Biology subjects

Rhoades, E.

Publications and source records attributed to Rhoades, E..

17 recordsLinked to original sources

bioRxiv2026

Genetic Code Expansion, Enzymatic Modification, and C-Terminal Labeling Enable Facile Production of Highly Modified α-Synuclein

-Synuclein (S), a protein that plays a central role in Parkinsons disease and related synucleinopathies, is an intrinsically disordered protein (IDP) whose functional interactions and aggregation behavior can be strongly influenced by post-translational modifications (PTMs). Phosphorylation, acetylation, and other PTMs regulate Ss interactions with lipid membranes and binding partners, whereas their dysregulation is associated with aggregation and neuronal toxicity. Despite significant progress through chemical and semi-synthetic approaches, investigating the combinatorial effects of PTMs has remained challenging due to the lack of accessible, site-specific methods. Here, we present an integrated strategy combining genetic code expansion, enzymatic modification, and intein-mediated click chemistry to generate S variants bearing multiple defined PTMs and a C-terminal fluorescent label. The resulting constructs enable direct evaluation of how individual and combined PTMs influence S structure, lipid binding, and cellular internalization. Our approach expands the molecular toolkit for dissecting PTM crosstalk in S and other aggregation-prone IDPs, advancing mechanistic understanding and supporting the development of therapeutic strategies for neurodegenerative disease.

biochemistry↗

bioRxiv2025

Low-order assemblies drive oncogenic RTK fusion signaling without condensation

Receptor tyrosine kinase (RTK) fusions are a large class of oncoproteins found in [~]5% of cancers. Key questions remain, however, about how RTK fusions transmit oncogenic signals, including how these largely cytoplasmic proteins activate downstream pathways that originate at the plasma membrane. Fusions are multimeric and can form mesoscale condensates in cancer cells, and condensation has been implicated as an essential mechanism to enable signal transmission from the cytoplasm. However, whether condensates play a causal role, or whether smaller diffuse assemblies are sufficient to transduce signals, has been challenging to establish. Here we apply advanced microscopy, single-cell analysis, and synthetic fusions to determine the principles by which multimerization and condensation drive signaling from cytoplasmic RTK fusions. For EML4-ALK, a prominent fusion that forms condensates, we found poor correlation between condensation and signaling. By contrast, EML4-ALK activity was abundant in the diffuse phase, and the kinetics of diffuse-phase activity aligned more closely with downstream Erk signaling than did kinetics of signaling within condensates. Synthetic RTK fusions showed that cytoplasmic ALK or RET fusion dimers--and even constitutively active monomers--were sufficient to induce strong Ras-Erk signaling despite the absence of condensates, and diffuse fusions were sufficient to transform cells in vitro and in subcutaneous tumor models. A panel of various other cancer-driving RTK fusions showed that low-order multimerization was universal across fusions, whereas mesoscale condensation was rare and did not correlate with signaling. Our results suggest that low-order fusion multimerization is sufficient to drive its phosphorylation, which is necessary and sufficient to trigger downstream oncogenic signaling.

cancer biology↗

bioRxiv2025

Specific Combinations of Physiological Tau Phosphorylation Regulate Tau-Microtubule Interactions in Developing Neurons

