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

Thermotolerance Requires Refolding of Aggregated Proteins by Substrate Translocation through the Central Pore of ClpB

AbstractCell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins

How flexible electrification can integrate fluctuating renewables

To phase out fossil fuels, energy systems must shift to renewable electricity as the main source of primary energy. In this paper, we analyze how electrification can support the integration of fluctuating renewables, like wind and PV, and mitigate the need for storage and thermal backup plants. Using a cost-minimizing model for system planning, we find substantial benefits of electricity demand in heating, transport, and industry adapting to supply. In Germany, flexible demand halves the residual peak load and the residual demand and reduces excess generation by 80%. Flexible operation of electrolyzers has the most significant impact accounting for 42% of the reduction in residual peak load and 59% in residual demand. District heating networks and BEVs also provide substantial flexibility, while the contribution of space and process heating is negligible. The results are robust to restrictions on the expansion of the transmission grid.ISSN:0360-5442ISSN:1873-678

Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum

Protein folding in the endoplasmic reticulum is a complex process whose malfunction is implicated in disease and aging. Using the cell’s endogenous sensor (the unfolded protein response), we identified several hundred yeast genes with roles in endoplasmic reticulum folding and systematically characterized their functional interdependencies by measuring unfolded protein response levels in double mutants. This strategy revealed multiple conserved factors critical for endoplasmic reticulum folding including an intimate dependence on the later secretory pathway, a previously uncharacterized six-protein transmembrane complex, and a co-chaperone complex that delivers tail-anchored proteins to their membrane insertion machinery. The use of a quantitative reporter in a comprehensive screen followed by systematic analysis of genetic dependencies should be broadly applicable to functional dissection of complex cellular processes from yeast to man

A Systematic Mammalian Genetic Interaction Map Reveals Pathways Underlying Ricin Susceptibility

Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs

The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins.

The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability

The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins.

The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability