Biology subjects

Alexander-Katz, A.

Publications and source records attributed to Alexander-Katz, A..

7 recordsLinked to original sources

bioRxiv2026

An intrinsically disordered adaptor protein controls the size and composition of EGFR condensates on membranes

Receptor phase separation is common in signaling pathways, yet mechanisms that limit condensate size and drive their dissolution remain poorly understood. For example, phosphorylated EGFR forms large micron-scale condensates in vitro, but assembles into smaller nanoscale clusters in cells, suggesting cellular regulation of condensate size. Here, we show that the intrinsically disordered adaptor protein GAB1 actively dissolves EGFR condensates and re-nucleates smaller, compositionally distinct assemblies in reconstituted supported lipid bilayers and simulations. Dissolution requires unphosphorylated tyrosines within the GAB1 intrinsically disordered region, and tyrosine phosphorylation acts as a regulatory switch that abolishes this activity. GAB1-nucleated assemblies require PH domain-driven oligomerization, which is inhibited by phosphoinositide lipids, linking membrane composition to nucleation. These results uncover two unexpected regulatory features in signaling pathways. First, unphosphorylated tyrosines may function broadly to regulate condensate size or stability, since similar tyrosine-based sequence features are found across hundreds of proteins, including many that function in signal transduction. Additionally, PH domains may play a broader role in protein oligomerization and receptor organization, as an AlphaFold screen reveals dozens of additional PH domains predicted to self-associate. Together, these results establish general mechanisms that function as modular tools for reprogramming signaling through receptor reorganization and may enable new approaches to target such pathways in disease.

biophysics↗

bioRxiv2022

The interplay between adsorption and aggregation of von Willebrand factor chains in shear flows

AO_SCPLOWBSTRACTC_SCPLOWVon Willebrand factor (VWF) is a giant extracellular glycoprotein that carries out a key adhesive function during primary hemostasis. Upon vascular injury and triggered by the shear of flowing blood, VWF establishes specific interactions with several molecular partners in order to anchor platelets to collagen on the exposed sub-endothelial surface. VWF also interacts with itself to form aggregates that, adsorbed on the surface, provide more anchor sites for the platelets. However, the interplay between elongation and subsequent exposure of cryptic binding sites, self-association, and adsorption on the surface, remained unclear for VWF. In particular, the role of shear flow in these three processes is not well understood. In this study, we address these questions by using Brownian dynamics simulations at a coarse-grained level of resolution. We considered a system consisting of multiple VWF-like self-interacting chains that also interact with a surface under a shear flow. By a systematic analysis, we reveal that chain-chain and chain-surface interactions coexist non-trivially to modulate the spontaneous adsorption of VWF and the posterior immobilization of secondary tethered chains. Accordingly, these interactions tune VWFs extension and its propensity to form shear-assisted functional adsorbed aggregates. Our data highlights the collective behavior VWF self-interacting chains have when bound to the surface, distinct from that of isolated or flowing chains. Furthermore, we show that the extension and the exposure to solvent have a similar dependence on shear flow, at a VWF-monomer level of resolution. Overall, our results highlight the complex interplay that exists between adsorption, cohesion, and shear forces and its relevance for the adhesive hemostatic function of VWF.

biophysics↗

bioRxiv2022

Probing weak lipid-lipid binding domain interactions with a mechanically transduced immunosorbent assay (METRIS)

Protein-lipid interactions constitute a very important class of biological interactions critical for multiple cell and tissue functions. It is believed that most lipid-protein interactions are very weak, with affinities in the 1mM-1M range. Here we study the interactions of multiple protein lipid binding domains with lipid membranes containing signaling lipids known as phosphatidylinositol phosphates (PIPs) using a new mechanically transduced immunosorbent assay (METRIS). We demonstrate that this assay can measure extremely weak interactions at PIP bilayer concentrations below 1%, which is close to the biological lipid concentration regime. In particular, we have studied the interaction of DrrAWT, DrrAK568A, PH-{delta}, and 2XFYVE as well as previously unexplored lipid binding domains such as Auxilin 1 (PTEN) and Auxilin 2 (GAK) against a wide palette of PIPs. Our results confirm that each of these domains interacts specifically with a PIP partner. In the case of Auxilin 1 and Auxilin 2, both proteins in the Clathrin endocytotic pathway, we find that their PTEN-like domain interacts specifically yet with ultra low affinity with PI3P and PI4P respectively. We have also found a new unknown medium-high affinity interaction between GAK with PI34P2. Our work, thus, provides a direct and robust method to measure and catalog protein lipid interactions which are important in many processes such as signaling and membrane sculpting. Furthermore, this assay can be extended in a straightforward manner to study other interactions such as ligand-receptor or antibody-antigen.

biophysics↗

bioRxiv2020

Multivalent polymers can control phase boundary, dynamics, and organization of liquid-liquid phase separation

