Biology subjects

Broichhagen, J.

Publications and source records attributed to Broichhagen, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Emergence of new function through evolutionary divergence of an intrinsically disordered region

Intrinsically disordered regions (IDRs) are major drivers of protein functional diversification, yet the molecular features that enable the emergence of new functions within disordered sequences remain poorly understood. FCHO1 and FCHO2 are paralogous pioneer proteins of clathrin-mediated endocytosis that share a conserved domain architecture but perform distinct cellular functions and cannot compensate for each other's loss. Here we show that functional divergence between these proteins is associated with the acquisition of transient structure within their disordered regions. Using nuclear magnetic resonance spectroscopy, we identify two highly populated -helical elements in the IDR of FCHO1 that are absent from FCHO2. One of these helices mediates FCHO1 self-association and drives intracellular assembly, whereas FCHO2 lacks this behavior. Introduction of the FCHO1 helix into FCHO2 is sufficient to confer self-association and cellular assembly, demonstrating that a transient structural element embedded within an IDR can act as a transferable functional module. Evolutionary analysis reveals that this helical propensity emerged following duplication of the ancestral FCHO gene and became progressively reinforced during evolution. Despite this divergence, the same region retains a conserved membrane-binding activity in both paralogs. Together, our findings show how acquisition of transient secondary structure within an intrinsically disordered region can generate new molecular behaviors while preserving ancestral functions, providing a mechanism for the functional specialization of paralogous proteins.

biophysics↗

bioRxiv2026

Tau spreading coordinates intercellular lipid flux enhancing neuronal resilience to lipid toxicity

The presence of intrinsically disordered proteins (IDPs) in the extracellular environment of the brain suggests that protein disorder may serve functions beyond those confined to the intracellular space. Tau, a prototypical IDP linked to tauopathies, a class of neurodegenerative diseases, is continuously released and spreads between cells in the brain, yet the biological significance of this extracellular phase remains unresolved. Here, we show that spreading tau coordinates neutral lipid homeostasis by promoting neuronal lipid efflux and enhancing resilience to lipid toxicity. Integrating transcriptomics and lipidomics, we demonstrate that tau spreading reprograms lipid transport pathways and triggers redistribution of triacylglycerol pools, remodeling neutral lipid metabolism. Mechanistically, cellular uptake of spreading tau drives its accumulation within neutral lipid-rich compartments and promotes neuronal lipid efflux. The exported lipids are enriched in peroxidized species that are subsequently transferred to astrocytes, reducing neuronal lipid stress and revealing a pathway through which tau regulates lipid homeostasis. Our findings reframe spreading tau as a regulator of intercellular lipid flux and position protein disorder as an active mediator of cell-to-cell communication in coordinating tissue metabolism. Loss of this homeostatic function is likely to contribute to the early metabolic dysfunction associated with tauopathy.

neuroscience↗

bioRxiv2026

HaloTag Ligand and HaloTag Protein engineering for a binary fluorescent turn-on probe

Protein labelling by covalent attachment of a specific substrate to a self-labelling protein tag has become a regular in the life sciences. Herein, we report the design of a two-component labelling system, comprised of a non-fluorescent difluorinated xanthene, called F2X, and a HaloTag mutant engineered for targeted reactivity towards F2X. Upon primary covalent locking of the ligand at the canonical aspartate residue, two proximal lysine residues located at the protein surface can undergo nucleophilic aromatic substitution with the F2X core, building a fluorescent rhodamine via triple-covalent fusion. We used a generalizable in silico pipeline for heuristic conformational sampling of covalent protein-ligand complexes to find suitable mutation sites, culminating in the curation of 7 double-lysine HaloTag mutants for targeted in vitro testing. Reaction with the best-performing mutant, HTPL161K_Q165K, is characterized by full protein mass spectrometry, fluorescence polarization fluorescence lifetime, and fluorescence anisotropy and rationalized by computational modelling. We showcase the system in single molecule microscopy, where obviation of post-labelling purification is a prime advantage when targeting recombinant proteins that may not be expressed in larger quantities, and employ F2X in living cells with reduced photobleaching. Lastly, a cell-impermeable version was obtained by means of sulfonation, exclusively targeting extracellularly exposed HTPKK fused to the neuromodulatory G protein-coupled receptor metabotropic glutamate receptor 2.