Tau phosphorylation is a defining feature of Alzheimers disease, yet it also plays an important physiological role in regulating functional interactions with microtubules (MTs), particularly during normal neuronal development. While individual tau phosphorylation sites have been well-studied, how combinatorial phosphorylation impacts taus behavior in both contexts is poorly understood. We hypothesized that contrasting developmental and pathological models of tau phosphorylation would yield insights into mechanisms of impaired neuronal resilience in tauopathies. We use transgenic PS19 tau mice to demonstrate that increases in individual tau phospho-epitopes with aging do not correlate with their enrichment in tau aggregates in these mice. Using seeded aggregation of tau in neurons, we observe that tau aggregate formation does not necessarily result in MT destabilization; instead, aggregation diminishes taus ability to counteract destabilization when MTs are externally perturbed. We identify that combinations of tau phospho-signatures are responsive to MT destabilization, and phosphorylation at residues S235 and S262 as particularly critical for regulation of interactions with MTs. Phosphorylation of these residues was associated both with significantly reduced binding to MTs, as well as impaired phosphatase response in aggregates. As a whole, this work provides mechanistic insight into phospho-regulation of tau function and prompts reconsideration of the canonical view that tau phosphorylation precedes MT destabilization in disease. Rather, we provide compelling evidence that phosphorylation of tau may be a response to changes in MT stability and that cell death may result from aggregated taus inability to function in this manner. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/669485v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@dd84e5org.highwire.dtl.DTLVardef@14001eaorg.highwire.dtl.DTLVardef@13db7a0org.highwire.dtl.DTLVardef@e26d16_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO Schematic model of our findings: Tau phosphorylation is dynamically regulated in specific combinatorial patterns in response to shifting MT growth cycles. C_FIG

neuroscience↗

bioRxiv2025

Genetic Code Expansion and Enzymatic Modifications as Accessible Methods for Studying Site-Specific Post-Translational Modifications of Alpha-Synuclein and Tau

Alpha-synuclein (S) and tau play important roles in the pathology of Parkinsons Disease and Alzheimers Disease, respectively, as well as numerous other neurodegenerative diseases. Both proteins are classified as intrinsically disordered proteins (IDPs), as they have no stable structure that underlies their function in healthy tissue, and both proteins are prone to aggregation in disease states. There is substantial interest in understanding the roles that post-translational modifications (PTMs) play in regulating the structural dynamics and function of S and tau monomers, as well as their propensity to aggregate. While there have been many valuable insights into site-specific effects of PTMs garnered through chemical synthesis and semi-synthesis, these techniques are often outside of the expertise of biochemistry and biophysics laboratories wishing to study S and tau. Therefore, we have assembled a primer on genetic code expansion and enzymatic modification approaches to installing PTMs into S and tau site-specifically, including isotopic labeling for NMR and fluorescent labeling for biophysics and microscopy experiments. These methods should be enabling for those wishing to study authentic PTMs in S or tau as well as the broader field of IDPs and aggregating proteins.

biochemistry↗

bioRxiv2025

Investigation of All Disease-Relevant Lysine Acetylation Sites in α-Synuclein Enabled by Non-canonical Amino Acid Mutagenesis

Aggregates of -synuclein (S) are hallmarks of synucleinopathies, including Parkinsons Disease (PD) and Multiple System Atrophy (MSA). We have recently shown that S lysine acetylation in the soluble monomer pool varies between healthy controls, PD, and MSA patients. We used non-canonical amino acid (ncAA) mutagenesis to express and purify all 12 currently known disease-relevant variants of S and studied their binding to membranes as well as their aggregation propensities. We found that acetylation of lysine 12, 43, and 80 had particularly strong effects. To understand the implications for acetylation of monomeric S found in healthy cells, we performed NMR experiments to study protein conformation and fluorescence correlation spectroscopy experiments to quantify lipid binding. We also investigated the effects of acetylation at lysine 12, 43, and 80 on fibril seeding in neurons. Collectively, our biochemical and cell biological investigations indicated that acetylation of K80 could inhibit aggregation without conferring negative effects on monomer function in healthy cells. Therefore, we studied the structures of fibrils with K80 acetylation through cryo-electron microscopy to uncover the structural basis for these effects. Finally, we identified inhibition of HDAC8 as a way of potentially increasing acetylation at K80 and other inhibitory sites for therapeutic benefit.

