Biology subjects

Hartl, F.-U.

Publications and source records attributed to Hartl, F.-U..

11 recordsLinked to original sources

bioRxiv2026

Q-DOAS: A Proximity Quenching Assay for Real-Time Detection of Early Protein Aggregation Events

Accurate measurement of protein aggregation is essential for studying neurodegenerative diseases. The standard ThT assay reports on amyloid formation but is blind to early oligomers and is prone to interference. We describe Q-DOAS, a plate-reader assay that quantifies protein self-assembly in real time via proximity-quenching of a single, site-specifically conjugated dye (BODIPY-TMR). Using mutant Huntingtin-exon 1 (mHTT-Ex1) and -Synuclein A53T, we show Q-DOAS detects pre-amyloid oligomers, yielding quantitative kinetic data compatible with mechanistic analysis. We demonstrate its utility to dissect mutational effects, screen for protein and small-molecule inhibitors, and quantify amyloid seeding activity in cellular and mouse models of Huntingtons disease. Q-DOAS also detects seeds in cerebrospinal fluid from Parkinsons disease patients without amplification. Q-DOAS provides a sensitive, robust, and scalable tool for studying the earliest events in amyloid pathologies and for advancing therapeutic development.

biochemistry↗

bioRxiv2025

Tryptoline Stereoprobe Elaboration Identifies Inhibitors of the GRPEL1-HSPA9 Chaperone Complex

Activity-based protein profiling has identified hundreds of proteins from diverse classes that react at specific cysteine residues with stereochemically defined electrophilic compounds (stereoprobes) in human cells. The structure-activity relationships underlying these stereoprobe-protein interactions, however, remain poorly understood. Here we show that the protein interaction landscape of tryptoline acrylamide stereoprobes can be profoundly altered by structural modifications distal to the acrylamide reactive group. The majority of stereoprobe liganding events occurred at non-orthosteric sites and mostly evaded assignment by the machine learning-based co-folding model Boltz-2, which instead tended to misplace the stereoprobes in orthosteric pockets (an outcome we term "orthostery burnout"). We found that stereoprobes reacting with C124 in the nucleotide exchange factor GRPEL1 disrupt interactions with the mitochondrial HSP70 chaperone HSPA9/mortalin, leading to impairments in mitochondrial protein import and induction of mitophagy. Our results highlight tryptoline acrylamides as a versatile source of covalent ligands targeting non-orthosteric sites on proteins, including tool compounds that perturb the mitochondrial HSP70 chaperone system.

biochemistry↗

bioRxiv2025

Mitochondrial proteostatic stress disrupts mitoribosome biogenesis and translation

Protein aggregation in various cellular compartments is a hallmark of proteostasis impairment linked to aging and numerous pathologies. Mitochondrial function depends on a balanced interplay of proteins imported from the cytosol as well as those synthesized on mitochondrial ribosomes (mitoribosomes). Here, we reveal an unexpected susceptibility of mitoribosome biogenesis to organellar proteostatic stress. Importing aggregation-prone proteins into yeast and human mitochondria triggered a chain of detrimental events involving extensive co-aggregation of newly-imported mitoribosome subunits and other RNA-binding proteins, as well as local disruption of mitochondrial cristae morphology. As a result, mitoribosome assembly and mitochondrial translation were severely impaired, leading to respiratory deficiency and, ultimately, loss of mitochondrial DNA. Surprisingly, dysfunction of mitochondrial HSP60 phenocopied the ribosome biogenesis defect and inhibition of translation, indicating a pronounced chaperone dependence of mitoribosome proteins. Declining mitochondrial translation likely contributes to aging and diseases associated with deficiencies in mitochondrial protein quality control machinery.

cell biology↗

bioRxiv2024

Hydrophobic tails enable diverse functions of the extracellular chaperone clusterin