synthetic biology↗

bioRxiv2026

A bio-orthogonal and covalent 5 kDa small protein tag

Precise and minimally perturbative protein labelling remains a key challenge for studying biomolecular function in living systems. Here, a minimal way to specifically label proteins based on SNAP-tag and intein-mediated protein splicing reaction is introduced. Termed CLUSTER (for Chemical Label-Unfold-Splice Technology Enables Recombination), this chimeric platform supports efficient labelling across diverse targets in living cells by retaining a fluorescent, 5 kDa sized peptide on a fusion protein of interest after splicing. A bacterial screening workflow was developed to optimize the reaction efficiency and construct design. Quantitative characterization using fluorescence polarization provides mechanistic insight into labelling efficiency and dynamics, while molecular dynamics simulations elucidate its stability, grasping the intricate nature of protein behaviour upon covalent labelling. This bio-orthogonal labelling technology allows for a versatile and minimally invasive approach for protein labelling, providing a powerful tool to probe protein behavior in native cellular systems.

synthetic biology↗

bioRxiv2026

Molecular assembly of the KCNQ1-KCNE1-BACE1 complex

We previously showed that the {beta}-secretase BACE1 directly modulates KCNQ1 channels through a non-proteolytic mechanism. Here, we dissect the molecular interplay among KCNQ1, its canonical auxiliary subunit KCNE1, and BACE1 using bimolecular fluorescence complementation (BiFC), electrophysiology, single-molecule pull-down (SiMPull), and a Forster resonance energy transfer (FRET)-based interaction assay. BiFC validated KCNQ1 homotetramerization and confirmed specific interactions of KCNQ1 with KCNE1 and with BACE1 at the plasma membrane. To map interaction determinants, we generated six KCNE1/BACE1 chimeras. BiFC and electrophysiological recordings revealed domain-specific contributions: BACE1s large extracellular domain primarily mediates its modulatory effects on KCNQ1 gating, while--consistent with previous reports--the KCNE1 transmembrane segment is necessary and sufficient to confer an IKs-like phenotype, and the KCNE1 intracellular domain tunes the voltage dependence of activation. Notably, a chimera combining the BACE1 extracellular region with KCNE1 transmembrane and intracellular regions produced an IKs-like current with additional BACE1-like slowing of activation, indicating functional additivity. Stoichiometry measurements by SiMPull and bleaching-step analysis demonstrated that KCNQ1 channel complexes predominantly recruit two BACE1 molecules. Although BACE1 oligomerizes at the plasma membrane, BiFC and FRET showed that KCNQ1 co-expression reduces BACE1 homomeric assembly, and this effect is unchanged by KCNE1 co-expression. Together, our data support a model in which BACE1 binds directly to KCNQ1, occupies a site distinct from KCNE1, and modulates KCNQ1 gating via its extracellular domain while remaining compatible with KCNE1 co-assembly.

biophysics↗

bioRxiv2026

Nanobodies equipped with HaloTag variants enable rapid and straightforward one-step immunofluorescence lifetime multiplexing

Multiplexed fluorescence imaging is limited by spectral overlap, whereas fluorescence lifetime provides an orthogonal encoding dimension. We replace genetically fused HaloTags with recombinant nanobody-HaloTag constructs applied as immunofluorescence reagents and determine a lifetime palette. Integrating lifetime and spectral encoding enables up to eight targets in a single acquisition and supports multiplexed imaging in cells and tissue using a simple OneStep labeling strategy compatible with fluorescent proteins, STED nanoscopy, and any standard laboratory antibody.

cell biology↗

bioRxiv2025

MRAP2 potentiates GPCR signaling by conserved mechanisms that are disrupted by obesity-associated genetic variants

Accessory proteins such as members of the melanocortin-2 receptor accessory protein family (MRAP) have been described to interact with and regulate the signaling of diverse G protein-coupled receptors (GPCRs), however, surprisingly little is known about the mechanisms by which they mediate these effects. MRAP2 modifies signaling of three distinct GPCRs, melanocortin receptor 4 (MC4R), MC3R and the ghrelin receptor (GHSR), which each play essential roles in appetite regulation. Human mutations in MRAP2 cause obesity with hyperglycaemia and hypertension, suggesting that its regulation of GPCRs is critical for maintaining metabolic homeostasis. However, the nature of MRAP2/GPCR complexes and whether there are shared mechanisms for complex assembly, critical structural regions or consistent effects on receptor signaling and trafficking remains unknown. Here we showed all three GPCRs preferentially interact with MRAP2 as 1:1 complexes and that MRAP2 binding disrupts GPCR homodimerization. MRAP2 interacts with the same receptor transmembrane regions to promote GPCR signaling, and the accessory protein impairs {beta}-arrestin-2 recruitment to prolong signaling and delay internalization. Deletion of the cytoplasmic region of MRAP2 impairs GPCR signaling by modulating receptor constitutive activity. Genetic variants in MRAP2 associated with overweight or obesity modulate the constitutive activity of all three GPCRs. Thus, MRAP2 regulates GPCR function using shared molecular mechanisms and these studies provide further evidence of the importance of GHSR constitutive activity.

