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Axel Mogk

Publications and source records attributed to Axel Mogk.

10 recordsLinked to original sources
PubMed2025

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 Hsf1's displacement from heat shock elements in target DNA. In apj1Δ 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. Apj1's 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.

Saccharomyces cerevisiae Proteins↗
PubMed2003

Poly-L-lysine enhances the protein disaggregation activity of ClpB.

The Hsp100 protein ClpB is a member of the AAA+ protein family that mediates the solubilization of aggregated proteins in cooperation with the DnaK chaperone system. Unstructured polypeptides such as casein or poly-L-lysine have been shown to stimulate the ATPase activity of ClpB and thus may both act as substrates. Here we compared the effects of alpha-casein and poly-L-lysine on the ATPase and chaperone activities of ClpB. alpha-Casein stimulated ATP hydrolysis by both AAA domains of ClpB and inhibited the ClpB-dependent solubilization of aggregated proteins if present in excess. In contrast, poly-L-lysine stimulated exclusively the ATPase activity of the second AAA domain and increased the disaggregation activity of ClpB. Thus poly-L-lysine does not act as substrate, but rather represents an effector molecule, which enhances the chaperone activity of ClpB.

Adenosine Triphosphatases↗
PubMed2003

Characterization of a trap mutant of the AAA+ chaperone ClpB.

The AAA+ protein ClpB mediates the solubilization of protein aggregates in cooperation with the DnaK chaperone system (KJE). The order of action of ClpB and KJE on aggregated proteins is unknown. We describe a ClpB variant with mutational alterations in the Walker B motif of both AAA domains (E279A/E678A), which binds but does not hydrolyze ATP. This variant associates in vitro and in vivo in a stable manner with protein substrates, demonstrating direct interaction of ClpB with protein aggregates for the first time. Substrate interaction is strictly dependent on ATP binding to both AAA domains of ClpB. The unique substrate binding properties of the double Walker B variant allowed to dissect the order of ClpB and DnaK action during disaggregation reactions. ClpB-E279A/E678A outcompetes the DnaK system for binding to the model substrate TrfA and inhibits the dissociation of small protein aggregates by DnaK only, indicating that ClpB acts prior to DnaK on protein substrates.

Adenosine Triphosphatases↗
PubMed2003

Refolding of substrates bound to small Hsps relies on a disaggregation reaction mediated most efficiently by ClpB/DnaK.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that bind denatured proteins in vitro, thereby facilitating their subsequent refolding by ATP-dependent chaperones. The mechanistic basis of this refolding process is poorly defined. We demonstrate that substrates complexed to sHsps from various sources are not released spontaneously. Dissociation and refolding of sHsp bound substrates relies on a disaggregation reaction mediated by the DnaK system, or, more efficiently, by ClpB/DnaK. While the DnaK system alone works for small, soluble sHsp/substrate complexes, ClpB/DnaK-mediated protein refolding is fastest for large, insoluble protein aggregates with incorporated sHsps. Such conditions reflect the situation in vivo, where sHsps are usually associated with insoluble proteins during heat stress. We therefore propose that sHsp function in cellular protein quality control is to promote rapid resolubilization of aggregated proteins, formed upon severe heat stress, by DnaK or ClpB/DnaK.

Animals↗
PubMed2003

Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization, ATP hydrolysis, and chaperone activity.

ClpB of Escherichia coli is an ATP-dependent ring-forming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the Hsp100/Clp subfamily of AAA+ proteins and is composed of an N-terminal domain and two AAA-domains that are separated by a "linker" region. Here we present a detailed structure-function analysis of ClpB, dissecting the individual roles of ClpB domains and conserved motifs in oligomerization, ATP hydrolysis, and chaperone activity. Our results show that ClpB oligomerization is strictly dependent on the presence of the C-terminal domain of the second AAA-domain, while ATP binding to the first AAA-domains stabilized the ClpB oligomer. Analysis of mutants of conserved residues in Walker A and B and sensor 2 motifs revealed that both AAA-domains contribute to the basal ATPase activity of ClpB and communicate in a complex manner. Chaperone activity strictly depends on ClpB oligomerization and the presence of a residual ATPase activity. The N-domain is dispensable for oligomerization and for the disaggregating activity in vitro and in vivo. In contrast the presence of the linker region, although not involved in oligomerization, is essential for ClpB chaperone activity.

