The Bacterial Hsp90 Chaperone: Cellular Functions and Mechanism of Action

Heat shock protein 90 (Hsp90) is a molecular chaperone that folds and remodels proteins, thereby regulating the activity of numerous substrate proteins. Hsp90 is widely conserved across species and is essential in all eukaryotes and in some bacteria under stress conditions. To facilitate protein remodeling, bacterial Hsp90 collaborates with the Hsp70 molecular chaperone and its cochaperones. In contrast, the mechanism of protein remodeling performed by eukaryotic Hsp90 is more complex, involving more than 20 Hsp90 cochaperones in addition to Hsp70 and its cochaperones. In this review, we focus on recent progress toward understanding the basic mechanisms of bacterial Hsp90-mediated protein remodeling and the collaboration between Hsp90 and Hsp70. We describe the universally conserved structure and conformational dynamics of these chaperones and their interactions with one another and with client proteins. The physiological roles of Hsp90 in Escherichia coli and other bacteria are also discussed. We anticipate that the information gained from exploring the mechanism of the bacterial chaperone system will provide a framework for understanding the more complex eukaryotic Hsp90 system. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Abstract 14895: Shear Stress-induced Endothelial Inflammatory Response and EndMT Promote Adverse Remodeling in a Microengineered Coronary-artery-on-a-chip

Background: Atherosclerotic lesions preferentially occur at bifurcations and branching points where blood flow exerts low and bidirectional oscillatory shear stress (OSS) to the vascular walls. This study aims to determine the role of OSS in the induction of inflammatory activation and phenotypic transition in coronary artery endothelial cells and investigate the interaction between endothelial cells and smooth muscle cells in a coronary-artery-on-a-chip. Methods and Results: A three-dimensional microengineered human coronary-artery-on-a-chip was developed and incorporated with OSS to mimic the flow patterns within the microenvironment of coronary artery in atheroprone regions. Human coronary artery endothelial cells (HCAECs) were cultured on the upper surface of a collagen I-coated membrane, and human coronary artery smooth muscle cells (HCASMCs) were seeded on the opposite side of the membrane. Single-cell RNA sequencing analysis revealed that HCAECs were segregated into four subgroups after being exposed to OSS for 24 h, and inflammatory response and EndMT-related genes were enriched simultaneously in most HCAECs. OSS-induced inflammatory response and EndMT in HCAECs were confirmed by immunoblotting and immunofluorescence, and these changes were mediated by the Notch1/p38 MAPK-NF-κB signaling pathways. Moreover, HCAECs exposed to OSS induced extracellular matrix (ECM) protein remodeling and proliferation in HCASMCs through a paracrine mechanism. Multiplex ELISA analysis identified that RANTES exhibited the greatest increase after OSS in HCAEC culture and played a major role in modulation of ECM protein remodeling in HCASMCs. Further, IL-37 was able to prevent OSS-induced inflammatory response and EndMT in HCAECs and thereby abrogated ECM protein remodeling and proliferation in HCASMCs. Conclusion: OSS provokes inflammatory response and EndMT in HCAECs through activating the Notch1/p38 MAPK-NF-κB signaling pathways. The novel findings demonstrate that OSS-induced endothelial inflammatory response and associated EndMT promote vascular adverse remodeling and that anti-inflammatory cytokine IL-37 may have therapeutic potential for suppressing endothelial changes and resultant vascular adverse remodeling.

Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates

When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In Escherichia coli and other eubacteria, the tmRNA system rescues stalled ribosomes and adds an ssrA tag or degron to the C-terminus of the incomplete protein, which directs degradation by the AAA+ ClpXP protease. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. C-terminal residues of the ssrA degron initially bind in the top of an otherwise closed ClpX axial channel and subsequently move deeper into an open channel. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. For longer degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies.

Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates

ABSTRACTWhen ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In E. coli and other eubacteria, the tmRNA system rescues stalled ribosomes and adds an ssrA tag or degron to the C-terminus of the incomplete protein, which directs degradation by the AAA+ ClpXP protease. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. C-terminal residues of the ssrA degron initially bind in the top of an otherwise closed ClpX axial channel and subsequently move deeper into an open channel. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. For longer-degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies.

Cryo-EM structure of catalytic ribonucleoprotein complex RNase MRP

RNase MRP is an essential eukaryotic ribonucleoprotein complex involved in the maturation of rRNA and the regulation of the cell cycle. RNase MRP is related to the ribozyme-based RNase P, but it has evolved to have distinct cellular roles. We report a cryo-EM structure of the S. cerevisiae RNase MRP holoenzyme solved to 3.0 Å. We describe the structure of this 450 kDa complex, interactions between its components, and the organization of its catalytic RNA. We show that while the catalytic center of RNase MRP is inherited from the ancestral enzyme RNase P, the substrate binding pocket of RNase MRP is significantly altered by the addition of unique RNA and protein elements, as well as by RNA-driven protein remodeling.One Sentence SummaryChanges in peripheral RNA elements and RNA-driven protein remodeling result in diversification of related catalytic RNPs

Functional and physical interaction between yeast Hsp90 and Hsp70

Heat shock protein 90 (Hsp90) is a highly conserved ATP-dependent molecular chaperone that is essential in eukaryotes. It is required for the activation and stabilization of more than 200 client proteins, including many kinases and steroid hormone receptors involved in cell-signaling pathways. Hsp90 chaperone activity requires collaboration with a subset of the many Hsp90 cochaperones, including the Hsp70 chaperone. In higher eukaryotes, the collaboration between Hsp90 and Hsp70 is indirect and involves Hop, a cochaperone that interacts with both Hsp90 and Hsp70. Here we show that yeast Hsp90 (Hsp82) and yeast Hsp70 (Ssa1), directly interact in vitro in the absence of the yeast Hop homolog (Sti1), and identify a region in the middle domain of yeast Hsp90 that is required for the interaction. In vivo results using Hsp90 substitution mutants showed that several residues in this region were important or essential for growth at high temperature. Moreover, mutants in this region were defective in interaction with Hsp70 in cell lysates. In vitro, the purified Hsp82 mutant proteins were defective in direct physical interaction with Ssa1 and in protein remodeling in collaboration with Ssa1 and cochaperones. This region of Hsp90 is also important for interactions with several Hsp90 cochaperones and client proteins, suggesting that collaboration between Hsp70 and Hsp90 in protein remodeling may be modulated through competition between Hsp70 and Hsp90 cochaperones for the interaction surface.