Multivalent polymers are a key structural component of many biocondensates. When interacting with their cognate binding proteins, multivalent polymers such as RNA and modular proteins have been shown to influence the liquid-liquid phase separation (LLPS) boundary to control condensate formation and to influence condensate dynamics after phase separation. Much is still unknown about the function and formation of these condensed droplets, but changes in their dynamics or phase separation are associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimers Disease. Therefore, investigation into how the structure of multivalent polymers relates to changes in biocondensate formation and maturation is essential to understanding and treating these diseases. Here, we use a coarse-grain, Brownian Dynamics simulation with reactive binding that mimics specific interactions in order to investigate the difference between non-specific and specific multivalent binding polymers. We show that non-specific binding interactions can lead to much larger changes in droplet formation at lower energies than their specific, valence-limited counterparts. We also demonstrate the effects of solvent conditions and polymer length on phase separation, and we present how modulating binding energy to the polymer can change the organization of a droplet in a three component system of polymer, binding protein, and solvent. Finally, we compare the effects of surface tension and polymer binding on the condensed phase dynamics, where we show that both lower protein solubilities and higher attraction/affinity of the protein to the polymer result in slower droplet dynamics. We hope this research helps to better understand experimental systems and provides additional insight into how multivalent polymers can control LLPS.

biophysics↗

bioRxiv2020

Polymer Stiffness Regulates Multivalent Binding and Liquid-Liquid Phase Separation

Multivalent binding is essential to many biological processes because it builds high affinity bonds by using several weak binding interactions simultaneously. Multivalent polymers have shown promise as inhibitors of toxins and other pathogens, and they are important components in the formation of biocondensates. Explaining how structural features of these polymers change their binding and subsequent control of phase separation is critical to designing better pathogen inhibitors and also to understanding diseases associated with membraneless organelles. In this work, we will examine the binding of a multivalent polymer to a small target. This scenario could represent a polymeric inhibitor binding to a toxic protein or RNA binding to an RNA-binding protein in the case of liquid-liquid phase separation. We use simulation and theory to show that flexible random-coil polymers bind more strongly than stiff rod-like polymers and that flexible polymers nucleate condensed phases at lower energies than their rigid analogues. We hope these results will provide insight into the rational design of polymeric inhibitors and improve understanding of membraneless organelles. Statement of SignificanceMultivalent polymers are essential for many biological systems, including targeting pathogens and controlling the formation of liquid-liquid phase separated biocondensates. Here, we explain how increasing polymer stiffness can reduce multivalent binding affinity to a small target such as a toxic protein and how modulating polymer stiffness can change the phase boundary for liquid-liquid phase separation. These results have implications for designing stronger pathogen inhibitors and provide insights on neurodegenerative diseases associated with abnormal biocondensate formation.

biophysics↗

bioRxiv2019

Unraveling the Complexity of Amyloid Polymorphism Using Gold Nanoparticles and Cryo-EM

The misfolding and self-assembly of proteins into {beta}-sheet-rich amyloid fibrils of various structures and morphologies is a hallmark of several neurodegenerative and systemic diseases. Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicity and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the use of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) to label the edges of synthetic, recombinant and native amyloid fibrils to assess amyloid morphological polymorphism using cryogenic transmission electron microscopy (cryo-EM). The fibrils studied were derived from amyloid proteins involved in disorders of the central nervous system (amyloid-{beta}, tau, -synuclein) and in systemic amyloidosis (a fragment of an immunoglobulin {lambda} light chain). The labeling efficiency enabled imaging and characterization of amyloid fibrils of different morphologies under hydrated conditions using cryo-EM. These NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including TEM with use of the negative stain or cryo-EM imaging. We also demonstrate the use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of an Alzheimers disease patient, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain (AL) amyloidosis. Analysis of the cryo-EM images of amyloids decorated with NPs shows exceptional homogeneity across the fibrils derived from human tissue in comparison to fibrils aggregated in vitro. The use of these NPs enabled us to gain novel insight into the structural features that distinguish amyloid fibrils formed in vivo from those formed in cell-free in vitro systems. Our findings demonstrate that these NPs represent a powerful tool for rapid imaging and profiling of amyloid morphological polymorphism in different types of samples, including those derived from complex biological aggregates found in human tissue and animal models of amyloid diseases. These advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM but also pave the way to elucidate the structural basis of amyloid strains and toxicity and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

neuroscience↗

bioRxiv2019

Computational Insights Into Avidity of Polymeric Multivalent Binders

Multivalent binding interactions are commonly found throughout biology to enhance weak monovalent binding such as between glycoligands and protein receptors. Designing multivalent polymers to bind to viruses and toxic proteins is a promising avenue for inhibiting their attachment and subsequent infection of cells. Several studies have focused on oligomeric multivalent inhibitors and how changing parameters such as ligand shape and size, and linker length and flexibility affect binding. However, experimental studies of how larger structural parameters of multivalent polymers such as degree of polymerization affect binding avidity to targets have mixed results with some finding an improvement with longer polymers and some finding no effect. Here, we use Brownian dynamics simulations to provide a theoretical understanding of how degree of polymerization affects the binding avidity of multivalent polymers. We show that longer polymers increase binding avidity to multivalent targets, but reach a limit in binding avidity at high degrees of polymerization. We also show that when interacting with multiple targets simultaneously, longer polymers are able to use inter-target interactions to promote clustering and improve binding efficiency. We expect our results to narrow the design space for optimizing the structure and effectiveness of multivalent inhibitors, as well as be useful to understand biological design strategies for multivalent binding.

bioengineering↗