Adenosine Triphosphatases↗
PubMed2003

MecA, an adaptor protein necessary for ClpC chaperone activity.

ClpC of Bacillus subtilis is an ATP-dependent HSP100Clp protein involved in general stress survival. A complex of ClpC with the protease ClpP and the adaptor protein MecA also controls competence development by regulated proteolysis of the transcription factor ComK. We investigated the in vitro chaperone activity of ClpC and found that the presence of MecA was crucial for the major chaperone activities of ClpC. In particular, MecA enabled ClpC to solubilize and refold aggregated proteins. Finally, in the presence of ClpP, MecA allowed the ClpC-dependent degradation of unfolded or heat-aggregated proteins. This study demonstrates that adaptor proteins like MecA through interaction with their cognate ClpC proteins can have a dual role in the protein quality-control network by rescuing, or together with ClpP, by degrading, aggregated proteins. MecA can thereby coordinate substrate targeting with ClpC activation, adding another layer to the regulation of HSP100/Clp protein activity.

Adenosine Triphosphatases↗
PubMed2003

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Chaperonin 60↗
PubMed2002

AAA+ proteins and substrate recognition, it all depends on their partner in crime.

Members of the AAA+ superfamily have been identified in all organisms studied to date. They are involved in a wide range of cellular events. In bacteria, representatives of this superfamily are involved in functions as diverse as transcription and protein degradation and play an important role in the protein quality control network. Often they employ a common mechanism to mediate an ATP-dependent unfolding/disassembly of protein-protein or DNA-protein complexes. In an increasing number of examples it appears that the activities of these AAA+ proteins may be modulated by a group of otherwise unrelated proteins, called adaptor proteins. These usually small proteins specifically modify the substrate recognition of their AAA+ partner protein. The occurrence of such adaptor proteins are widespread; representatives have been identified not only in Escherichia coli but also in Bacillus subtilis, not to mention yeast and other eukaryotic organisms. Interestingly, from the currently known examples, it appears that the N domain of AAA+ proteins (the most divergent region of the protein within the family) provides a common platform for the recognition of these diverse adaptor proteins. Finally, the use of adaptor proteins to modulate AAA+ activity is, in some cases, an elegant way to redirect the activity of an AAA+ protein towards a particular substrate without necessarily affecting other activities of that AAA+ protein while, in other cases, the adaptor protein triggers a complete switch in AAA+ activity.

ATP-Dependent Proteases↗
PubMed2002

Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implications for their applicability in biotechnology.

The amount of a native protein reflects an equilibrium of protein synthesis, de novo folding and protein stability. Stress situations, like heat shock, or overproduction of a protein can cause an imbalance in this equilibrium, resulting in protein aggregation. Molecular chaperones control protein folding processes and protect misfolded proteins from aggregation in all cells. Since protein aggregation is frequently observed upon synthesis of heterologous proteins in E. coli, molecular chaperones have been applied in biotechnology by their co-overproduction with the desired protein. While increasing protein solubility in some cases, this approach has not been generally successful. Recent findings demonstrate, that protein aggregation, even in case of inclusion bodies, must not be a dead end in the life cycle of a protein. Such resolubilization of aggregated proteins is mediated by a bi-chaperone system consisting of ClpB and DnaK, the prokaryotic representatives of the Hsp100 and Hsp70 families. The disaggregation capacity of this bi-chaperone system has now been demonstrated in vitro and in vivo for a wide variety of aggregated proteins and offers a new perspective to increase the solubility of proteins of interest.

Biotechnology↗