Functional cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex.

A functional association is uncovered between the ribosome-associated trigger factor (TF) chaperone and the ClpXP degradation complex. Bioinformatic analyses demonstrate conservation of the close proximity of tig, the gene coding for TF, and genes coding for ClpXP, suggesting a functional interaction. The effect of TF on ClpXP-dependent degradation varies based on the nature of substrate. While degradation of some substrates are slowed down or are unaffected by TF, surprisingly, TF increases the degradation rate of a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA-tagged proteins. Globally, TF acts to enhance the degradation of about 2% of newly synthesized proteins. TF is found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone-protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP.

ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores.

Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.

Multiple functionalities of molecular chaperones revealed through systematic mapping of their interaction networks.

Chaperones are a highly interactive group of proteins that function globally in many cellular processes involved in maintaining protein homeostasis. Traditional biochemical assays typically do not provide a complete view of the intricate networks through which chaperones collaborate to promote proteostasis. Recent advances in high-throughput systematic analyses of chaperone interactions have uncovered that chaperones display a remarkable cooperativity in their interactions with numerous client proteins. This cooperativity has been found to be a fundamental aspect of a properly functioning cell. Aberrant formation or improper regulation of these interactions can easily lead to disease states. Herein, we provide an overview of the use of large-scale interaction assays, whether physical (protein-protein) or genetic (epistatic), to study chaperone interaction networks. Importantly, we discuss the ongoing need for such studies to determine the mechanisms by which protein homeostasis is controlled in the cell.

Acyldepsipeptide Analogs Dysregulate Human Mitochondrial ClpP Protease Activity and Cause Apoptotic Cell Death.

Acyldepsipeptides (ADEPs) are potential antibiotics that dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease, leading to bacterial cell death. Here, we identified ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP, causing the protease to non-specifically degrade model substrates. Dysregulation of HsClpP activity by ADEP was found to induce cytotoxic effects via activation of the intrinsic, caspase-dependent apoptosis. ADEP-HsClpP co-crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighboring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a therapeutic potential for ADEPs in cancer treatment.

Reversible inhibition of the ClpP protease via an N-terminal conformational switch.

Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation by proteases. In most prokaryotes and in chloroplasts and mitochondria, protein degradation is performed by the caseinolytic protease ClpP, a tetradecamer barrel-like proteolytic complex. Dysregulating ClpP function has shown promise in fighting antibiotic resistance and as a potential therapy for acute myeloid leukemia. Here we use methyl-transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, biochemical assays, and molecular dynamics simulations to characterize the structural dynamics of ClpP from Staphylococcus aureus (SaClpP) in wild-type and mutant forms in an effort to discover conformational hotspots that regulate its function. Wild-type SaClpP was found exclusively in the active extended form, with the N-terminal domains of its component protomers in predominantly ß-hairpin conformations that are less well-defined than other regions of the protein. A hydrophobic site was identified that, upon mutation, leads to unfolding of the N-terminal domains, loss of SaClpP activity, and formation of a previously unobserved split-ring conformation with a pair of 20-Å-wide pores in the side of the complex. The extended form of the structure and partial activity can be restored via binding of ADEP small-molecule activators. The observed structural plasticity of the N-terminal gates is shown to be a conserved feature through studies of Escherichia coli and Neisseria meningitidis ClpP, suggesting a potential avenue for the development of molecules to allosterically modulate the function of ClpP.

Systems analysis of the genetic interaction network of yeast molecular chaperones.

Molecular chaperones are typically promiscuous interacting proteins that function globally in the cell to maintain protein homeostasis. Recently, we had carried out experiments that elucidated a comprehensive interaction network for the core 67 chaperones and 15 cochaperones in the budding yeast Saccharomyces cerevisiae [Rizzolo et al., Cell Rep., 2017, 20, 2735-2748]. Here, the genetic (i.e. epistatic) interaction network obtained for chaperones was further analyzed, revealing that the global topological parameters of the resulting network have a more central role in mediating interactions in comparison to the rest of the proteins in the cell. Most notably, we observed Hsp10, Hsp70 Ssz1 chaperone, and Hsp90 cochaperone Cdc37 to be the main drivers of the network architecture. Systematic analysis on the physicochemical properties for all chaperone interactors further revealed the presence of preferential domains and folds that are highly interactive with chaperones such as the WD40 repeat domain. Further analysis with established cellular complexes revealed the involvement of R2TP chaperone in quaternary structure formation. Our results thus provide a global overview of the chaperone network properties in yeast, expanding our understanding of their functional diversity and their role in protein homeostasis.