Clusterin, a conserved secretory glycoprotein abundant in blood plasma and cerebrospinal fluid, functions as a molecular chaperone and apolipoprotein (Wyatt et al. 2013, Raulin et al. 2022). Dysregulation of clusterin is linked to late-onset Alzheimers disease, cardiovascular pathology and cancer (Rohne et al. 2016, Satapathy and Wilson 2021, Wilson et al. 2023). Despite its prominent role in extracellular proteostasis, the chaperone mechanism of clusterin has remained unclear. Here we present crystal structures of human clusterin, revealing a discontinuous three-domain architecture. Structure-based mutational analysis demonstrated that two intrinsically disordered, hydrophobic peptide tails enable diverse clusterin activities. Resembling the N-terminal substrate binding regions of so-called small heat shock proteins, these sequences mediate clusterins chaperone function in suppressing amyloid-{beta}, tau and -synuclein aggregation. In conjunction with highly conserved surface areas, the tail segments also participate in clusterin binding to very low density lipoprotein receptor (VLDLR) and cellular uptake. Moreover, the disordered tails cooperate with an adjacent amphipathic helix in lipoprotein formation, but remain accessible for chaperone function in the lipoprotein complex. The remarkable versatility of these sequences allows clusterin to function alone or bound to lipid in maintaining solubility of aberrant extracellular proteins and facilitating their clearance by endocytosis and lysosomal degradation.

molecular biology↗

bioRxiv2023

Autophagy preferentially degrades non-fibrillar polyQ aggregates

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntingtons disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

cell biology↗

bioRxiv2023

Single-molecule digital sizing of proteins in solution

Proteins constitute the molecular machinery of life and exert their biological function by interacting with other proteins, as well as by assembling into biomolecular complexes and higher order structures. Characterizing the sizes, interactions, and assembly states of proteins is thus key for understanding the normal functional behavior of proteins and for elucidating aberrant processes and interactions that can lead to dysfunction and disease. However, the physical characterization of proteins has remained a challenging problem due to the inherent compositional heterogeneity of protein mixtures as well as the polydisperse nature of protein complexes. Here, we address this challenge by demonstrating measurements of molecular diffusivity of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. The approach, termed single-molecule microfluidic diffusional sizing (smMDS), allows individual molecules to be counted directly, that is, in a digital manner, to enable calibration-free single-molecule diffusional-sizing-based monitoring of protein hydrodynamic radii even within heterogenous multicomponent mixtures. Applying smMDS to a variety of protein systems, we show that the high sensitivity provided by smMDS enables ultrasensitive sizing of proteins down to the femtomolar concentration range. We further demonstrate the applicability of the approach towards affinity profiling of protein interactions at the single-molecule level and illustrate the potential of smMDS in resolving different assembly states of high- and low-molecular weight protein oligomers. Furthermore, we highlight the digital nature of the detection process by sizing multiple protein species within complex aggregation mixtures. Finally, we apply the approach to characterize nanoscale clusters of a phase separating protein system. Taken together, smMDS constitutes a versatile approach for digital, in-solution characterization of the sizes, interactions, and assembly states of proteins. We anticipate that smMDS will facilitate the discovery of new biomolecular mechanisms of proteins and will find broad applicability in the analysis of protein complexes in the biological, biophysical, and biomedical sciences, and beyond.

biophysics↗

bioRxiv2023

NEMO reshapes the protein aggregate interface and promotes aggrephagy by co-condensation with p62

NEMO is a ubiquitin-binding protein which regulates canonical NF-{kappa}B pathway activation in innate immune signaling, cell death regulation and host-pathogen interactions. Here we identified an NF-{kappa}B-independent function of NEMO in proteostasis regulation by promoting autophagosomal clearance of protein aggregates. NEMO-deficient cells accumulate misfolded proteins upon proteotoxic stress and are vulnerable to proteostasis challenges. Moreover, a patient with a mutation in the NEMO gene resulting in defective binding of NEMO to linear ubiquitin chains, developed a widespread mixed brain proteinopathy, including -synuclein, tau and TDP-43 pathology. NEMO amplifies linear ubiquitylation at -synuclein aggregates and promotes the local concentration of p62 into foci. In vitro, NEMO lowers the threshold concentrations required for ubiquitin-dependent phase transition of p62. In summary, NEMO reshapes the aggregate surface for efficient autophagosomal clearance by providing a mobile phase at the aggregate interphase favoring co-condensation with p62.

cell biology↗

bioRxiv2022

Resolving chaperone-assisted protein folding on the ribosome at the peptide level

