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This collection of short essays utilizes a narrative approach to present the RISE framework, with four pillars centered on promoting student Resilience, Identity, Strength in Scholarship and Empowerment. Each short essay aims to draw attention to the spectrum of challenges that students currently face during their higher education journeys in Canada and what innovative solutions have been or could be implemented to address these adversities in accordance with RISE. The application of the framework to evaluate and reorient learning environments in Canadian higher education holds immense potential to help all students grow as collaborative thinkers, partners, and leaders that will leave academic settings well prepared for their life as leaders in their community. Dans cet article qui rassemble de courts essais, nous utilisons une approche narrative pour présenter le cadre RISE en l’appuyant sur quatre piliers permettant de promouvoir la Résilience, l’Identité, la Solidité universitaire et l’Émancipation. Chacun des courts essais vise à mettre en relief, d’une part, l’éventail des défis auxquels les étudiants font face actuellement dans l’enseignement supérieur au Canada et, d’autre part, les solutions novatrices qui ont été mises en œuvre – ou qui pourraient l’être – pour s’attaquer à ces difficultés dans le contexte de RISE. L’application de ce cadre dans l’examen et la reconfiguration des environnements d’apprentissage de l’enseignement supérieur au Canada est pleine de promesses. Voilà qui pourrait aider tous les étudiants à devenir des penseurs collaboratifs, des partenaires et des leaders qui, lorsqu’ils quitteront l’université, seront prêts à agir comme des chefs de file de leur communauté.

Pseudomonas aeruginosa is a human pathogen that relies on quorum sensing to establish infections. The PqsE quorum-sensing protein is required for P. aeruginosa virulence factor production and infection. PqsE has a reported enzymatic function in the biosynthesis of the quorum-sensing autoinducer called PQS. However, this activity is redundant because, in the absence of PqsE, this role is fulfilled by alternative thioesterases. Rather, PqsE drives P. aeruginosa pathogenic traits via a protein-protein interaction with the quorum-sensing receptor/transcription factor RhlR, an interaction that enhances the affinity of RhlR for target DNA sequences. PqsE catalytic activity is dispensable for interaction with RhlR. Thus, the virulence function of PqsE can be decoupled from its catalytic function. Here, we present an immunoprecipitation-mass spectrometry method employing enhanced green fluorescent protein-PqsE fusions to define the protein interactomes of wild-type PqsE and the catalytically inactive PqsE(D73A) variant in P. aeruginosa and their dependence on RhlR. Several proteins were identified to have specific interactions with wild-type PqsE while not forming associations with PqsE(D73A). In the Δ rhlR strain, an increased number of specific PqsE interactors were identified, including the partner autoinducer synthase for RhlR, called RhlI. Collectively, these results suggest that specific protein-protein interactions depend on PqsE catalytic activity and that RhlR may prevent proteins from interacting with PqsE, possibly due to competition between RhlR and other proteins for PqsE binding. Our results provide a foundation for the identification of the in vivo PqsE catalytic function and, potentially, new proteins involved in P. aeruginosa quorum sensing. IMPORTANCE Pseudomonas aeruginosa causes hospital-borne infections in vulnerable patients, including immunocompromised individuals, burn victims, and cancer patients undergoing chemotherapy. There are no effective treatments for P. aeruginosa infections, which are usually broadly resistant to antibiotics. Animal models show that, to establish infection and to cause illness, P. aeruginosa relies on an interaction between two proteins, namely, PqsE and RhlR. There could be additional protein-protein interactions involving PqsE, which, if defined, could be exploited for the design of new therapeutic strategies to combat P. aeruginosa. Here, we reveal previously unknown protein interactions in which PqsE participates, which will be investigated for potential roles in pathogenesis.

