Biology subjects

Gomes, G.-N. W.

Publications and source records attributed to Gomes, G.-N. W..

5 recordsLinked to original sources

bioRxiv2025

ApoE lipidation, not isoform, is the key modulator of Aβ interaction, uptake, and cytotoxicity

BackgroundInherited variations in the Apolipoprotein E (APOE) gene are the largest genetic determinant for late-onset Alzheimers Disease, with the APOE{varepsilon}4 allele conferring the highest risk. While APOE was shown to modulate amyloid beta (A{beta}) pathology in a genotype-specific manner (APOE{varepsilon}4>APOE{varepsilon}3>APOE{varepsilon}2), it remains an important open question whether these differences are due directly to isoform-specific interactions between ApoE and A{beta} or indirect effects on A{beta} clearance. This is further complicated because ApoE exists in lipidated or unlipidated states, which influence its biochemical properties. To disentangle how single ApoE mutations confer vastly different effects on A{beta} pathology, we investigated how both the ApoE isoform and lipidation modulate its interaction with inert and cytotoxic A{beta} species, and its effect on A{beta} uptake and cytotoxicity in human astrocytes. MethodsWe prepared A{beta} in distinct aggregation states (monomers, oligomers, and fibrils) and ApoE in different lipidation states to characterize their size and affinity for one another using fluorescence correlation spectroscopy and fluorescence polarization, respectively. We then utilized flow cytometry and live cell imaging to quantify the uptake of A{beta} in different aggregation states by primary and immortalized human astrocytes in the presence of different isoforms of lipidated or unlipidated ApoE. ResultsThis study revealed that ApoE lipidation, not isoform, has the biggest impact on its interaction with A{beta}, on the uptake of A{beta} by astrocytes, and on A{beta}-induced cytotoxicity. Specifically, unlipidated ApoE preferentially interacts with A{beta} oligomers and fibrils, which substantially inhibits their uptake by astrocytes. Conversely, lipidated ApoE showed no interaction with A{beta} oligomers and had a reduced ability to inhibit A{beta} uptake. ConclusionsOur observations provide important molecular details that suggest previously observed ApoE isoform-specific differences in AD risk are likely driven primarily by in vivo differences in ApoE lipidation, rather than biophysical differences between ApoE isoforms in their interaction with A{beta}. We propose that the impaired lipidation of ApoE4 in the brain increases levels of unlipidated ApoE, which then bind toxic A{beta} oligomers and reduce their clearance by astrocytes. These findings underscore the therapeutic potential of interventions aimed at increasing ApoE lipidation and decreasing interactions between toxic A{beta} oligomers and ApoE.

biophysics↗

bioRxiv2025

Consensus DNA Inhibits p53 Aggregation but Fails to Rescue Mutants

The tumor suppressor p53 plays a crucial role in regulating gene expression under cellular stress. Somatic mutations to its DNA-binding domain are common in lethal cancers and lead to lost regulatory function and pathological hallmarks, such as misfolded p53 aggregates and amyloid-like fibrils. Despite decades of these observations, the molecular determinants driving p53 aggregation and its role in cancer incidence, progression, and lethality remain poorly defined. Identifying these determinants, however, is critical for developing therapeutic interventions that modulate protein solubility in cancer and understanding disparities in cancer outcomes. Therefore, we investigated whether consensus DNA sequences that stabilize wild-type p53 are sufficient to regulate the aggregation of oncogenic p53 mutants with reduced binding affinities. Using a combination of probe-based and label-free spectroscopic and microscopic techniques, we examined how consensus (p21, Bax) and non-consensus (p21-scramble, Poly-GC) DNA oligonucleotides regulate the aggregation of wild-type p53 in comparison to three cancer-associated mutants (R248Q, R273H, and R175H). We find that equimolar p21 consensus sequences significantly inhibits wild-type p53 aggregation, while other DNA sequences do not. In contrast, oncogenic p53 mutants evaded DNA regulation of protein aggregation, and some oligonucleotides even enhanced aggregation at low concentrations, which suggests concentration-dependent DNA-p53 interactions. These findings emphasize that DNA response elements are sufficient for regulating wild-type p53 aggregation in solution. However, key somatic mutations in cancer promote aggregation at the expense of DNA-binding, directly leading to the loss of p53 solubility through biochemical interactions. Taken together, these observations suggest that potent regulators of p53 aggregation should aim to restore affinity between oncogenic p53 mutants and regulatory DNA to minimize the pathological hallmarks of lethal tumors. Significance StatementOur research investigates whether DNA sequences that bind tumor suppressor protein p53 regulate its aggregation behavior in wild-type and somatically mutated forms linked to cancer. While specific DNA sequences regulate p53 aggregation, protein mutations that reduce affinity for consensus DNA or destabilize p53s conformation result in persistent, likely dysfunctional aggregates, even in the presence of regulatory nucleotides. Unsurprisingly, p53 and its mutants exhibit increased aggregation when there is substantially more protein to DNA, suggesting insufficient regulatory DNA or excess unregulated p53 may inevitably lead to pathological aggregation. These findings clarify our understanding of p53s molecular behavior and suggest that cancer-linked somatic mutations enable p53 to evade DNAs regulatory effects and subsequently aggregate, leading to downstream losses in function.

