Biology subjects

Ulloa-Aguirre, A.

Publications and source records attributed to Ulloa-Aguirre, A..

3 recordsLinked to original sources

bioRxiv2023

Differential effects of follicle-stimulating hormone glycoforms on the transcriptome profile of cultured rat granulosa cells as disclosed by RNA-seq

It has been documented that variations in glycosylation on glycoprotein hormones, confer distinctly different biological features to the corresponding glycoforms when multiple in vitro biochemical readings are analyzed. We here applied next generation RNA sequencing to explore changes in the transcriptome of rat granulosa cells exposed for 0, 6, and 12 h to 100 ng/ml of four highly purified follicle-stimulating hormone (FSH) glycoforms, each exhibiting different glycosylation patterns: human pituitary FSH18/21 and equine FSH (eqFSH) (hypo-glycosylated), and human FSH24 and chinese-hamster ovary cell-derived human recombinant FSH (recFSH) (fully-glycosylated). Total RNA from triplicate incubations was prepared from FSH glycoform-exposed cultured granulosa cells obtained from DES-pretreated immature female rats, and RNA libraries were sequenced in a HighSeq 2500 sequencer (2 x 125 bp paired-end format, 10-15 x 106 reads/sample). The computational workflow focused on investigating differences among the four FSH glycoforms at three levels: gene expression, enriched biological processes, and perturbed pathways. Among the top 200 differentially expressed genes, only 4 (0.6%) were shared by all 4 glycoforms at 6 h, whereas 118 genes (40%) were shared at 12 h. Follicle-stimulating hormone glycocoforms stimulated different patterns of exclusive and associated up regulated biological processes in a glycoform and time-dependent fashion with more shared biological processes after 12 h of exposure and fewer treatment-specific ones, except for recFSH, which exhibited stronger responses with more specifically associated processes at this time. Similar results were found for down-regulated processes, with a greater number of processes at 6 h or 12 h, depending on the particular glycoform. In general, there were fewer downregulated than upregulated processes at both 6 h and 12 h, with FSH18/21 exhibiting the largest number of down-regulated associated processes at 6 h while eqFSH exhibited the greatest number at 12 h. Signaling cascades, largely linked to cAMP-PKA, MAPK, and PI3/AKT pathways were detected as differentially activated by the glycoforms, with each glycoform exhibiting its own molecular signature. These data extend previous observations demonstrating glycosylation-dependent differential regulation of gene expression and intracellular signaling pathways triggered by FSH in granulosa cells. The results also suggest the importance of individual FSH glycoform glycosylation for the conformation of the ligand-receptor complex and induced signalling pathways.

genomics↗

bioRxiv2023

Searching deeply into the conformational space of glycoprotein hormone receptors. Molecular dynamics of the human follitropin and lutropin receptors within a bilayer of (SDPC) poly-unsaturated lipids

