Biology subjects

Mogk, A.

Publications and source records attributed to Mogk, A..

9 recordsLinked to original sources

bioRxiv2026

Small-molecule activators of the Staphylococcus aureus ClpC/ClpP AAA+ protease

Summary/AbstractThe central AAA+ ClpC/ClpP protease in Gram-positive bacteria is crucial for virulence and stress resistance and has been recognized as drug target. Natural cyclic peptides deregulate the essential Mycobacterium tuberculosis ClpC1 and cause cell death. Similarly, overactivated mutants of the non-essential Staphylococcus aureus ClpC homologue cause uncontrolled proteolysis and severe toxicity in vivo. However, no chemical modulators of S. aureus ClpC have been described. Here, using a biochemical high-throughput screen we identify eight chemically distinct bona fide small molecules that robustly stimulate ClpC ATPase and proteolytic activity in vitro. Structural, computational, and mutational analyses define two ligandable regulatory sites within the ClpC N-terminal domain (NTD) as compound targets: a conserved hydrophobic groove and an allosteric pArg1 pocket, both engaged in substrate recognition. These findings establish S. aureus ClpC as chemically targetable and provide mechanistic insight into its regulatory architecture, enabling future development and optimization of chemical probes to deregulate AAA+ protease control.

biochemistry↗

bioRxiv2025

Structure of the central Staphylococcus aureus AAA+ protease MecA/ClpC/ClpP

Bacterial AAA+ proteases are composed of a AAA+ partner (e.g. ClpC) and an associated peptidase (e.g. ClpP). They represent ATP-fuelled and self-compartmentalized proteolytic machines that are crucial for stress resistance and virulence. ClpC requires cooperation with adaptor proteins such as MecA for activation and complex formation with ClpP. Here, we present the cryo-EM structure of the MecA/ClpC/ClpP complex from the major pathogen Staphylococcus aureus. MecA forms a dynamic crown on top of the ClpC/ClpP complex with its substrate-binding domain positioned near the ClpC pore site, likely facilitating substrate transfer. ClpC/ClpP complex formation involves ClpC P-loops and ClpP N-terminal {beta}-hairpins, which insert into the central ClpC threading channel and contact sites next to the ClpC ATPase center. ClpC and ClpP interactions are asymmetric and dictated by the activity states of ClpC ATPase subunits. ClpP binding increases ClpC ATPase and threading activities in a {beta}-hairpin dependent manner, illuminating an allosteric pathway in the cooperation of ATPase and peptidase components in bacterial AAA+ proteases.

biochemistry↗

bioRxiv2025

Nuclear and cytosolic J-domain proteins provide synergistic control of Hsf1 at distinct phases of the heat shock response

The heat shock response (HSR) is the major defense mechanism against proteotoxic stress in the cytosol and nucleus of eukaryotic cells. Initiation and attenuation of the response are mediated by stress-dependent regulation of heat shock transcription factors (HSFs). Saccharomyces cerevisiae encodes a single HSF (Hsf1), facilitating the analysis of HSR regulation. Hsf1 is repressed by Hsp70 chaperones under non-stress conditions, and becomes activated under proteotoxic stress, directly linking protein damage and its repair to the HSR. J-domain proteins (JDPs) are essential for targeting of Hsp70s to their substrates, yet the specific JDP(s) regulating Hsf1 and connecting protein damage to HSR activation remain unclear. Here we show that the yeast nuclear JDP Apj1 primarily controls the attenuation phase of the HSR by promoting Hsf1s displacement from heat shock elements in target DNA. In apj1{Delta} cells, HSR attenuation is significantly impaired. Additionally, yeast cells lacking both Apj1 and the major JDP Ydj1 exhibit increased HSR activation even in non-stress conditions, indicating their distinct regulatory roles. Apj1s role in both nuclear protein quality control and Hsf1 regulation underscores its role in directly linking nuclear proteostasis to HSR regulation. Together these findings establish the nucleus as key stress-sensing signaling hub.

molecular biology↗

bioRxiv2024

Reprograming of the ubiquitin ligase Ubr1 by intrinsically disordered Roq1 through cooperating multifunctional motifs

One way cells control the speed and specificity of protein degradation is by regulating the activity of ubiquitin ligases. Upon proteotoxic stress in yeast, the intrinsically disordered protein Roq1 binds the ubiquitin ligase Ubr1 as a pseudosubstrate, thereby modulating the degradation of substrates of the N-degron pathway and promoting the elimination of misfolded proteins. The mechanism underlying this reprograming of Ubr1 is unknown. Here, we show that Roq1 controls Ubr1 by means of two cooperating multifunctional motifs. The N-terminal arginine and a short hydrophobic motif of Roq1 interact with Ubr1 as part of a heterobivalent binding mechanism. Via its N-terminal arginine, Roq1 regulates the ubiquitination of various N-degron substrates and folded proteins. Via its hydrophobic motif, Roq1 accelerates the ubiquitination of misfolded proteins. These findings reveal how a small, intrinsically disordered protein with a simple architecture engages parallel channels of communication to reprogram a functionally complex ubiquitin ligase.

biochemistry↗

bioRxiv2023

The Listeria monocytogenes persistence factor ClpL is a potent stand-alone disaggregase

Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during e.g. food-processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.

biochemistry↗

bioRxiv2023

Structural basis of aggregate binding by the AAA+ disaggregase ClpG

Severe heat stress causes massive loss of essential proteins by aggregation necessitating a cellular activity that rescues aggregated proteins. This activity is executed by ATP-dependent, ring-forming, hexameric AAA+ disaggregases. Little is known about the recognition principles of stress-induced protein aggregates. How can disaggregases specifically target aggregated proteins while avoiding binding to soluble non-native proteins? Here, we determined by NMR spectroscopy the core structure of the aggregate targeting N1 domain of the bacterial AAA+ disaggregase ClpG, which confers extreme heat resistance to bacteria. N1 harbors a Zn2+-coordination site that is crucial for structural integrity and disaggregase functionality. Conserved hydrophobic N1 residues located on a {beta}-strand were found crucial for aggregate targeting and disaggregation activity. Mixing experiments with N1-truncated AAA+ hexamers revealed that a minimal number of four N1 domains must be present in a AAA+ ring for high disaggregation activity. We suggest that multiple N1 domains increase substrate affinity through avidity effects. These findings define the recognition principle of a protein aggregate by a disaggregase, involving simultaneous contacts with multiple hydrophobic substrate patches located in close vicinity on an aggregate surface. This binding mode ensures selectivity for aggregated proteins while sparing soluble, non-native protein structures from disaggregase activity.

biochemistry↗

bioRxiv2019

Disassembly of Tau fibrils by the human Hsp70 disaggregation machinery generates small seeding-competent species

The accumulation of amyloid Tau aggregates is implicated in Alzheimers disease and other Tauopathies. Molecular chaperones are known for their function in maintaining protein homeostasis by preventing the formation or promoting the disaggregation of amorphous and amyloid protein aggregates. Here we show that an ATP-dependent human chaperone system disassembles Tau fibrils in vitro. This function is mediated by the core chaperone Hsc70, assisted by specific co-chaperones, in particular class B J-domain proteins and an Hsp110-type NEF. Recombinant fibrils assembled from all six Tau isoforms as well as Sarkosyl-resistant Tau aggregates extracted from cell culture were processed by the Hsp70 disaggregation machinery, demonstrating the ability of this machinery to recognize a broad range of Tau aggregates. Chaperone treatment released monomeric, and small oligomeric Tau species, which induced the aggregation of self-propagating Tau species in a Tau cell culture model. We infer from these results that the activity of the Hsp70 disaggregation machinery is a double-sided sword as it attempts to eliminate Tau amyloids but with the price of generating new seeds. The Hsp70 disaggregase therefore has a crucial function in the Tau propagation cycle, rendering it a potential drug target in Tauopathies.

molecular biology↗

bioRxiv2019

HSP110 dependent HSP70 disaggregation machinery mediates prion-like propagation of amyloidogenic proteins in metazoa

The gradual accumulation and prion-like propagation of -synuclein and other amyloidogenic proteins is associated with devastating neurodegenerative diseases. The metazoan disaggregation machinery, a specific combination of HSP70 and its co-chaperones, is able to disassemble -synuclein fibrils in vitro, but the physiological consequence in vivo is unknown. To explore this, we used Caenorhabditis elegans models that exhibit pathological features of -synuclein, such as misfolding, intercellular spreading and toxicity. We inhibited the HSP70 disaggregase by depleting the crucial component HSP-110 and monitored the effect on -synuclein related phenotypes. The knockdown of HSP-110 not only impaired HSP70 disaggregation activity and prevented the resolubilization of amorphous heat shock induced firefly luciferase aggregates, but also compromised the cellular folding capacity. In stark contrast, HSP-110 depletion reduced -synuclein foci formation, cell-to-cell transmission and toxicity. Similar effects were observed for a polyQ model substrate, confirming that inhibition of HSP70 disaggregation function mitigates amyloid toxicity. These data demonstrate that the metazoan HSP70 disaggregation complex plays a critical role in the prion-like propagation of amyloid-type conformers. Therefore, the HSP70 disaggregation activity is a double-edged sword as it is essential for the maintenance of cellular proteostasis while being involved in the generation of toxic amyloid-type protein species.

cell biology↗

bioRxiv2017

Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control

Ring-forming AAA+ chaperones exert ATP-fueled substrate unfolding by threading through a central pore. This activity is potentially harmful requiring mechanisms for tight repression and substrate-specific activation. The AAA+ chaperone ClpC with the peptidase ClpP forms a bacterial protease essential to virulence and stress resistance. The adaptor MecA activates ClpC by targeting substrates and stimulating ClpC ATPase activity. We show how ClpC is repressed in its ground state by determining ClpC cryo-EM structures with and without MecA. ClpC forms large two-helical assemblies that associate via head-to-head contacts between coiled-coil middle domains (MDs). MecA converts this resting state to an active planar ring structure by binding to MD interaction sites. Loss of ClpC repression in MD mutants causes constitutive activation and severe cellular toxicity. These findings unravel an unexpected regulatory concept executed by coiled-coil MDs to tightly control AAA+ chaperone activity.

biochemistry↗