Biology subjects

Le, V. Q.

Publications and source records attributed to Le, V. Q..

5 recordsLinked to original sources

bioRxiv2026

Heterotypic interactions and sequence features modulate cellular reflectin condensate dynamics

Reflectin proteins drive dynamic structural coloration in cephalopods by organizing into dense intracellular lamellar structures that dictate local refractive index. While reconstituted reflectins readily undergo liquid-liquid phase separation in vitro, these assemblies frequently undergo dynamic arrest, vitrifying into non-dynamic condensates. Here, we investigate how primary sequence features, post-translational modifications, and heterotypic interactions regulate the material properties of reflectin condensates within the environment of mammalian HeLa cells. Using confocal microscopy and fluorescence recovery after photobleaching (FRAP), we demonstrate that canonical block copolymeric A-type reflectins readily form dynamically arrested condensates, with the linker blocks primarily responsible for the observed arrest. In contrast, non-canonical B/C reflectin variants exhibit significantly greater fluidity and rapid recovery kinetics. We show that phosphomimetic substitutions progressively fluidize some reflectin condensates. Lastly, we find that heterotypic condensates composed of canonical and non-canonical reflectins in combinations associated with reversible iridescence in squid substantially enhance canonical mobility. Our findings establish a biophysical framework in which phosphorylation and heterotypic mixing cooperatively suppress dynamic arrest, enabling the reversible material transitions required for active cephalopod camouflage and communication.

biophysics↗

bioRxiv2023

De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8

The RGD (Arg-Gly-Asp)-binding integrins v{beta}6 and v{beta}8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of v{beta}6 and v{beta}8 with high selectivity. The v{beta}6 and v{beta}8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7[A] root-mean-square deviation (RMSD) to the computational design models; the designed v{beta}6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-v{beta}6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the v{beta}8 inhibitor maintains the constitutively fixed extended-closed v{beta}8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the v{beta}6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

biochemistry↗

bioRxiv2021

Protection of the prodomain 1-helix correlates with latency in the transforming growth factor- family

The 33 members of the transforming growth factor beta (TGF-{beta}) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of prodomains that are non-covalently bound to the growth factor (GF). The pro-complexes of some members are latent and require activation steps to release the GF for signaling. Why some members are latent while others are non-latent is incompletely understood, but crystal structures and hydrogen-deuterium exchange (HDX) of four family members have begun to unravel how latency is regulated. Here, we extend this understanding by comparing pro-complex conformation in negative stain EM (nsEM) and HDX of ActA, BMP7, BMP9, BMP10, GDF8, TGF-{beta}1, and TGF-{beta}2. nsEM revealed that family members varied in either adopting cross-armed, open-armed, or V-armed configurations. Latency was achieved in both cross-armed and V-armed but not open-armed conformations. HDX revealed remarkably varying patterns of exchange between family members, consistent with large prodomain sequence divergence. We observed a strong correlation between latency and protection of the prodomain 1-helix from exchange, which in latent members coincided with greater buried surface area of the 1-helix and more hydrogen and cation-pi bonds from the prodomain fastener and GF to the 1-helix. Strong sequence conservation of the 1-helix and fastener only in latent members suggests that similar interactions are conserved and sufficient to confer latency. Moreover, most members exhibited rapid exchange in the unstructured "association region" at the prodomain N-terminus, highlighting their availability for interacting with factors that may regulate latency and extracellular storage.

biophysics↗

bioRxiv2018

Characteristics of the mitochondrial and cellular uptake of MPP+, as probed by the fluorescent mimic, 4′I-MPP+

The discovery that 1-methyl-4-phenylpyridinium (MPP+) selectively destroys dopaminergic neurons and causes Parkinsons disease (PD) symptoms in mammals has strengthened the environmental hypothesis of PD. The current model for the dopaminergic toxicity of MPP+ is centered on the uptake into dopaminergic neurons, accumulation into the mitochondria, inhibition of the complex-I leading to ATP depletion, increased reactive oxygen species (ROS) production, and apoptotic cell death. However, some aspects of this mechanism and the details of the cellular and mitochondrial accumulation of MPP+ are still poorly understood. The aim of this study was to characterize a structural and functional MPP+ mimic which is suitable to study the cellular distribution and mitochondrial uptake of MPP+ in live cells and use it to identify the molecular details of these processes to advance the understanding of the mechanism of the selective dopaminergic toxicity of MPP+. Here we report the characterization of the fluorescent MPP+ derivative, 1-methyl-4-(4-iodophenyl)pyridinium (4I-MPP+), as a suitable candidate for this purpose. Using this novel probe, we show that cytosolic/mitochondrial Ca2+ play a critical role through sodium-calcium exchanger (NCX) in the mitochondrial and cellular accumulation of MPP+ suggesting for the first time that MPP+ and related mitochondrial toxins may also exert their toxic effects through the perturbation of Ca2+ homeostasis in dopaminergic cells. We also found that the specific mitochondrial NCX (mNCX) inhibitors protect dopaminergic cells from the MPP+ and 4I-MPP+ toxicity, most likely through the inhibition of the mitochondrial uptake, which could potentially be exploited for the development of pharmacological agents to protect the central nervous system (CNS) dopaminergic neurons from PD-causing environmental toxins.

neuroscience↗

bioRxiv2017

Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation

Growth differentiation factor 8 (GDF8)/Myostatin is a latent TGF{-}{beta} family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid{-}cleaved GDF8 pro{-}complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro{-}complexes reveals a V{-}shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring{-}like, cross{-}armed conformation of latent TGF{-}{beta}1. Surprisingly, Tolloid{-}cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-{beta}1 to the 1-helix, latency lasso, and {beta}1 strand in the prodomain and to the {beta}6-7 strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.

biochemistry↗