molecular biology↗

bioRxiv2025

A silicon rhodamine-fused glibenclamide to label and detect malaria-infected red blood cells

The malaria parasite Plasmodium falciparum affects the lives of millions of people worldwide every year. The detection of replicating parasites within human red blood cells is of paramount importance, requiring appropriate diagnostic tools. Herein, we design and apply a silicon rhodamine-fused glibenclamide (SiR-glib). We first test this far-red fluorescent, fluorogenic and endoplasmic reticulum-targeting sulfonylurea in mammalian cells and pancreatic tissues, before characterizing its labeling performance in red blood cells infected with the asexual developmental stages of Plasmodium falciparum. We further combine SiR-glib with a portable smartphone-based microscope to easily and rapidly identify parasitized red blood cells, providing proof of principle for diagnostic use in rural endemic areas without major healthcare facilities.

biochemistry↗

bioRxiv2025

Structural Diversity of Metabotropic Glutamate Receptor/Beta-Arrestin Coupling

Beta-arrestins ({beta}-arrs) are cytosolic proteins which mediate G protein-coupled receptor (GPCR) desensitization, endocytosis, and signaling. Despite the widespread physiological roles of {beta}-arr coupling, the molecular basis of GPCR/{beta}-arr interaction has been studied primarily in monomeric family A GPCRs. Here we take an integrative biophysical and structural approach to uncover molecular diversity in {beta}-arr coupling to the neuromodulatory metabotropic glutamate receptors (mGluRs), prototypical, dimeric family C GPCRs. We find, using a new single molecule pulldown assay, that mGluRs couple to {beta}-arrs with a 2:1 or 2:2 stoichiometry via a combination of "tail" and "core" interactions. Using single molecule FRET analysis, we also find that {beta}-arr1 stabilizes active conformations of mGluR8. Cryo-EM structures of mGluR8 alone or with either G proteins or {beta}-arr1 reveal transducer-specific mGluR8 active states and, in combination with molecular dynamics simulations, define the positioning of mGluR8-bound {beta}-arr1, supporting a steric mechanism of mGluR desensitization involving interactions with both subunits and the lipid bilayer. Finally, combinatorial mutagenesis enables the identification of a landscape of homo- and hetero-dimeric mGluR/{beta}-arr complexes, including mGluR/{beta}-arr1/{beta}-arr2 megacomplexes, providing a framework for family C GPCR/{beta}-arr coupling and expanding the known range of GPCR/transducer coupling modes.

biophysics↗

bioRxiv2024

The MRAP2 accessory protein directly interacts with melanocortin-3 receptor to enhance signaling

The central melanocortin system links nutrition to energy expenditure, with melanocortin-4 receptor (MC4R) controlling appetite and food intake, and MC3R regulating timing of sexual maturation, rate of linear growth and lean mass accumulation. Melanocortin-2 receptor accessory protein-2 (MRAP2) is a single transmembrane protein that interacts with MC4R to potentiate its signalling, and human mutations in MRAP2 cause obesity. Previous studies have been unable to consistently show whether MRAP2 affects MC3R activity. Here we used single-molecule pull-down (SiMPull) to confirm that MC3R and MRAP2 interact in HEK293 cells. Analysis of fluorescent photobleaching steps showed that MC3R and MRAP2 readily form heterodimers most commonly with a 1:1 stoichiometry. Human single-nucleus and spatial transcriptomics show MRAP2 is co-expressed with MC3R in hypothalamic neurons with important roles in energy homeostasis and appetite control. Functional analyses showed MRAP2 enhances MC3R cAMP signalling, impairs {beta}-arrestin recruitment, and reduces internalization in HEK293 cells. Structural homology models revealed putative interactions between the two proteins and alanine mutagenesis of five MRAP2 and three MC3R transmembrane residues significantly reduced MRAP2 effects on MC3R signalling. Finally, we showed genetic variants in MRAP2 that have been identified in individuals that are overweight or obese prevent MRAP2s enhancement of MC3R-driven signalling. Thus, these studies reveal MRAP2 as an important regulator of MC3R function and provide further evidence for the crucial role of MRAP2 in energy homeostasis.

