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 hallmark of Mycobacterium tuberculosis is its ability to persist for a long-term in host granulomas, in a non-replicating and drug-tolerant state, and later awaken to cause disease. To date, the cellular factors and the molecular mechanisms that mediate entry into the persistence phase are poorly understood. Remarkably, M. tuberculosis possesses a very high number of toxin-antitoxin (TA) systems in its chromosome, 79 in total, regrouping both well-known (68) and novel (11) families, with some of them being strongly induced in drug-tolerant persisters. In agreement with the capacity of stress-responsive TA systems to generate persisters in other bacteria, it has been proposed that activation of TA systems in M. tuberculosis could contribute to its pathogenesis. Herein, we review the current knowledge on the multiple TA families present in this bacterium, their mechanism, and their potential role in physiology and virulence.

Summary: Molecular chaperones are highly conserved in all free‐living organisms. There are many types of chaperones, and most are conveniently grouped into families. Genome sequencing has revealed that many organisms contain multiple members of both the DnaK (Hsp70) family and their partner J‐domain protein (JDP) cochaperone, belonging to the DnaJ (Hsp40) family. Escherichia coli K‐12 encodes three Hsp70 genes and six JDP genes. The coexistence of these chaperones in the same cytosol suggests that certain chaperone–cochaperone interactions are permitted, and that chaperone tasks and their regulation have become specialized over the course of evolution. Extensive genetic and biochemical analyses have greatly expanded knowledge of chaperone tasking in this organism. In particular, recent advances in structure determination have led to significant insights of the underlying complexities and functional elegance of the Hsp70 chaperone machine.

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.

Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones. J-domain containing proteins (JDPs) are the largest family of Hsp70 co-chaperones and play a determining role functionally specifying and directing Hsp70 functions. Many features of JDPs are not understood; however, a number of JDP experts gathered at a recent CSSI-sponsored workshop in Gdansk (Poland) to discuss various aspects of J-domain protein function, evolution, and structure. In this report, we present the main findings and the consensus reached to help direct future developments in the field of Hsp70 research.

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