Castration-resistant prostate cancer (CRPC) is associated with an increased reliance on heat shock protein 70 (HSP70), but it is not clear what other protein homeostasis (proteostasis) factors might be involved. To address this question, we performed functional and synthetic lethal screens in four prostate cancer cell lines. These screens confirmed key roles for HSP70, HSP90, and their co-chaperones, but also suggested that the mitochondrial chaperone, HSP60/HSPD1, is selectively required in CRPC cell lines. Knockdown of HSP60 does not impact the stability of androgen receptor (AR) or its variants; rather, it is associated with loss of mitochondrial spare respiratory capacity, partly owing to increased proton leakage. Finally, transcriptional data revealed a correlation between HSP60 levels and poor survival of prostate cancer patients. These findings suggest that re-wiring of the proteostasis network is associated with CRPC, creating selective vulnerabilities that might be targeted to treat the disease., Competing Interests: Declaration of interests J.E.G. holds patents to the use of HSP70 inhibitors and is a member of the Cell Chemical Biology Scientific Advisory Board. The authors declare no other competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pseudomonas aeruginosa is an opportunistic pathogen that causes disease in immunocompromised individuals and individuals with underlying pulmonary disorders. P. aeruginosa virulence is controlled by quorum sensing (QS), a bacterial cell-cell communication mechanism that underpins transitions between individual and group behaviors. In P. aeruginosa, the PqsE enzyme and the QS receptor RhlR directly interact to control the expression of genes involved in virulence. Here, we show that three surface-exposed arginine residues on PqsE comprise the site required for interaction with RhlR. We show that a noninteracting PqsE variant [PqsE(NI)] possesses catalytic activity, but is incapable of promoting virulence phenotypes, indicating that interaction with RhlR, and not catalysis, drives these PqsE-dependent behaviors. Biochemical characterization of the PqsE-RhlR interaction coupled with RNA-seq analyses demonstrates that the PqsE-RhlR complex increases the affinity of RhlR for DNA, enabling enhanced expression of genes encoding key virulence factors. These findings provide the mechanism for PqsE-dependent regulation of RhlR and identify a unique regulatory feature of P. aeruginosa QS and its connection to virulence. IMPORTANCE Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of molecules called autoinducers (AI). QS is required for virulence in the human pathogen Pseudomonas aeruginosa, which can cause fatal infections in patients with underlying pulmonary disorders. In this study, we determine the molecular basis for the physical interaction between two virulence-driving QS components, PqsE and RhlR. We find that the ability of PqsE to bind RhlR correlates with virulence factor production. Since current antimicrobial therapies exacerbate the growing antibiotic resistance problem because they target bacterial growth, we suggest that the PqsE-RhlR interface discovered here represents a new candidate for targeting with small molecule inhibition. Therapeutics that disrupt the PqsE-RhlR interaction should suppress virulence. Targeting bacterial behaviors such as QS, rather than bacterial growth, represents an attractive alternative for exploration because such therapies could potentially minimize the development of resistance.

Prion protein (PrP) adopts either a helical conformation (PrP C ) or an alternative, beta sheet-rich, misfolded conformation (PrP Sc ). The PrP Sc form has the ability to "infect" PrP C and force it into the misfolded state. Accumulation of PrP Sc is associated with a number of lethal neurodegenerative disorders, including Creutzfeldt-Jacob disease (CJD). Knockout of PrP C protects cells and animals from PrP Sc infection; thus, there is interest in identifying factors that regulate PrP C stability, with the therapeutic goal of reducing PrP C levels and limiting infection by PrP Sc . Here, we assembled a short-hairpin RNA (shRNA) library composed of 25+ shRNA sequences for each of 133 protein homeostasis (aka proteostasis) factors, such as molecular chaperones and co-chaperones. This Proteostasis shRNA Library was used to identify regulators of PrP C stability in HEK293 Hu129M cells. Strikingly, the screen identified a number of Hsp70 family members and their co-chaperones as putative targets. Indeed, a chemical pan-inhibitor of Hsp70s reduced PrP C levels and limited conversion to PrP Sc in N2a cells. These results implicate specific proteostasis sub-networks, especially the Hsp70 system, as potential new targets for the treatment of CJD. More broadly, the Proteostasis shRNA Library might be a useful tool for asking which proteostasis factors are important for a given protein.

Understanding the selectivity of a small molecule for its target(s) in cells is an important goal in chemical biology and drug discovery. One powerful way to address this question is with dominant negative (DN) mutants, in which an active site residue in the putative target is mutated. While powerful, this approach is less straightforward for allosteric sites. Here, we introduce tryptophan scanning mutagenesis as an expansion of this idea. As a test case, we focused on the challenging drug target, heat shock cognate protein 70 (Hsc70), and its allosteric inhibitor JG-98. Structure-based modelling predicted that mutating Y149W in human Hsc70 or Y145W in the bacterial ortholog DnaK would place an indole side chain into the allosteric pocket normally occupied by the compound. Indeed, we found that the tryptophan mutants acted as if they were engaged with JG-98. We then used DnaK Y145W to suggest that this protein may be an anti-bacterial target. Indeed, we found that DnaK inhibitors have minimum inhibitory concentration (MIC) values <0.125 μg mL -1 against several pathogens, including multidrug-resistant Staphylococcus aureus (MRSA) strains. We propose that tryptophan scanning mutagenesis may provide a distinct way to address the important problem of target engagement., Competing Interests: The authors claim no competing financial interests., (This journal is © The Royal Society of Chemistry.)

Legionella pneumophila (L.p.), the microbe responsible for Legionnaires' disease, secretes ∼300 bacterial proteins into the host cell cytosol. A subset of these proteins affects a wide range of post-translational modifications (PTMs) to disrupt host cellular pathways. L.p. has 5 conserved eukaryotic-like Ser/Thr effector kinases, LegK1-4 and LegK7, which are translocated during infection. Using a chemical genetic screen, we identified the Hsp70 chaperone family as a direct host target of LegK4. Phosphorylation of Hsp70s at T495 in the substrate-binding domain disrupted Hsp70's ATPase activity and greatly inhibited its protein folding capacity. Phosphorylation of cytosolic Hsp70 by LegK4 resulted in global translation inhibition and an increase in the amount of Hsp70 on highly translating polysomes. LegK4's ability to inhibit host translation via a single PTM uncovers a role for Hsp70 in protein synthesis and directly links it to the cellular translational machinery., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hsp70 chaperones bind to various protein substrates for folding, trafficking, and degradation. Considerable structural information is available about how prokaryotic Hsp70 (DnaK) binds substrates, but less is known about mammalian Hsp70s, of which there are 13 isoforms encoded in the human genome. Here, we report the interaction between the human Hsp70 isoform heat shock cognate 71-kDa protein (Hsc70 or HSPA8) and peptides derived from the microtubule-associated protein Tau, which is linked to Alzheimer's disease. For structural studies, we used an Hsc70 construct (called BETA) comprising the substrate-binding domain but lacking the lid. Importantly, we found that truncating the lid does not significantly impair Hsc70's chaperone activity or allostery in vitro Using NMR, we show that BETA is partially dynamically disordered in the absence of substrate and that binding of the Tau sequence GKVQIINKKG (with a K D = 500 nm) causes dramatic rigidification of BETA. NOE distance measurements revealed that Tau binds to the canonical substrate-binding cleft, similar to the binding observed with DnaK. To further develop BETA as a tool for studying Hsc70 interactions, we also measured BETA binding in NMR and fluorescent competition assays to peptides derived from huntingtin, insulin, a second Tau-recognition sequence, and a KFERQ-like sequence linked to chaperone-mediated autophagy. We found that the insulin C-peptide binds BETA with high affinity ( K D < 100 nm), whereas the others do not ( K D > 100 μm). Together, our findings reveal several similarities and differences in how prokaryotic and mammalian Hsp70 isoforms interact with different substrate peptides., (© 2018 Taylor et al.)

Protein-protein interactions (PPIs) are an important category of putative drug targets. Improvements in high-throughput screening (HTS) have significantly accelerated the discovery of inhibitors for some categories of PPIs. However, methods suitable for screening multiprotein complexes ( e.g. those composed of three or more different components) have been slower to emerge. Here, we explored an approach that uses reconstituted multiprotein complexes (RMPCs). As a model system, we chose heat shock protein 70 (Hsp70), which is an ATP-dependent molecular chaperone that interacts with co-chaperones, including DnaJA2 and BAG2. The PPIs between Hsp70 and its co-chaperones stimulate nucleotide cycling. Thus, to re-create this ternary protein system, we combined purified human Hsp70 with DnaJA2 and BAG2 and then screened 100,000 diverse compounds for those that inhibited co-chaperone-stimulated ATPase activity. This HTS campaign yielded two compounds with promising inhibitory activity. Interestingly, one inhibited the PPI between Hsp70 and DnaJA2, whereas the other seemed to inhibit the Hsp70-BAG2 complex. Using secondary assays, we found that both compounds inhibited the PPIs through binding to allosteric sites on Hsp70, but neither affected Hsp70's intrinsic ATPase activity. Our RMPC approach expands the toolbox of biochemical HTS methods available for studying difficult-to-target PPIs in multiprotein complexes. The results may also provide a starting point for new chemical probes of the Hsp70 system., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)