molecular biology↗

bioRxiv2024

Endogenous tagging of Munc13-1 with a SNAP tag as a tool for monitoring synapse nanoarchitecture

ABSTRACTSynaptic function is governed by highly regulated protein machineries, whose abundance and spatial localization change continually. Studies to determine the dynamic changes in synaptic proteins nanoarchitecture typically rely on immunolabeling, or on the expression of fluorescent proteins. The former employs chemical fluorophores and signal amplification, but requires fixation. The latter enables monitoring of proteins in live microscopy, but uses suboptimal fluorophores. Self-labeling tags have been introduced to combine the advantages of these two approaches, and here we introduce a knock-in mouse line where the essential presynaptic protein Munc13-1 is endogenously fused to the self-labeling SNAP tag. We demonstrate efficient Munc13-1-SNAP labeling in fixed neurons and brain sections by various SNAP dyes, as well as by a novel far-red and cell impermeable compound, SBG-SiR-d12. We introduce and characterize SBG-SiR-d12 here as a highly-efficient dye for SNAP-tag labeling of extracellular epitopes, and of intracellular proteins in fixed and permeabilized tissue. Finally, we show that Munc13-1-SNAP can be labeled in living neurons and monitored through live-cell imaging using confocal- and super resolution microscopy. We conclude that the Unc13aSNAP mouse line is a useful tool for the analysis of presynaptic nanoarchitectural dynamics, with a potential for wide adoption.

neuroscience↗

bioRxiv2024

A one-step protocol to generate impermeable fluorescent HaloTag substrates for in situ live cell application and super-resolution imaging

Communication between cells is largely orchestrated by proteins on the cell surface, which allow information transfer across the cell membrane. Super-resolution and single-molecule visualization of these proteins can be achieved by genetically grafting HTP (HaloTag Protein) into the protein of interest followed by brief incubation of cells with a dye-HTL (dye-linked HaloTag Ligand). This approach allows for use of cutting-edge fluorophores optimized for specific optical techniques or a cell-impermeable dye-HTL to selectively label surface proteins without labeling intracellular copies. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Traditional methods to eliminate cell permeability face synthetic bottlenecks and risk altering photophysical properties. Here we report that dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed. This simple, one-step method requires no purification and is compatible with both the original HTL and second-generation HTL.2, the latter offering accelerated labeling. We validate such compounds, termed dye-SHTL ( dye shuttle) conjugates, in live cells via widefield microscopy, demonstrating exclusive membrane staining of extracellular HTP fusion proteins. In transduced primary hippocampal neurons, we label mGluR2, a neuromodulatory G protein-coupled receptor (GPCR), with dyes optimized for stimulated emission by depletion (STED) super-resolution microscopy, allowing unprecedented accuracy in distinguishing surface and receptors from those in internal compartments of the presynaptic terminal, important in neural communication. This approach offers broad utility for surface-specific protein labelling.

pharmacology and toxicology↗

bioRxiv2024

Melanocortin system activates carotid body arterial chemoreceptors in hypertension

BackgroundThe bodys internal milieu is controlled by a system of interoceptors coupled to motor outflows that drive compensatory adaptive responses. These include the arterial chemoreceptors, best known for sensing arterial oxygen. In cardiometabolic diseases, such as essential hypertension, the carotid bodies (CB) exhibit heightened reflex sensitivity and tonic activity without an apparent stimulus. The mechanisms behind CB sensitization in these conditions are not well understood. MethodsGuided by functional genomics, a range of functional assays is used to interrogate downstream intracellular and interorgan signalling pathways involved in arterial chemosensory function. ResultsHere, we report the presence of the Melanocortin 4 receptor (MC4R) in the mammalian CB and show its elevated expression in experimental hypertension. We demonstrate that melanocortin agonists activate arterial chemosensory cells, modulating CB chemosensory afferent drive to influence both resting and chemoreflex-evoked sympathetic and ventilatory activity. Transcriptional analysis of hypertensive CB implicates the activation of the Mash1 (Ascl1) regulatory network in driving elevated Mc4r expression. ConclusionsCollectively, our data indicate a primarily pathophysiological role of melanocortin signalling in arterial chemosensation, contributing to excess sympathetic activity in cardiometabolic disease.