The cellular environment is critical for efficient protein maturation, but how proteins fold during biogenesis remains poorly understood. We used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. On the ribosome, the nascent polypeptide folds via structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing C-terminal sequences remain confined in the ribosome exit tunnel. We find that partially-folded nascent chains recruit the chaperone Trigger factor, which uses a large composite hydrophobic/hydrophilic interface to engage folding intermediates without disrupting their structure. In addition, we comprehensively mapped dynamic interactions between the nascent chain and ribosomal proteins, tracing the path of the emerging polypeptide during synthesis. Our work provides a high-resolution description of de novo protein folding dynamics, thereby revealing new mechanisms by which cellular factors shape the conformational search for the native state.

biochemistry↗

bioRxiv2022

The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds

Amyloid-like aggregates of the microtubule-associated protein Tau are associated with several neurodegenerative disorders including Alzheimers disease. The existence of cellular machinery for the removal of such aggregates has remained unclear, as specialized disaggregase chaperones are thought to be absent in mammalian cells. Here we show in cell culture and in neurons that the AAA+ chaperone VCP is recruited to ubiquitylated Tau fibrils, resulting in their efficient disaggregation. Aggregate clearance depends on the functional cooperation of VCP with Hsp70 and the ubiquitin-proteasome machinery. Inhibition of VCP activity stabilizes large Tau aggregates, and is accompanied by a reduction in the amount of Tau species competent of prion- like aggregate seeding in recipient cells. Thus, disaggregation by VCP generates seeding-active Tau as byproduct. These findings identify VCP as a core component of the machinery for the removal of neurodegenerative disease aggregates and suggest that its activity can be associated with enhanced aggregate spreading in tauopathies.

cell biology↗

bioRxiv2020

The Hsc70 Disaggregation Machinery RemovesMonomer Units Directly from α-Synuclein FibrilEnds

Molecular chaperones contribute to the maintenance of cellular protein homeostasis through a wide range of mechanisms, including the assistance of de novo protein folding, the rescue of misfolded proteins, and the prevention of amyloid formation. Chaperones of the Hsp70 family have a striking capability of disaggregating otherwise irreversible aggregate structures such as amyloid fibrils that accumulate during the development of neurodegenerative diseases. However, the mechanisms of this key emerging functionality remain largely unknown. Here, we bring together microfluidic measurements with kinetic analysis and show that that the Hsp70 protein heat chock complement Hsc70 together with its two co-chaperones DnaJB1 and the nucleotide exchange factor Apg2 is able to completely reverse the aggregation process of alpha-synuclein, associated with Parkinsons disease, back to its soluble monomeric state. Moreover, we show that this reaction proceeds with first order kinetics in a process where monomer units are taken off directly from the fibril ends. Our results demonstrate that all components of the chaperone triad are essential for fibril disaggregation. Lastly, we quantify the interactions between the three chaperones as well as between the chaperones and the fibrils in solution, yielding both binding stoichiometries and dissociation constants. Crucially, we find that the stoichiometry of Hsc70 binding to fibrils suggests Hsc70 clustering at the fibril ends. Taken together, our results show that the mechanism of action of the Hsc70-DnaJB1-Apg2 chaperone system in disaggregating -synuclein fibrils involves the removal of monomer units without any intermediate fragmentation steps. These findings are fundamental to our understanding of the suppression of amyloid proliferation early in life and the natural clearance mechanisms of fibrillar deposits in Parkinsons disease, and inform on the possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies and related neurodegenerative diseases.

biophysics↗

bioRxiv2020

Multiple pathways of toxicity induced by C9orf72 dipeptide repeat aggregates and G4C2 RNA in a cellular model

The most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia is a G4C2 repeat expansion in the C9orf72 gene. This expansion gives rise to translation of aggregating dipeptide repeat (DPR) proteins, including poly-GA as the most abundant species. However, gain of toxic function effects have been attributed to either the DPRs or the pathological G4C2 RNA. Here we analyzed in a cellular model the relative toxicity of DPRs and RNA. Cytoplasmic poly-GA aggregates, generated in the absence of G4C2 RNA, interfered with nucleocytoplasmic protein transport, but had little effect on cell viability. In contrast, nuclear poly-GA was more toxic, impairing nucleolar protein quality control and protein biosynthesis. Production of the G4C2 RNA strongly reduced viability independent of DPR translation and caused pronounced inhibition of nuclear mRNA export and protein biogenesis. Thus, while the toxic effects of G4C2 RNA predominate, DPRs exert additive effects that may contribute to pathology.

biochemistry↗