Mitochondria Tether Protein Trash to Rejuvenate Cellular Environments

Protein damage segregates asymmetrically in dividing yeast cells, rejuvenating daughters at the expense of mother cells. Zhou et al. now show that newly synthesized proteins are particularly prone to aggregation and describe a mechanism that tethers aggregated proteins to mitochondria. This association constrains aggregate mobility, effectively retaining and sorting toxic aggregates away from younger cells

The Escherichia coli trigger factor

AbstractE. coli trigger factor is an abundant cytosolic protein originally iDAntified by its ability to maintain the precursor of a secretory protein in a translocation competent form. Recent studies shed new light on the function of this protein. Trigger factor was found to be a peptidyl-prolyl-cisltrans-isomerase capable of catalysing protein folding in vitro, to associate with nascent cytosolic and secretory polypeptiDA chains, and to cooperate with the GroEL chaperone in promoting proteolysis of an unstable polypeptiDA in vivo. These findings suggest roles for trigger factor in various folding processes of secretory as well as cytosolic proteins

Unscrambling an egg: protein disaggregation by AAA+ proteins

A protein quality control system, consisting of molecular chaperones and proteases, controls the folding status of proteins and prevents the aggregation of misfolded proteins by either refolding or degrading aggregation-prone species. During severe stress conditions this protection system can be overwhelmed by high substrate load, resulting in the formation of protein aggregates. In such emergency situations, Hsp104/ClpB becomes a key player for cell survival, as it has the extraordinary capacity to rescue proteins from an aggregated state in cooperation with an Hsp70 chaperone system. The ring-forming Hsp104/ClpB chaperone belongs to the AAA+ protein superfamily, which in general drives the assembly and disassembly of protein complexes by ATP-dependent remodelling of protein substrates. A disaggregation activity was also recently attributed to other eubacterial AAA+ proteins, while such an activity has not yet been identified in mammalian cells. In this review, we report on new insights into the mechanism of protein disaggregation by AAA+ proteins, suggesting that these chaperones act as molecular crowbars or ratchets

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

AbstractThe 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 α-casein and poly-L-lysine on the ATPase and chaperone activities of ClpB. α-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

Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli

<p>Abstract</p> <p>Background</p> <p>The overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful. We now assessed the effects of combined overproduction of the functionally cooperating chaperone network of the <it>E. coli </it>cytosol on the solubility of recombinant proteins.</p> <p>Results</p> <p>A two-step procedure was found to show the strongest enhancement of solubility. In a first step, the four chaperone systems GroEL/GroES, DnaK/DnaJ/GrpE, ClpB and the small HSPs IbpA/IbpB, were coordinately co-overproduced with recombinant proteins to optimize <it>de novo </it>folding. In a second step, protein biosynthesis was inhibited to permit chaperone mediated refolding of misfolded and aggregated proteins <it>in vivo</it>. This novel strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold.</p> <p>Conclusion</p> <p>The engineered <it>E. coli </it>strains and the two-step procedure presented here led to a remarkable increase in the solubility of a various recombinant proteins and should be applicable to a wide range of target proteins produced in biotechnology.</p

Common and specific mechanisms of AAA+ proteins involved in protein quality control

Abstract A protein quality control system, consisting of molecular chaperones and proteases, controls the folding status of proteins and mediates the refolding or degradation of misfolded proteins. Ring-forming AAA+ (ATPase associated with various cellular activities) proteins play crucial roles in both processes by co-operating with either peptidases or chaperone systems. Peptidase-associated AAA+ proteins bind substrates and thread them through their axial channel into the attached proteolytic chambers for degradation. In contrast, the AAA+ protein ClpB evolved independently from an interacting peptidase and co-operates with a cognate Hsp70 (heat-shock protein 70) chaperone system to solubilize and refold aggregated proteins. The activity of this bi-chaperone system is crucial for the survival of bacteria, yeast and plants during severe stress conditions. Hsp70 acts at initial stages of the disaggregation process, enabling ClpB to extract single unfolded polypeptides from the aggregate via a threading activity. Although both classes of AAA+ proteins share a common threading activity, it is apparent that their divergent evolution translates into specific mechanisms, reflecting adaptations to their respective functions. The ClpB-specific M-domain (middle domain) represents such an extra feature that verifies ClpB as the central disaggregase in vivo. M-domains act as regulatory devices to control both ClpB ATPase activity and the Hsp70-dependent binding of aggregated proteins to the ClpB pore, thereby coupling the Hsp70 chaperone activity with the ClpB threading motor to ensure efficient protein disaggregation

Cooperative amyloid fibre binding and disassembly by the Hsp70 disaggregase

Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly. Here we use atomic force microscopy to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography and total internal reflection fluorescence microscopy reveal fibrils with regions of densely bound chaperones, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~ 4 subunits per second. The observed unidirectional bursts of Hsc70 loading and unravelling may be explained by differences between the two ends of the polar fibre structure