Biology subjects

Fallon, L.

Publications and source records attributed to Fallon, L..

5 recordsLinked to original sources

bioRxiv2026

Aggregation of misfolded proteins in the sperm head impairs preimplantation embryo development

Paternal contributions to embryogenesis extend beyond DNA, yet the molecular cargo carried by sperm and its impact on development remain poorly defined. Aggresomes (AGG), cytoplasmic inclusions formed by misfolded proteins, are present in mammalian gametes, but their functional consequences are unclear. Here, we show that excessive AGG content in bovine sperm head compromises preimplantation embryo development. Using image-based flow cytometry, sperm from 32 sires were classified into low-, moderate-, and high-AGG groups. AGG levels were unrelated to sire age and did not affect in vitro capacitation or acrosome remodeling. However, embryos derived from high-AGG sires exhibited reduced blastocyst formation, delayed cleavage timing, and a higher incidence of developmental arrest at the 4-6 cell stage. Embryos from high-AGG sires also accumulated more AGG during development, showed elevated reactive oxygen species (ROS) levels, and displayed altered mitophagy dynamics. Supplementation with an ER stress inhibitor temporarily improved cleavage but did not enhance overall blastocyst formation, indicating a limited and stage-specific effect. In vivo, embryos from high-AGG sires showed lower transferable quality compared with those from low-AGG sires. These findings establish sperm head AGG content as a novel paternal determinant of embryo quality. By linking sperm-borne misfolded protein aggregates to disrupted developmental pathways in the resulting embryo, our study reveals a previously unrecognized mechanism of paternal influence on fertility and suggests new opportunities for molecular screening for male fertility. Significance StatementSperm contribute more to the embryo than DNA alone, yet the consequences of sperm-borne molecular cargo for early development remain largely unknown. We show that aggregates of misfolded proteins in the sperm head, a marker of disrupted protein quality control, impair preimplantation embryo development in cattle. Sires with elevated sperm aggregate content produce embryos that cleave later, arrest more frequently, and reach the blastocyst stage at lower rates, both in vitro and in vivo. These embryos carry greater aggregate loads, show heightened oxidative stress, and display dysregulated mitochondrial clearance. Our findings establish paternal proteostasis as a determinant of embryo quality and identify a class of sperm defects invisible to conventional semen analysis, opening new avenues for molecular fertility screening.

cell biology↗

bioRxiv2024

Alternative Conformations of lncRNAs Identified Through Structural Deconvolution of SHAPE- and DMS-MaP Datasets

The biological function of many classes of RNAs depend on their structures, which can exist as structural ensembles, rather than a single minimum free energy fold. In the past decade, long noncoding RNAs (lncRNAs) have emerged as functional transcripts in gene regulation that behave through their primary sequences and the structures they adopt. Chemical probing experiments, like selective 2-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), and dimethyl sulfate-MaP (DMS-MaP), facilitate the characterization of RNA secondary structure both inside and outside the cell. But chemical probing experiments yield an average reactivity profile, representative of all the structures a particular RNA transcript adopts at the time of chemical probing, weighted by their relative populations. Chemical probing experiments often struggle to identify coexisting conformations a lncRNA might sample. Computational methods (DRACO, DREEM, DANCE-MaP) have been developed to identify alternate conformations of RNAs by deconvoluting chemical probing data. In this work, we investigate the propensity for lncRNAs to sample multiple structured states, and find each of the studied lncRNAs possess coexisting folds. We discuss the implications of lncRNAs harboring multiple structures and how it may contribute to their multifunctionality in regulating biological processes.