biochemistry↗

bioRxiv2024

Structural Insights into the Role of the Proline Rich Region in Tau Function

Tau is a microtubule-associated protein that plays an important role in modulating axonal microtubules in neurons. Intracellular tau aggregates are found in a broad class of disorders, including Alzheimers disease, termed tauopathies. Tau is an intrinsically disordered protein, and its structural disorder appears to be critical to its microtubule-related functions. Tubulin binding sites are found in taus proline-rich region (PRR), microtubule binding repeats (MTBR: R1-R4), and pseudo-repeat, R'. While many post-translational modifications have been identified on tau, phosphorylation sites, which both regulate tubulin dimer and microtubule interactions and are correlated with disease, cluster with high frequency within the PRR. Here, we use fluorescence correlation spectroscopy and structural mass spectrometry techniques to characterize the impact of phosphomimic mutations in the PRR on tubulin dimer binding and probe the structure of the PRR-tubulin dimer complex. We find that phosphomimics cumulatively diminish tubulin dimer binding and slow microtubule polymerization. Additionally, we map two [~]15 residue regions of the PRR as primary tubulin dimer binding sites and propose a model in which PRR enhances lateral interactions between tubulin dimers, complementing the longitudinal interactions observed for MTBR. Together these measurements provide insight into the previously overlooked relevance of taus PRR in functional interactions with tubulin. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/614010v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@360dd3org.highwire.dtl.DTLVardef@c5d75aorg.highwire.dtl.DTLVardef@bd76eeorg.highwire.dtl.DTLVardef@cb433c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

The C-terminus of alpha-Synuclein regulates its dynamic cellular internalization by Neurexin 1 beta

The aggregation of the disordered neuronal protein, -Synuclein (S), is the primary pathological feature of Parkinsons disease. Current hypotheses favor cell-to-cell spread of S species as underlying disease progression, driving interest in identifying the molecular and cellular species involved in cellular internalization of S. Prior work from our lab identified the chemically specific interaction between S and the pre-synaptic adhesion protein neurexin 1{beta} (N1{beta}) to be capable of driving cellular internalization of both monomer and aggregated forms of S. Here we explore the physical basis of N1{beta}-driven internalization of S. Specifically, we show that spontaneous internalization of S by SH-SY5Y and HEK293 cells expressing N1{beta} requires essentially all of the membrane-binding domain of S; S constructs truncated beyond residue 90 bind to N1{beta} in the plasma membrane of HEK cells, but are not internalized. Interestingly, prior to internalization, S and N1{beta} co-diffuse rapidly in the plasma membrane. S constructs that are not internalized show very slow mobility themselves, as well as slow N1{beta} diffusion. Finally, we find that truncated S is capable of blocking internalization of full-length S. Our results draw attention to the potential therapeutic value of blocking S-N1{beta} interactions.

biophysics↗

bioRxiv2023

Nanomolar interactions of alpha-synuclein fibrils to tau determined by FCS

Age-related neurodegenerative disorders like Alzheimers disease (AD) and Parkinsons disease (PD) are characterized by deposits of protein aggregates, or amyloid, in various regions of the brain. Traditionally, aggregation of a single protein was observed to be correlated with these different pathologies: tau in AD and -synuclein (S) in PD. However, there is increasing evidence that the pathologies of these two diseases overlap, and the individual proteins may promote each others aggregation. Both tau and S are intrinsically disordered proteins (IDPs), lacking stable secondary and tertiary structure under physiological conditions. In this study we used a combination of biochemical and biophysical techniques to interrogate the interaction of tau with both soluble and fibrillar S. Fluorescence correlation spectroscopy (FCS) was used to assess the interactions of specific domains of fluorescently labeled tau with full length and C-terminally truncated S in both monomeric and fibrillar forms. We found that full-length tau as well as individual tau domains interact with monomer S weakly, but this interaction is much more pronounced with S seeds. This interaction does not impact tau aggregation or fibril formation. These findings provide insight into the nature of interactions between tau and S as well as the domains responsible.