Computational Analysis of the Chaperone Interaction Networks.

We provide computational protocols to identify chaperone interacting proteins using a combination of both physical (protein-protein) and genetic (gene-gene or epistatic) interaction data derived from the published large-scale proteomic and genomic studies for the budding yeast Saccharomyces cerevisiae. Using these datasets, we discuss bioinformatic analyses that can be employed to build comprehensive high-fidelity chaperone interaction networks. Given that many proteins typically function as complexes in the cell, we highlight various step-wise approaches for combining both the genetic and physical interaction datasets to decipher intra- and inter-connections for distinct chaperone- and non-chaperone-containing complexes in the network. Together, these informatics procedures will aid in identifying protein complexes with distinctive functional specializations in the cell that yield a very broad and diverse set of interactions. The described procedures can also be leveraged to datasets from other eukaryotes, including humans.

Features of the Chaperone Cellular Network Revealed through Systematic Interaction Mapping.

A comprehensive view of molecular chaperone function in the cell was obtained through a systematic global integrative network approach based on physical (protein-protein) and genetic (gene-gene or epistatic) interaction mapping. This allowed us to decipher interactions involving all core chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. Our analysis revealed the presence of a large chaperone functional supercomplex, which we named the naturally joined (NAJ) chaperone complex, encompassing Hsp40, Hsp70, Hsp90, AAA+, CCT, and small Hsps. We further found that many chaperones interact with proteins that form foci or condensates under stress conditions. Using an in vitro reconstitution approach, we demonstrate condensate formation for the highly conserved AAA+ ATPases Rvb1 and Rvb2, which are part of the R2TP complex that interacts with Hsp90. This expanded view of the chaperone network in the cell clearly demonstrates the distinction between chaperones having broad versus narrow substrate specificities in protein homeostasis.

Novel function discovery with GeneMANIA: a new integrated resource for gene function prediction in Escherichia coli.

MOTIVATION: The model bacterium Escherichia coli is among the best studied prokaryotes, yet nearly half of its proteins are still of unknown biological function. This is despite a wealth of available large-scale physical and genetic interaction data. To address this, we extended the GeneMANIA function prediction web application developed for model eukaryotes to support E.coli. RESULTS: We integrated 48 distinct E.coli functional interaction datasets and used the GeneMANIA algorithm to produce thousands of novel functional predictions and prioritize genes for further functional assays. Our analysis achieved cross-validation performance comparable to that reported for eukaryotic model organisms, and revealed new functions for previously uncharacterized genes in specific bioprocesses, including components required for cell adhesion, iron-sulphur complex assembly and ribosome biogenesis. The GeneMANIA approach for network-based function prediction provides an innovative new tool for probing mechanisms underlying bacterial bioprocesses. CONTACT: gary.bader@utoronto.ca; mohan.babu@uregina.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454.

We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.

The PDGF signaling pathway controls multiple steroid-producing lineages.

The platelet-derived growth factor (PDGF) signaling pathway regulates numerous lineages of mesenchymal cell origin during development and in the adult. The transcriptional targets of this pathway have been shown to be required in several PDGF-dependent processes, but the roles of these targets in specific tissues is just beginning to be identified. In this study, we show that five different PDGF target genes are essential for male and/or female fertility. Mutations in each of these five different genes lead to defects in the steroid-producing cells in the testis and/or ovary and altered hormone production, suggesting that the PDGF pathway controls steroidogenesis through these genes in both sexes. Furthermore, conditional mutations of both PDGF receptors revealed a requirement in steroid-producing cells in multiple organs, including the testis, ovary, and adrenal cortex. Therefore, PDGF signaling may constitute a common mechanism in the control of multiple steroidogenic lineages.