The production of newly synthesized proteins is a key process of protein homeostasis that initiat... more The production of newly synthesized proteins is a key process of protein homeostasis that initiates the biosynthetic flux of proteins and thereby determines the composition, stability and functionality of the proteome. Protein synthesis is highly regulated on multiple levels to adapt the proteome to environmental and physiological challenges such as aging and proteotoxic conditions. Imbalances of protein folding conditions are sensed by the cell that then trigger a cascade of signaling pathways aiming to restore the protein folding equilibrium. One regulatory node to rebalance proteostasis upon stress is the control of protein synthesis itself. Translation is reduced as an immediate response to perturbations of the protein folding equilibrium that can be observed in the cytosol as well as in the organelles such as the endoplasmatic reticulum and mitochondria. As reduction of protein synthesis is linked to life span increase, the signaling pathways regulating
protein synthesis might be putative targets for treatments of age-related diseases. Eukaryotic cells have evolved acomplex system for protein synthesis regulation and this review will summarize cellular strategies to regulate mRNA
translation upon stress and its impact on longevity.
Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different p... more Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different proteolytic machines, including ClpAP. The ClpA component is a hexameric AAA+ (ATPase associated with various cellular activities) chaperone that utilizes the energy of ATP to control substrate recognition and unfolding. The precise role of the N-domains of ClpA in this process, however, remains elusive. Here, we have analysed the role of five highly conserved basic residues in the N-domain of ClpA by monitoring the binding, unfolding and degradation of several different substrates, including short unstructured peptides, tagged and untagged proteins. Interestingly, mutation of three of these basic residues within the N-domain of ClpA (H94, R86 and R100) did not alter substrate degradation. In contrast mutation of two conserved arginine residues (R90 and R131), flanking a putative peptide-binding groove within the N-domain of ClpA, specifically compromised the ability of ClpA to unfold and degrade selected substrates but did not prevent substrate recognition, ClpS-mediated substrate delivery or ClpP binding. In contrast, a highly conserved tyrosine residue lining the central pore of the ClpA hexamer was essential for the degradation of all substrate types analysed, including both folded and unstructured proteins. Taken together, these data suggest that ClpA utilizes two structural elements, one in the N-domain and the other in the pore of the hexamer, both of which are required for efficient unfolding of some protein substrates.
The mitochondrial matrix of mammalian cells contains several different ATP-dependent proteases, i... more The mitochondrial matrix of mammalian cells contains several different ATP-dependent proteases, including CLPXP, some of which contribute to protein maturation and quality control. Currently however, the substrates and the physiological roles of mitochondrial CLPXP in humans, has remained elusive. Similarly, the mechanism by which these ATP-dependent proteases recognize their substrates currently remains unclear. Here we report the characterization of a Walker B mutation in human CLPX, in which the highly conserved glutamate was replaced with alanine. This mutant protein exhibits improved interaction with the model unfolded substrate casein and several putative physiological substrates in vitro. Although this mutant lacks ATPase activity, it retains the ability to mediate casein degradation by hCLPP, in a fashion similar to the small molecule ClpP-activator, ADEP. Our functional dissection of hCLPX structure, also identified that most model substrates are recognized by the N-terminal domain, although some substrates bypass this step and dock, directly to the pore-1 motif. Collectively these data reveal, that despite the difference between bacterial and human CLPXP complexes, human CLPXP exhibits a similar mode of substrate recognition and is deregulated by ADEPs.
The cellular proteostasis network integrates the protein folding and clearance machineries in mul... more The cellular proteostasis network integrates the protein folding and clearance machineries in multiple sub-cellular compartments of the eukaryotic cell. The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. A distinctive feature of the ER is its tightly controlled redox homeostasis necessary for the formation of inter- and intra-molecular disulphide bonds. Employing genetically encoded in vivo sensors reporting on the redox state in an organelle-specific manner, we show in the nematode Caenorhabditis elegans that the redox state of the ER is subject to profound changes during worm lifetime. In young animals, the ER is oxidizing and this shifts towards reducing conditions during ageing, whereas in the cytosol the redox state becomes more oxidizing with age. Likewise, the redox state in the cytosol and the ER change in an opposing manner in response to proteotoxic challenges in C. elegans and in HeLa cells revealing conservation of red...
Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathop... more Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are f...
Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly un... more Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly under stress conditions. Calibrated Western blot analyses were used to estimate the approximate numbers of heat-inducible Clp molecules within a single cell. According to these numbers, the different Clp ATPases do not seem to compete for the proteolytic subunit ClpP. Coimmunoprecipitation experiments revealed the predicted specific ClpX-ClpP, ClpC-ClpP, and ClpE-ClpP interactions. ClpE and ClpX are rapidly degraded in wild-type cells during permanent heat stress but remained almost stable in a clpP mutant, suggesting ClpP-dependent degradation. In particular, ClpCP appeared to be involved in the degradation of the short-lived ClpE ATPase, indicating a negative "autoregulatory" circuit for this particular Clp ATPase at the posttranslational level. Analysis of the half-life of stress-inducible clp mRNAs during exponential growth and heat shock revealed precise regulation of the sy...
In Bacillus subtilis, the alternative sigma factor sigma(B) is activated in response to environme... more In Bacillus subtilis, the alternative sigma factor sigma(B) is activated in response to environmental stress or energy depletion. The general stress regulon under the control of sigma(B) provides the cell with multiple stress resistance. Experiments were designed to determine how activated sigma(B) replaces sigma(A) as a constituent of the RNA polymerase holoenzyme. Studies of the transcription of the sigma(A)-dependent stress gene clpE under sigma(B)-inducing conditions showed that expression was higher in a sigB mutant background than in the wild type. The relative affinities of sigma(A) and sigma(B) for binding to the core RNA polymerase (E) were determined by means of indirect surface plasmon resonance. The results showed that the affinity of sigma(B) for E was 60-fold lower than that of sigma(A). Western blot analyses with antibodies against sigma(A), sigma(B), and E showed that, after exposure to ethanol stress, the concentration of sigma(B) was only twofold higher than those ...
The production of newly synthesized proteins is a key process of protein homeostasis that initiat... more The production of newly synthesized proteins is a key process of protein homeostasis that initiates the biosynthetic flux of proteins and thereby determines the composition, stability and functionality of the proteome. Protein synthesis is highly regulated on multiple levels to adapt the proteome to environmental and physiological challenges such as aging and proteotoxic conditions. Imbalances of protein folding conditions are sensed by the cell that then trigger a cascade of signaling pathways aiming to restore the protein folding equilibrium. One regulatory node to rebalance proteostasis upon stress is the control of protein synthesis itself. Translation is reduced as an immediate response to perturbations of the protein folding equilibrium that can be observed in the cytosol as well as in the organelles such as the endoplasmatic reticulum and mitochondria. As reduction of protein synthesis is linked to life span increase, the signaling pathways regulating
protein synthesis might be putative targets for treatments of age-related diseases. Eukaryotic cells have evolved acomplex system for protein synthesis regulation and this review will summarize cellular strategies to regulate mRNA
translation upon stress and its impact on longevity.
Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different p... more Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different proteolytic machines, including ClpAP. The ClpA component is a hexameric AAA+ (ATPase associated with various cellular activities) chaperone that utilizes the energy of ATP to control substrate recognition and unfolding. The precise role of the N-domains of ClpA in this process, however, remains elusive. Here, we have analysed the role of five highly conserved basic residues in the N-domain of ClpA by monitoring the binding, unfolding and degradation of several different substrates, including short unstructured peptides, tagged and untagged proteins. Interestingly, mutation of three of these basic residues within the N-domain of ClpA (H94, R86 and R100) did not alter substrate degradation. In contrast mutation of two conserved arginine residues (R90 and R131), flanking a putative peptide-binding groove within the N-domain of ClpA, specifically compromised the ability of ClpA to unfold and degrade selected substrates but did not prevent substrate recognition, ClpS-mediated substrate delivery or ClpP binding. In contrast, a highly conserved tyrosine residue lining the central pore of the ClpA hexamer was essential for the degradation of all substrate types analysed, including both folded and unstructured proteins. Taken together, these data suggest that ClpA utilizes two structural elements, one in the N-domain and the other in the pore of the hexamer, both of which are required for efficient unfolding of some protein substrates.
The mitochondrial matrix of mammalian cells contains several different ATP-dependent proteases, i... more The mitochondrial matrix of mammalian cells contains several different ATP-dependent proteases, including CLPXP, some of which contribute to protein maturation and quality control. Currently however, the substrates and the physiological roles of mitochondrial CLPXP in humans, has remained elusive. Similarly, the mechanism by which these ATP-dependent proteases recognize their substrates currently remains unclear. Here we report the characterization of a Walker B mutation in human CLPX, in which the highly conserved glutamate was replaced with alanine. This mutant protein exhibits improved interaction with the model unfolded substrate casein and several putative physiological substrates in vitro. Although this mutant lacks ATPase activity, it retains the ability to mediate casein degradation by hCLPP, in a fashion similar to the small molecule ClpP-activator, ADEP. Our functional dissection of hCLPX structure, also identified that most model substrates are recognized by the N-terminal domain, although some substrates bypass this step and dock, directly to the pore-1 motif. Collectively these data reveal, that despite the difference between bacterial and human CLPXP complexes, human CLPXP exhibits a similar mode of substrate recognition and is deregulated by ADEPs.