biochemistry↗

bioRxiv2023

Semi-synthetic CoA-alpha-Synuclein Constructs Trap N-terminal Acetyltransferase NatB for Binding Mechanism Studies

N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases (NATs). A major member of this enzyme family, NatB, acts on much of the human proteome, including -synuclein (S), a synaptic protein that mediates vesicle trafficking. NatB acetylation of S modulates its lipid vesicle binding properties and amyloid fibril formation, which underlies its role in the pathogenesis of Parkinsons disease. Although the molecular details of the interaction between human NatB (hNatB) and the N-terminus of S have been resolved, whether the remainder of the protein plays a role in interacting with the enzyme is unknown. Here we execute the first synthesis, by native chemical ligation, of a bisubstrate inhibitor of NatB consisting of coenzyme A and full-length human S, additionally incorporating two fluorescent probes for studies of conformational dynamics. We use cryo-electron microscopy (cryo-EM) to characterize the structural features of the hNatB/inhibitor complex and show that, beyond the first few residues, S remains disordered when in complex with hNatB. We further probe changes in the S conformation by single molecule Forster resonance energy transfer (smFRET) to reveal that the C-terminus expands when bound to hNatB. Computational models based on the cryo-EM and smFRET data help to explain the conformational changes and their implications for hNatB substrate recognition and specific inhibition of the interaction with S. Beyond the study of S and NatB, these experiments illustrate valuable strategies for the study of challenging structural biology targets through a combination of protein semi-synthesis, cryo-EM, smFRET, and computational modeling.

biochemistry↗

bioRxiv2020

Tau avoids the GTP cap at growing microtubule plus ends

Plus-end tracking proteins (+TIPs) are a group of proteins that associate with the growing end of microtubules and mediate important cellular functions including neural development and cell division. Work in recent years has shown that the majority of +TIPs are directed to the plus-end through a family of end binding proteins (EBs), which preferentially bind the stabilizing cap of GTP-tubulin present during microtubule growth, versus weaker binding to GDP-tubulin in the proximal microtubule. One question yet to be addressed is whether there may exist other microtubule associated proteins (MAPs) that preferentially bind specific nucleotide states of tubulin. Here we report that the neuronal MAP tau, which is enriched in axons where it promotes microtubule growth and bundling, preferentially binds GDP-tubulin (KD = 0.26 {micro}M) over GMPCPP-tubulin (KD = 1.1 {micro}M) in vitro as well as GTP-tubulin at the tips of growing microtubules in vitro and in vivo. This nucleotide preference causes tau binding to lag behind the growing microtubule plus-end by about 100-200 nm both in vitro and in live cells. Thus, tau is a microtubule tip avoiding protein, establishing a new class of tip avoiding MAPs, and acts primarily by suppressing microtubule shortening rather than promoting growth. We speculate that neurological disease-relevant tau mutations may exert their phenotype by their failure to properly recognize GDP-tubulin, thus displacing +TIPs, such as EB3, and their associated activities into abnormal locations in the neuron.

cell biology↗

bioRxiv2019

Structural characterization of tau in fuzzy tau:tubulin complexes

Tau is a neuronal microtubule (MT) associated protein of significant interest due to its association with several neurodegenerative disorders. Taus intrinsic disorder and the dynamic nature of its interactions with tubulin and MTs make its structural characterization challenging. Here we use an environmentally sensitive fluorophore as a site-specific probe of tau bound to soluble tubulin. Comparison of our results with recently published tau:MT cryo-EM model reveals structural similarities between tubulin- and MT-bound tau. Analysis of residues across the repeat regions reveal a hierarchy in tubulin occupancy, which may be relevant to taus ability to differentiate between tubulin and MTs. As binding to soluble tubulin is a critical first step in MT polymerization, our characterization of the structural features of tau in dynamic, fuzzy tau:tubulin assemblies advances our understanding of how tau functions in the cell and how function may be disrupted in disease.