bioinformatics↗

bioRxiv2024

RNA Chemical Probing Reagents and Protein Amino Acids: a Double-Edged Sword

RNA chemical probing experiments are a broadly used method for revealing the structure of RNA, as well as for identifying protein binding sites. This is beneficial for expanding our understanding of biological processes governed by protein-RNA complex interactions, as well as facilitating the identification of complex inhibiting molecules. The reagents commonly used in chemical probing experiments are highly reactive, methylating or acylating flexible RNA nucleotides. The highly reactive nature of the chemical probes means that they can also react with nucleophilic amino acid side chains, and subsequently affect protein-RNA binding events. We combine molecular dynamics (MD) simulations, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), and nuclear magnetic resonance (NMR) experiments to show that commonly used RNA chemical probes react with protein amino acids, and demonstrate that this effect alters protein-RNA binding interactions through binding shift assays. We discuss the implications of this phenomenon in elucidating the protein-RNA interaction interface using chemical probing experiments. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/586119v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@13378baorg.highwire.dtl.DTLVardef@3b0114org.highwire.dtl.DTLVardef@1c3f967org.highwire.dtl.DTLVardef@12afaec_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Receptor binding may directly activate the fusion machinery in coronavirus spike glycoproteins

SARS-CoV-2, the causative agent of the COVID-19 pandemic, is an enveloped RNA virus. Trimeric spike glycoproteins extend outward from the virion; these class I viral membrane fusion proteins mediate entry of the virus into a host cell and are the dominant antigen for immune response. Cryo-EM studies have generated a large number of structures for the spike either alone, or bound to the cognate receptor ACE2 or antibodies, with the three receptor binding domains (RBDs) seen closed, open, or in various combinations. Binding to ACE2 requires an open RBD, and is believed to trigger the series of dramatic conformational changes in the spike that lead to the shedding of the S1 subunit and transition of the spring-loaded S2 subunit to the experimentally observed post-fusion structure. The steps following ACE2 binding are poorly understood despite extensive characterization of the spike through X-ray, cryo-EM, and computation. Here, we use all-atom simulations, guided by analysis of 81 existing experimental structures, to develop a model for the structural and energetic coupling that connects receptor binding to activation of the membrane fusion machinery.

molecular biology↗

bioRxiv2020

The flexibility of ACE2 in the context of SARS-CoV-2 infection

The COVID-19 pandemic has swept over the world in the past months, causing significant loss of life and consequences to human health. Although numerous drug and vaccine developments efforts are underway, many questions remain outstanding on the mechanism of SARS-CoV-2 viral association to angiotensin-converting enzyme 2 (ACE2), its main host receptor, and entry in the cell. Structural and biophysical studies indicate some degree of flexibility in the viral extracellular Spike glycoprotein and at the receptor binding domain-receptor interface, suggesting a role in infection. Here, we perform all-atom molecular dynamics simulations of the glycosylated, full-length membrane-bound ACE2 receptor, in both an apo and spike receptor binding domain (RBD) bound state, in order to probe the intrinsic dynamics of the ACE2 receptor in the context of the cell surface. A large degree of fluctuation in the full length structure is observed, indicating hinge bending motions at the linker region connecting the head to the transmembrane helix, while still not disrupting the ACE2 homodimer or ACE2-RBD interfaces. This flexibility translates into an ensemble of ACE2 homodimer conformations that could sterically accommodate binding of the spike trimer to more than one ACE2 homodimer, and suggests a mechanical contribution of the host receptor towards the large spike conformational changes required for cell fusion. This work presents further structural and functional insights into the role of ACE2 in viral infection that can be exploited for the rational design of effective SARS-CoV-2 therapeutics. Statement of SignificanceAs the host receptor of SARS-CoV-2, ACE2 has been the subject of extensive structural and antibody design efforts in aims to curtail COVID-19 spread. Here, we perform molecular dynamics simulations of the homodimer ACE2 full-length structure to study the dynamics of this protein in the context of the cellular membrane. The simulations evidence exceptional plasticity in the protein structure due to flexible hinge motions in the head-transmembrane domain linker region and helix mobility in the membrane, resulting in a varied ensemble of conformations distinct from the experimental structures. Our findings suggest a dynamical contribution of ACE2 to the spike glycoprotein shedding required for infection, and contribute to the question of stoichiometry of the Spike-ACE2 complex.

biophysics↗