Biology subjects

Abriata, L. A.

Publications and source records attributed to Abriata, L. A..

21 records · Page 2Linked to original sources

bioRxiv2020

3D architecture and structural flexibility revealed in the subfamily of large glutamate dehydrogenases by a mycobacterial enzyme

Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs180) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH180 (mL-GDH180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs.

microbiology↗

bioRxiv2020

Site-specific phosphorylation of Huntingtin exon 1 recombinant proteins enabled by the discovery of novel kinases

Posttranslational modifications (PTMs) within the first 17 amino acids (Nt17) of exon1 of the Huntingtin protein (Httex1) play important roles in modulating its cellular properties and functions in health and disease. In particular, phosphorylation of threonine and serine residues (T3, S13, and/or S16) has been shown to inhibit Htt aggregation in vitro and inclusion formation in cellular and animal models of Huntingtons disease (HD). In this manuscript, we describe a new and simple methodology for producing milligram quantities of highly pure wild type or mutant Httex1 proteins that are site-specifically phosphorylated at T3 or at both S13 and S16. This advance was enabled by 1) the discovery and validation of novel kinases that efficiently phosphorylate Httex1 at S13 and S16 (TBK1), at T3 (GCK) or T3 and S13 (TNIK and HGK); and, 2) the development of an efficient methodology for producing recombinant native Httex1 proteins using a SUMO-fusion expression and purification strategy. As proof of concept, we demonstrate how this method can be applied to produce Httex1 proteins that are both site- specifically phosphorylated and fluorescently labeled or isotopically labeled. Together, these advances should increase access to these valuable tools and expand the range of methods and experimental approaches that can be used to elucidate the mechanisms by which phosphorylation influences Httex1 structure, aggregation, interactome and function(s) in health and disease.

biochemistry↗

bioRxiv2018

Structural model of Cyc2, the primary electron acceptor of Acidithiobacillus ferrooxidans respiratory chain, as a modular cytochrome - β-barrel fusion protein, and mechanistic proposals based on this model

Acidithiobacillus ferrooxidans oxidizes Fe(II) to Fe(III) to feed electrons into its respiratory chain. The primary electron acceptor of this complex system is Cyc2, an outer membrane protein of unknown structure. This work proposes a feasible model of Cyc2s global structure, based on homology modeling, residue-residue coevolution data, bioinformatics predictions and limited knowledge about Cyc2s function. The proposal is that the sequence segment spanning residues ~30 to ~90 folds as a cytochrome-like domain that contains a heme group which would presumably bind and oxidize external Fe(II), whereas the remaining segment from residue ~90 until the end adopts a {beta}-barrel fold similar to that of most outer membrane proteins. Such model differs strongly from a published model, but is backed up by more data and is more compatible with the known topology of outer membrane proteins and with Cyc2s function of internalizing reducing equivalents. The small size of the cytochrome-like domain would allow it to reside inside, and/or slide through, the {beta}-barrel domain, thus communicating in a controlled fashion the extracellular medium with the periplasm to import electrons through the outer membrane. All the models discussed are provided as PyMOL session files in the Supporting Information and can be visualized online at http://lucianoabriata.altervista.org/modelshome.html

biophysics↗