biophysics↗

bioRxiv2019

Regulation of tau’s proline rich region by its N-terminal domain

Tau is an intrinsically disordered, microtubule-associated protein with a role in regulating microtubule dynamics. Despite intensive research, the molecular mechanisms of taumediated microtubule polymerization are poorly understood. Here we use single molecule fluorescence to investigate the role of taus N-terminal domain (NTD) and proline rich region (PRR) in regulating interactions of tau with soluble tubulin. Both full-length tau isoforms and truncated variants are assayed for their ability to bind soluble tubulin and stimulate microtubule polymerization. We describe a novel role for taus PRR as an independent tubulin-binding domain with polymerization capacity. In contrast to the relatively weak tubulin interactions distributed throughout the microtubule binding repeats (MTBR), resulting in heterogeneous tau:tubulin complexes, the PRR binds tubulin tightly and stoichiometrically. Moreover, we demonstrate that interactions between the PRR and MTBR are reduced by the NTD through a conserved conformational ensemble. Based on our data, we propose that taus PRR can serve as a core tubulin-binding domain, while the MTBR enhances polymerization capacity by increasing the local tubulin concentration. The NTD negatively regulates tubulin-binding interactions of both of these domains. This study draws attention to the central role of the PRR in tau function, as well as providing mechanistic insight into tau-mediated polymerization of tubulin.\n\nSignificance StatementTau is an intrinsically disordered, microtubule associated protein linked to a number of neurodegenerative disorders. Here we identify taus proline rich region as having autonomous tubulin binding and polymerization capacity, which is enhanced by the flanking microtubule binding repeats. Moreover, we demonstrate that taus N-terminal domain negatively regulates both binding and polymerization. We propose a novel model for tau-mediated polymerization whereby the proline rich region serves as a core tubulin-binding domain, while the microtubule binding repeats increase the local concentration. Our work draws attention to the importance of the proline rich region and N-terminal domain in tau function, and highlights the proline rich region as a putative target for the development of therapeutics.

biophysics↗

bioRxiv2019

Polyphosphate initiates tau aggregation through intra- and intermolecular scaffolding

The aggregation and deposition of tau is a hallmark of a class of neurodegenerative diseases called tauopathies. Despite intensive study, cellular and molecular factors that trigger tau aggregation are not well understood. Here we provide evidence for two mechanisms relevant to the initiation of tau aggregation in the presence of cytoplasmic polyphosphates (polyP): changes in the conformational ensemble of monomer tau and noncovalent cross-linking of multiple tau monomers. We identified conformational changes throughout full-length tau, most notably diminishment of long-range interactions between the termini coupled with compaction of the microtubule binding and proline rich regions. We found that while the proline rich and microtubule binding regions both contain polyP binding sites, the proline rich region is a requisite for compaction of the microtubule binding region upon binding. Additionally, both the magnitude of the conformational change and the aggregation of tau are dependent on the chain length of the polyP polymer. Longer polyP chains are more effective at intermolecular, noncovalent cross-linking of tau. These observations provide an understanding of the initial steps of tau aggregation through interaction with a physiologically relevant aggregation inducer.

biophysics↗

bioRxiv2019

A novel small molecule screening platform for disrupting toxic tau oligomers in cells

Tauopathies, including Alzheimers disease, are a group of neurodegenerative disorders characterized by pathological aggregation of the microtubule binding protein tau. Recent studies suggest that toxic tau oligomers, which are soluble and distinct from insoluble beta-sheet fibrils, are central players in neuronal cell death. To exploit this new therapeutic window, we engineered two first-in-class FRET based biosensors that monitor tau conformations in cells. Because this new technology platform operates in cells, it enables high-throughput screening of small molecules that target tau oligomers while avoiding the uncertainties of idiosyncratic in vitro preparations of tau assemblies from purified protein. We found a small molecule, MK-886, that disrupts tau oligomers and reduces tau-induced cell cytotoxicity with nanomolar potency. Using SPR and an advanced single-molecule FRET technique, we show that MK-886 directly binds to tau and specifically perturbs the folding of tau monomer in the proline-rich and microtubule-binding regions. Furthermore, we show that MK-886 accelerates the tau aggregation lag phase using a thioflavin-T assay, implying that the compound stabilizes a non-toxic, on-pathway oligomer. The technology described here should generalize to the study and targeting of conformational ensembles within the aggregation pathways of most intrinsically disordered proteins.

