

evolutionary biology↗

bioRxiv2024

Post-transcriptional regulation by the CCR4-NOT deadenylase complex maintains redox homeostasis in insulin biosynthesis in mouse pancreatic β cells

Pancreatic {beta} cells synthesize insulin to maintain glucose homeostasis. In diabetes, elevated blood glucose and insulin resistance compel {beta} cells to produce insulin, and hence {beta} cells are vulnerable to oxidative stress by glucotoxicity. In insulin biosynthesis, the conversion of proinsulin to insulin is initiated by forming disulfide bonds in proinsulin for oxidative protein folding. Insulin content and insulin secretion in {beta} cells are decreased by deletion of CNOT7, a catalytic subunit of the CCR4-NOT complex, and accompanied by increased proinsulin, implying the impaired conversion of proinsulin to insulin. We found that PRDX4 essential for disulfide bond formation in proinsulin, is reduced in Cnot7-KO {beta} cells. Moreover, protein expression of CNOT8, a paralog of CNOT7, is increased in Cnot7-KO {beta} cells and binds to Prdx4 mRNA via MSI2. Here, we demonstrate the post-transcriptional regulation of Prdx4 mRNA by the CCR4-NOT complex to maintain oxidative-reductive homeostasis in insulin biosynthesis. HIGHLIGHTSO_LIInsulin content and secretion are decreased in Cnot7-KO pancreatic {beta} cells. C_LIO_LIPRDX4, needed for proinsulin folding, is decreased in Cnot7-KO pancreatic islets. C_LIO_LIThe conversion of proinsulin to insulin is impaired in Cnot7-KO {beta} cells. C_LIO_LICNOT8, but not CNOT7, interacts with MSI2 that binds to Prdx4 mRNA C_LI

physiology↗

bioRxiv2022

Automated phenol-chloroform extraction of high molecular weight genomic DNA for use in long-read single-molecule sequencing.

In order to automate the genome sequencing pipeline in our laboratory, we programmed a dual-arm anthropomorphic robot, the Robotic Biology Institutes Maholo LabDroid, to perform organic solvent-based genomic DNA extraction from cell lysates. To the best of our knowledge, this is the first time that automation of phenol-chloroform extraction has been reported. We achieved routine extraction of high molecular weight genomic DNA (>100 kb) from diverse biological samples including algae cultured in sea water, bacteria, whole insects, and human cell lines. The results of pulse-field electrophoresis size analysis and the N50 sequencing metrics of reads obtained from Nanopore MinION runs verified the presence of intact DNA suitable for direct sequencing. We present the workflow that can be used to program similar robots and discuss the problems and solutions we encountered in developing the workflow. The protocol can be adapted to analogous methods such as RNA extraction, and there is ongoing work to incorporate further post-extraction steps such as library construction. This work shows the potential for automated robotic workflows to free molecular biological researchers from manual interventions in routine experimental work. A time-lapse movie of the entire automated run can be seen at http://doi.org/10.6084/m9.figshare.17097065

genomics↗

bioRxiv2021

Protein and substrate flexibility contribute to enzymatic specificity in human and bacterial methionine adenosyltransferases

Protein conformational change can facilitate the binding of non-cognate substrates and underlie promiscuous activities. However, the contribution of substrate conformational dynamics to this process is comparatively poorly understood. Here we analyse human (hMAT2A) and Escherichia coli (eMAT) methionine adenosyltransferases that have identical active sites but different substrate specificity. In the promiscuous hMAT2A, non-cognate substrates bind in a stable conformation to allow catalysis. In contrast, non-cognate substrates rarely sample stable productive binding modes in eMAT owing to altered mobility in the enzyme active site. Different cellular concentrations of substrate likely drove the evolutionary divergence of substrate specificity in these orthologs. The observation of catalytic promiscuity in hMAT2A led to the detection of a new human metabolite, methyl thioguanosine, that is produced at elevated level in a cancer cell line. This work establishes that identical active sites can result in different substrate specificity owing to the effects of substrate and enzyme dynamics.

biochemistry↗