neuroscience↗

bioRxiv2024

Projection-Targeted Photopharmacology Reveals Distinct Anxiolytic Roles for Presynaptic mGluR2 in Prefrontal- and Insula-Amygdala Synapses

Dissecting how membrane receptors regulate neural circuit function is critical for deciphering basic principles of neuromodulation and mechanisms of therapeutic drug action. Classical pharmacological and genetic approaches are not well-equipped to untangle the roles of specific receptor populations, especially in long-range projections which coordinate communication between brain regions. Here we use viral tracing, electrophysiological, optogenetic, and photopharmacological approaches to determine how presynaptic metabotropic glutamate receptor 2 (mGluR2) activation in the basolateral amygdala (BLA) alters anxiety-related behavior. We find that mGluR2-expressing neurons from the ventromedial prefrontal cortex (vmPFC) and posterior insular cortex (pIC) preferentially target distinct cell types and subregions of the BLA to regulate different forms of avoidant behavior. Using projection-specific photopharmacological activation, we find that mGluR2-mediated presynaptic inhibition of vmPFC-BLA, but not pIC-BLA, connections can produce long-lasting decreases in spatial avoidance. In contrast, presynaptic inhibition of pIC-BLA connections decreased social avoidance, novelty-induced hypophagia, and increased exploratory behavior without impairing working memory, establishing this projection as a novel target for the treatment of anxiety disorders. Overall, this work reveals new aspects of BLA neuromodulation with therapeutic implications while establishing a powerful approach for optical mapping of drug action via photopharmacology. Highlights- Basolateral amygdala is a key site for anxiolytic action of mGluR2 agonism - BLA receives dense Grm2+ inputs from ventromedial prefrontal cortex and posterior insular cortex - Photoactivation of mGluR2 has distinct anxiolytic effects in vmPFC-BLA and pIC-BLA synapses - Grm2+ vmPFC and pIC projections target medial and lateral BLA subregions, respectively

neuroscience↗

bioRxiv2023

Deuteration provides a general strategy to enhance azobenzene-based photopharmacology

Herein, we present deuterated azobenzene photoswitches as a general means of enhancing photopharmacological molecules. Deuteration can improve azobenzene performance in terms of light sensitivity, photoswitch efficiency, and photoswitch kinetics with minimal alteration to the underlying structure of the photopharmacological ligand. We report synthesized deuterated azobenzene-based ligands for the optimized optical control of ion channel and G protein-coupled receptor function in live cells, setting the stage for the straightforward, widespread adoption of this approach.

biochemistry↗

bioRxiv2023

A neuronal circuit driven by GLP-1 in the olfactory bulb regulates insulin secretion

Glucagon-like peptide 1 (GLP-1) stimulates insulin secretion and holds significant pharmacological potential. Nevertheless, the regulation of energy homeostasis by centrally-produced GLP-1 remains partially understood. Preproglucagon cells, known to release GLP-1, are found in the olfactory bulb (OB). We demonstrate that activating GLP-1 receptors (GLP-1R) in the OB stimulates insulin secretion in response to oral glucose in lean and diet-induced obese mice. This is associated with reduced noradrenaline content in the pancreas and blocked by an 2-adrenergic receptor agonist, highlighting the functional implication of the sympathetic nervous system (SNS). Inhibiting GABAA receptors in the paraventricular nucleus of the hypothalamus (PVN), the control centre of the SNS, abolishes the enhancing effect on insulin secretion induced by OB GLP-1R. Therefore, OB GLP-1-dependent regulation of insulin secretion relies on a relay within the PVN. These findings identify a novel top-down neural mechanism engaged by OB GLP-1 signaling to control insulin secretion via the SNS.

physiology↗

bioRxiv2023

Direct optical measurement of intra-molecular distances down to the Angstrom scale

Optical investigations of nanometer distances between proteins, their subunits, or other biomolecules have been the exclusive prerogative of Forster Resonance Energy Transfer (FRET) microscopy for decades. Here we show that MINFLUX fluorescence nanoscopy measures intra-molecular distances down to 1 nm - and in planar projections down to 1 Angstrom - directly, linearly, and with Angstrom precision. Our method is validated by quantifying well-characterized 1-to-10 nm distances in polypeptides and proteins. Moreover, we visualize the orientations of immunoglobulin subunits and reveal specific configurations of a histidine kinase domain dimer. Our results open the door for examining proximities and interactions of macromolecules under physiological conditions.

biophysics↗

bioRxiv2022

Real-time library search increases cross-link identification depth across all levels of sample complexity

Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition. Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.

systems biology↗