neuroscience↗

bioRxiv2018

α-Synuclein binds extracellular complex N-linked glycans

Cell-to-cell transmission of toxic forms of -Synuclein (S) is thought to underlie disease progression in Parkinsons disease. S in humans is constitutively N-terminally acetylated (Sacetyl), although the impact of this modification is relatively unexplored. Here we report that Sacetyl is more effective at inducing intracellular aggregation in primary neurons than unmodified S (Sun). We identify complex N-linked glycans as binding partners for Sacetyl, and demonstrate that cellular internalization of Sacetyl is reduced significantly upon cleavage of extracellular N-linked glycans, but not other carbohydrates. We verify binding of Sacetyl to N-linked glycans in vitro, using both isolated glycans and cell-derived proteoliposomes. Finally, we identify neurexin l{beta}, a neuronal glycoprotein, as capable of driving glycan-dependent uptake of Sacetyl. Importantly, our results are specific to Sacetyl as Sun does not demonstrate sensitivity for N-linked glycans. Our study identifies extracellular N-linked glycans, and neurexin l{beta} specifically, as key modulators of neuronal uptake of physiological Sacetyl drawing attention to the potential therapeutic value of Sacetyl-glycan interactions.

biophysics↗

bioRxiv2017

Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA and actin filaments

We used fluorescence spectroscopy and electron microscopy to determine how binding of ATP, nucleation-promoting factors (NPF), actin monomers and actin filaments change the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C-termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Forster resonance energy transfer (FRET) efficiency (ETeff) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide binding clefts of the Arp subunits. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ETeff between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected a different structural change that involves bringing ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. Each of the ligands that activates Arp2/3 complex changes the structure in different ways, each leading progressively to fully activated Arp2/3 complex on the side of a filament.

biophysics↗

bioRxiv2016

Structurally distinct oligomers of islet amyloid polypeptide mediate toxic and non-toxic membrane poration

Peptide mediated gain-of-toxic function is central to pathology in Alzheimers, Parkinsons and diabetes. In each system, self-assembly into oligomers is observed and can also result in poration of artificial membranes. Structural requirements for poration and the relationship of structure to cytotoxicity is unaddressed. Here, we focus on islet amyloid polypeptide (IAPP) mediated loss of insulin secreting cells in diabetics. Newly developed methods enable structure-function inquiry to focus on intracellular oligomers composed of hundreds of IAPP. The key insights are that porating oligomers are internally dynamic, grow in discrete steps and are not canonical amyloid. Moreover, two class of pores coexist; an IAPP-specific ligand establishes that only one is cytotoxic. Toxic rescue occurs by stabilizing non-toxic poration without displacing IAPP from mitochondria. These insights illuminate cytotoxic mechanism in diabetes and also provide a generalizable approach for inquiry applicable to other partially ordered protein assemblies.\n\nHighlightsO_LIThe peptide amyloid precursor, IAPP, forms two classes of membrane porating oligomers.\nC_LIO_LIThe two classes have a >100-fold difference in pore size with the large pore form correlated with mitochondrial depolarization and toxicity.\nC_LIO_LIA drug-like molecule distinguishes between the two oligomer classes and rescues toxicity by stabilizing non-toxic poration without displacing IAPP from the mitochondria.\nC_LIO_LIThe mechanism of pore-forming oligomer assembly includes stepwise coalescence of smaller, dynamic assemblies.\nC_LI

cell biology↗