AbstractGlycoprotein receptors are a subfamily of G-protein coupled receptors, including the follicle hormone (FSH) receptor (FSHR), thyroid-stimulating hormone receptor (TSH), and luteinizing/chorionic gonadotrophin hormone receptor (LHCGR). These receptors display common structural features such as a prominent extracellular domain, with a leucine-rich repeats (LRR) stabilized by {beta}-sheets, a long and flexible loop known as the hinge region (HR), and the transmembrane (TM) domain with seven -helices interconnected by intra- and extracellular loops. Binding of the ligand to the LRR resembles a hand coupling transversally to the - and {beta}-subunits of the hormone, with the thumb being the HR. The structure of the complex of FSHR-FSH suggests an activation mechanism in which Y335 at the HR binds into a pocket between the - and {beta}-chains of the hormone, leading to an adjustment of the extracellular loops. In this study, we performed molecular dynamics (MD) simulations to identify the conformational changes for the FSHR and LHCGR. We set up an FSHR structure as predicted by AlphaFold (AF-P23945); for the LHCGR structure we took the cryo-electron microscopy structure for the active state (PDB:7FII) as initial coordinates. Specifically, the flexibility of the HR domain and the correlated motions of the RLL and TMD were analyzed. From the conformational changes of the LRR, TMD, and HR we explored the conformational landscape by means of MD trajectories in all-atom approximation, including a membrane of polyunsaturated phospholipids. The distances and procedures here defined may be useful to propose reaction coordinates to describe diverse processes such as the active-to-inactive transition, to identify intermediaries suited for allosteric regulation, and biased binding to cellular transducers in a selective activation strategy. Author summaryIn the present study, we describe the results from a computational microscopy perspective (also known as molecular dynamics simulation) at the atomistic resolution for the two gonadotropin hormone receptors, the follicle-stimulant hormone receptor and the luteinizing/chorionic gonadotropin hormone receptor, which are essential for reproduction in humans. Several dysfunctional mutations in these receptors, leading to reproductive failure, have been detected in the clinical arena. To better understand the process whereby these two receptors perform their signaling tasks, triggering an intracellular response upon binding of their cognate agonist at the extracellular side, we assembled the receptor structures in a membrane bilayer of phospholipids with water molecules as solvent at both sides of the membrane. The systems included nearly 200 thousand atoms, each moving around at 300 kelvin and 1 bar given the interactions (attractive or repulsive forces) from each other. As the motion equations are solved in each time step (at femtoseconds time scale), the system evolves over time during hundreds of nanoseconds (millions of time steps) for three independent replicates. The receptor conformation, therefore, may display non-random motions due to the stability of specific structures in the complex molecular environment, including the hydrophobic membrane core, the bilayer interfaces, and the aqueous medium. From analysis of simulation trajectories and structural changes of the receptors, we could identify the main conformational changes exhibited by each receptor explored in a model cellular environment. We discussed the roll of the hinge domain at the extracellular domain in triggering the receptor conformational changes, as well as differences in the dynamics between these receptors in terms of the flexibility of the structures. Importantly, we proposed relative distances among the different receptor domains as parameters to characterize conformational intermediaries along a transition of states. Understanding of the signaling process in gonadotropin hormone receptors could be useful to explore new strategies for the modulation of the receptor functions, the bias of signaling pathways, or the selective binding of agonists.

biophysics↗

bioRxiv2022

Reshaping the dynamics of follicle-stimulant hormone receptor models in polyunsaturated lipid bilayers. Calculation of conformational free energy landscapes of α-helical domains from all-atom MD simulations.

G-coupled-protein receptors (GPCR) are conspicuous target molecules for novel therapeutic drugs due to their role as mediators of cellular responses. Structural biology of GPCR revealed that intracellular signaling stimulated by extracellular ligands involves subtle conformational changes of the receptor during activation. Nonetheless, transitions among intermediates evolve in an intricate and rough free energy landscape of the conformational space. Experimental evidence suggests that the membrane environment is an active modulator of the receptor dynamics; therefore, the lipid composition may facilitate conformational transitions towards productive signaling states. In this study, we setup molecular dynamics simulations to examine the conformational dynamics of the transmembrane domains, in the context of a membrane of polyunsaturated phospholipid molecules, for a homology model of the human follicle-stimulating-hormone receptor (FSHR) and the crystal structure of the Lumi intermediate of the squid rhodopsin (LSRh). The conformational dynamics of the -helical domains of LSRh was consistent with interactions stabilizing the crystal structure, which remained well preserved in the membrane environment. In contrast, conformations in the FSHR model evolved towards stable states in the membrane environment. To assess the relevance of the conformational dynamics in the FSHR model, dihedral restraints were imposed for the helical domains on top of the force field. This strategy was implemented to reoptimize the interhelical interactions probably overlooked in the modeling process. The conformational dynamics in the helical domains was evaluated by the TM-score, contact maps, principal components analysis of C atoms at the helical domains, and projections of the conformational free energy on principal components. The roughness of the conformational landscape in the FSHR model without dihedral restraints, suggested that alternative interhelical conformational states were populated, whereas imposing restraints led to a dominant conformational state. Template-based models of GPCR, with reoptimized interhelical interactions using dihedral restraints, may enhance the identification of binding sites for potential therapeutic drugs.

biophysics↗