Cellular chaperone networks prevent potentially toxic protein aggregation and ensure proteome integrity. Here, we used Escherichia coli as a model to understand the organization of these networks, focusing on the cooperation of the DnaK system with the upstream chaperone Trigger factor (TF) and the downstream GroEL. Quantitative proteomics revealed that DnaK interacts with at least ~700 mostly cytosolic proteins, including ~180 relatively aggregation-prone proteins that utilize DnaK extensively during and after initial folding. Upon deletion of TF, DnaK interacts increasingly with ribosomal and other small, basic proteins, while its association with large multidomain proteins is reduced. DnaK also functions prominently in stabilizing proteins for subsequent folding by GroEL. These proteins accumulate on DnaK upon GroEL depletion and are then degraded, thus defining DnaK as a central organizer of the chaperone network. Combined loss of DnaK and TF causes proteostasis collapse with disruption of GroEL function, defective ribosomal biogenesis, and extensive aggregation of large proteins.

The intracellular free Ca 2؉ concentration and redox status of murine fibroblasts exposed to prefibrillar aggregates of the HypF N-terminal domain have been investigated in vitro and in vivo using a range of fluorescent probes. Aggregate entrance into the cytoplasm is followed by an early rise of reactive oxygen species and free Ca 2؉ levels and eventually by cell death. Such changes correlate directly with the viability of the cells and are not observed when cell are cultured in the presence of reducing agents or in Ca 2؉ -free media. In addition, moderate cell stress following exposure to the aggregates was found to be fully reversible. The results show that the cytotoxicity of prefibrillar aggregates of HypF-N, a protein not associated with clinical disease, has the same fundamental origin as that produced by similar types of aggregates of proteins linked with specific amyloidoses. These findings suggest that misfolded proteinaceous aggregates stimulate generic cellular responses as a result of the exposure of regions of the structure (such as hydrophobic residues and the polypeptide main chain) that are buried in the normally folded proteins. They also support the idea that a higher number of degenerative pathologies than previously known might be considered as protein deposition diseases.

The combination of high-throughput sequencing and in vivo crosslinking approaches leads to the progressive uncovering of the complex interdependence between cellular transcriptome and proteome. Yet, the molecular determinants governing interactions in protein-RNA networks are not well understood. Here we investigated the relationship between the structure of an RNA and its ability to interact with proteins. Analysing in silico, in vitro and in vivo experiments, we find that the amount of double-stranded regions in an RNA correlates with the number of protein contacts. This relationship —which we call structure-driven protein interactivity— allows classification of RNA types, plays a role in gene regulation and could have implications for the formation of phase-separated ribonucleoprotein assemblies. We validate our hypothesis by showing that a highly structured RNA can rearrange the composition of a protein aggregate. We report that the tendency of proteins to phase-separate is reduced by interactions with specific RNAs.

Amyloid fibril formation is a process that represents an essential feature of the chemistry of proteins and plays a central role in human pathology and the biology of living organisms. In this Account, we shall describe some of the recent results on the sequence and structural determinants of protein aggregation. We shall describe the factors that govern aggregation of unfolded peptides and proteins. We shall then try to summarize the factors that pertain to the aggregation of partially structured states and will show that even fully folded states of proteins have an ability to aggregate into at least early oligomers with no need to undergo substantial conformational changes.

Protein biogenesis is tightly linked to protein quality control (PQC). The role of PQC machinery in recognizing faulty polypeptides is becoming increasingly understood. Molecular chaperones and cytosolic and vacuolar degradation systems collaborate to detect, repair, or hydrolyze mutant, damaged, and mislocalized proteins. On the other hand, the contribution of PQC to cofactor bindingrelated enzyme maturation remains largely unexplored, although the loading of a cofactor represents an all-or-nothing transition in regard to the enzymatic function and thus must be surveyed carefully. Combining proteomics and biochemical analysis, we demonstrate here that cells are able to detect functionally immature wild-type enzymes. We show that PQC-dedicated ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP) recognizes and marks for degradation not only a mutant protein but also its wild-type variant as long as the latter remains cofactor free. A distinct structural feature, the protruding C-terminal tail, which appears in both the mutant and wild-type polypeptides, contributes to recognition by CHIP. Our data suggest that relative insufficiency of apoprotein degradation caused by cofactor shortage can increase amyloidogenesis and aggravate protein aggregation disorders.apoprotein | ubiquitin ligase | protein aggregation

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Proteins and RNAs assemble in membrane-less organelles that organize intracellular spaces and regulate biochemical reactions. The ability of proteins and RNAs to form condensates is encoded in their sequences, yet it is unknown which domains drive the phase separation (PS) process and what are their specific roles. Here, we systematically investigated the human and yeast proteomes to find regions promoting condensation. Using advanced computational methods to predict the PS propensity of proteins, we designed a set of experiments to investigate the contributions of Prion-Like Domains (PrLDs) and RNA-binding domains (RBDs). We found that one PrLD is sufficient to drive PS, whereas multiple RBDs are needed to modulate the dynamics of the assemblies. In the case of stress granule protein Pub1 we show that the PrLD promotes sequestration of protein partners and the RBD confers liquid-like behaviour to the condensate. Our work sheds light on the fine interplay between RBDs and PrLD to regulate formation of membrane-less organelles, opening up the avenue for their manipulation.

The conversion of peptides and proteins into highly ordered and intractable aggregates is associated with a range of debilitating human diseases and represents a widespread problem in biotechnology. Protein engineering studies carried out in vitro have shown that mutations promote aggregation when they either destabilize the native state of a globular protein or accelerate the conversion of unfolded or partially folded conformations into oligomeric structures. We have extended such studies to investigate protein aggregation in vivo where a number of additional factors able to modify dramatically the aggregation behavior of proteins are present. We have expressed, in Escherichia coli cells, an E. coli protein domain, HypF-N. The results for a range of mutational variants indicate that although mutants with a conformational stability similar to that of the wild-type protein are soluble in the E. coli cytosol, variants with single point mutations predicted to destabilize the protein invariably aggregate after expression. We show, however, that aggregation of destabilized variants can be prevented by incorporating multiple mutations designed to reduce the intrinsic propensity of the polypeptide chain to aggregate; in the cases discussed here, this is achieved by an increase in the net charge of the protein. These results suggest that the principles being established to rationalize aggregation behavior in vitro have general validity for situations in vivo where aggregation has both biotechnological and medical relevance.