biophysics↗

bioRxiv2023

Delineating Structural Propensities of the 4E-BP2 Protein via Integrative Modelling and Clustering

The intrinsically disordered 4E-BP2 protein regulates mRNA cap-dependent translation through the interaction with the predominantly folded eukaryotic initiation factor 4E (eIF4E). Phosphorylation of 4E-BP2 dramatically reduces eIF4E binding, in part by stabilizing a binding- incompatible folded domain (REF). Here, we used a Rosetta-based sampling algorithm optimized for IDRs to generate initial ensembles for two phospho forms of 4E-BP2, non- and five-fold phosphorylated (NP and 5P, respectively), with the 5P folded domain flanked by N- and C-terminal IDRs (N-IDR and C-IDR, respectively). We then applied an integrative Bayesian approach to obtain NP and 5P conformational ensembles that agree with experimental data from nuclear magnetic resonance, small-angle X-ray scattering and single-molecule Forster resonance energy transfer (smFRET). For the NP state, inter-residue distance scaling and 2D maps revealed the role of charge segregation and pi interactions in driving contacts between distal regions of the chain ([~]70 residues apart). The 5P ensemble shows prominent contacts of the N-IDR region with the two phosphosites in the folded domain, pT37 and pT46, and, to a lesser extent, delocalized interactions with the C-IDR region. Agglomerative hierarchical clustering led to partitioning of each of the two ensembles into four clusters, with different global dimensions and contact maps. This helped delineate an NP cluster that, based on our smFRET data, is compatible with the eIF4E-bound state. 5P clusters were differentiated by interactions of C-IDR with the folded domain and of the N-IDR with the two phosphosites in the folded domain. Our study provides both a better visualization of fundamental structural poses of 4E-BP2 and a set of falsifiable insights on intrachain interactions that bias folding and binding of this protein.

biophysics↗

bioRxiv2020

Ligand Modulation of the Conformational Dynamics of the A2A Adenosine Receptor Revealed by Single-Molecule Fluorescence

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra-and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A Receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Forster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes [≥] 3 ms. Local conformational dynamics around residue 229 on TM6 was measured using Fluorescence Correlation Spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 s) and slow (200-300 s) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y288, and Y290) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 229 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

biophysics↗

bioRxiv2020

Integrating smFRET, SAXS and NMR data to infer structural ensembles of an intrinsically-disordered protein

Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes structural characterization challenging, but of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. We have used an integrative modelling approach to understand how conformational restraints imposed by the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Forster Resonance Energy Transfer (smFRET) reach concordance on structural ensembles for Sic1 and phosphorylated Sic1 (pSic1). To resolve apparent discrepancies between smFRET and SAXS, we integrated SAXS data with non-smFRET (NMR) data and reserved the new smFRET data for Sic1 and pSic1 as an independent validation. The consistency of the SAXS/NMR restrained ensembles with smFRET, which was not guaranteed a priori, indicates that the perturbative effects of NMR or smFRET labels on the Sic1 and pSic1 ensembles are minimal. Furthermore, the mutual agreement with such a diverse set of experimental data suggest that details of the generated ensembles can now be examined with a high degree of confidence to reveal distinguishing features of Sic1 vs. pSic1. From the experimentally well supported ensembles, we find they are consistent with independent biophysical models of Sic1s ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modelling in calculating and drawing biological conclusions from IDP conformational ensembles.

biophysics↗