The cellular proteostasis network integrates the protein folding and clearance machineries in mul... more The cellular proteostasis network integrates the protein folding and clearance machineries in multiple sub-cellular compartments of the eukaryotic cell. The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. A distinctive feature of the ER is its tightly controlled redox homeostasis necessary for the formation of inter- and intra-molecular disulphide bonds. Employing genetically encoded in vivo sensors reporting on the redox state in an organelle-specific manner, we show in the nematode Caenorhabditis elegans that the redox state of the ER is subject to profound changes during worm lifetime. In young animals, the ER is oxidizing and this shifts towards reducing conditions during ageing, whereas in the cytosol the redox state becomes more oxidizing with age. Likewise, the redox state in the cytosol and the ER change in an opposing manner in response to proteotoxic challenges in C. elegans and in HeLa cells revealing conservation of red...
Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathop... more Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are f...
Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly un... more Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly under stress conditions. Calibrated Western blot analyses were used to estimate the approximate numbers of heat-inducible Clp molecules within a single cell. According to these numbers, the different Clp ATPases do not seem to compete for the proteolytic subunit ClpP. Coimmunoprecipitation experiments revealed the predicted specific ClpX-ClpP, ClpC-ClpP, and ClpE-ClpP interactions. ClpE and ClpX are rapidly degraded in wild-type cells during permanent heat stress but remained almost stable in a clpP mutant, suggesting ClpP-dependent degradation. In particular, ClpCP appeared to be involved in the degradation of the short-lived ClpE ATPase, indicating a negative "autoregulatory" circuit for this particular Clp ATPase at the posttranslational level. Analysis of the half-life of stress-inducible clp mRNAs during exponential growth and heat shock revealed precise regulation of the sy...
In Bacillus subtilis, the alternative sigma factor sigma(B) is activated in response to environme... more In Bacillus subtilis, the alternative sigma factor sigma(B) is activated in response to environmental stress or energy depletion. The general stress regulon under the control of sigma(B) provides the cell with multiple stress resistance. Experiments were designed to determine how activated sigma(B) replaces sigma(A) as a constituent of the RNA polymerase holoenzyme. Studies of the transcription of the sigma(A)-dependent stress gene clpE under sigma(B)-inducing conditions showed that expression was higher in a sigB mutant background than in the wild type. The relative affinities of sigma(A) and sigma(B) for binding to the core RNA polymerase (E) were determined by means of indirect surface plasmon resonance. The results showed that the affinity of sigma(B) for E was 60-fold lower than that of sigma(A). Western blot analyses with antibodies against sigma(A), sigma(B), and E showed that, after exposure to ethanol stress, the concentration of sigma(B) was only twofold higher than those ...
The ring-forming AAA+ chaperone ClpB cooperates with the DnaK chaperone system to reactivate aggr... more The ring-forming AAA+ chaperone ClpB cooperates with the DnaK chaperone system to reactivate aggregated proteins. With the assistance of DnaK, ClpB extracts unfolded polypeptides from aggregates via substrate threading through its central channel. Here we analyze the processing of mixed aggregates consisting of protein fusions of misfolded and native domains. ClpB-DnaK reactivated all aggregated fusion proteins with similar efficiency, without unfolding native domains, demonstrating that partial threading of the misfolded moiety is sufficient to solubilize aggregates. Reactivation by ClpB-DnaK occurred even when two stably folded domains flanked the aggregated moiety, indicating threading of internal substrate segments. In contrast with the related AAA+ chaperone ClpC, ClpB lacks a robust unfolding activity, enabling it to sense the conformational state of substrates. ClpB rings are highly unstable, which may facilitate dissociation from trapped substrates during threading.
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protein synthesis might be putative targets for treatments of age-related diseases. Eukaryotic cells have evolved acomplex system for protein synthesis regulation and this review will summarize cellular strategies to regulate mRNA
translation upon stress and its impact on longevity.
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protein synthesis might be putative targets for treatments of age-related diseases. Eukaryotic cells have evolved acomplex system for protein synthesis regulation and this review will summarize cellular strategies to regulate mRNA
translation upon stress and its impact on longevity.