RESUMO
The opportunistic pathogen Pseudomonas aeruginosa causes antibiotic-resistant, nosocomial infections in immuno-compromised individuals and is a high priority for antimicrobial development. Key to pathogenicity in P. aeruginosa are biofilm formation and virulence factor production. Both traits are controlled by the cell-to-cell communication process called quorum sensing (QS). QS involves the synthesis, release, and population-wide detection of signal molecules called autoinducers. We previously reported that the activity of the RhlR QS transcription factor depends on a protein-protein interaction with the hydrolase, PqsE, and PqsE catalytic activity is dispensable for this interaction. Nonetheless, the PqsE-RhlR interaction could be disrupted by the substitution of an active site glutamate residue with tryptophan [PqsE(E182W)]. Here, we show that disruption of the PqsE-RhlR interaction via either the E182W change or alteration of PqsE surface residues that are essential for the interaction with RhlR attenuates P. aeruginosa infection in a murine host. We use crystallography to characterize the conformational changes induced by the PqsE(E182W) substitution to define the mechanism underlying disruption of the PqsE-RhlR interaction. A loop rearrangement that repositions the E280 residue in PqsE(E182W) is responsible for the loss of interaction. We verify the implications garnered from the PqsE(E182W) structure using mutagenic, biochemical, and additional structural analyses. We present the next generation of molecules targeting the PqsE active site, including a structure of the tightest binding of these compounds, BB584, in complex with PqsE. The findings presented here provide insights into drug discovery against P. aeruginosa with PqsE as the target.
Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Biofilmes , Domínio Catalítico , Humanos , Camundongos , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/metabolismo , Percepção de QuorumRESUMO
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 KD = 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 (KD < 100 nm), whereas the others do not (KD > 100 µm). Together, our findings reveal several similarities and differences in how prokaryotic and mammalian Hsp70 isoforms interact with different substrate peptides.
Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas tau/metabolismo , Trifosfato de Adenosina/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSC70/genética , Humanos , Ligação Proteica , Conformação Proteica , Proteínas tau/química , Proteínas tau/genéticaRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Descoberta de Drogas , Proteínas de Choque Térmico HSP40/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Preparações Farmacêuticas/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Ligação ProteicaRESUMO
Hetero-multinuclear, platinum/ruthenium species were synthesized and tested for their effect on the motility of A549 (nonsmall cell lung) and MDA-MB-231 (breast) cancer cells and for their ability to inhibit DNA mobility using gel electrophoresis. It was found that the Ru(2)Pt trinuclear species [Na(2)]{[Ru(III)Cl(4)(DMSO-S)(-µ-pyz)](2)Pt(II)Cl(2)}, AH197, was much more efficient at inhibiting cell motility than [C(3)N(2)H(5)][Ru(III)Cl(4)(DMSO-S)(C(3)N(2)H(4))], NAMI-A, while the dinuclear RuPt species [K][Ru(III)Cl(4)(DMSO-S)(-µ-pyz)Pt(II)(DMSO-S)Cl(2)], IT127, was slightly better than NAMI-A. However, the dinuclear species retarded the electrophoretic mobility of DNA greater than both the trinuclear complex and cisplatin. The metal complexes and their respective BSA protein/metal adducts were studied by X-ray absorption spectroscopy. The spectra led to the conclusion that BSA donor atoms have substituted for the chloride ligands and perhaps the DMSO ligands.
Assuntos
Antineoplásicos/farmacologia , Compostos Organometálicos/farmacologia , Platina/química , Rutênio/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Conformação Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Soroalbumina Bovina/química , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Comunicação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Pseudomonas aeruginosa/genética , Percepção de Quorum/fisiologia , Virulência , Fatores de Virulência/genéticaRESUMO
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 ΔrhlR 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.
Assuntos
Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa , Animais , Humanos , Pseudomonas aeruginosa/metabolismo , Mapas de Interação de Proteínas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Percepção de Quorum/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Antibacterianos/metabolismoRESUMO
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.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Masculino , Chaperonas Moleculares/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Proteostase , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismoRESUMO
Prion protein (PrP) adopts either a helical conformation (PrPC) or an alternative, beta sheet-rich, misfolded conformation (PrPSc). The PrPSc form has the ability to "infect" PrPC and force it into the misfolded state. Accumulation of PrPSc is associated with a number of lethal neurodegenerative disorders, including Creutzfeldt-Jacob disease (CJD). Knockout of PrPC protects cells and animals from PrPSc infection; thus, there is interest in identifying factors that regulate PrPC stability, with the therapeutic goal of reducing PrPC levels and limiting infection by PrPSc. 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 PrPC 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 PrPC levels and limited conversion to PrPSc 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.
Assuntos
Síndrome de Creutzfeldt-Jakob/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Priônicas/metabolismo , Proteostase , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Estabilidade ProteicaRESUMO
Pseudomonas aeruginosa is an opportunistic human pathogen that causes fatal infections. There exists an urgent need for new antimicrobial agents to combat P. aeruginosa. We conducted a screen for molecules that bind the virulence-controlling protein PqsE and characterized hit compounds for inhibition of PqsE enzymatic activity. The binding conformations of two inhibitory molecules, BB391 and BB393, were identified by crystallography, and inhibitor binding was mimicked by the substitution of PqsE residues E182 and S285 with tryptophan. Comparison of the inhibitor-mimetic mutations to the catalytically inactive PqsE D73A protein demonstrated that catalysis is not responsible for the role PqsE plays in driving virulence factor production. Rather, the PqsE E182W protein fails to interact with the quorum-sensing receptor, RhlR, and our results suggest that it is this interaction that is responsible for promoting virulence factor production in P. aeruginosa. These findings provide a new route for drug discovery efforts targeting PqsE.
Assuntos
Mimetismo Molecular , Mutação , Pseudomonas aeruginosa/genética , Percepção de Quorum , Fatores de Virulência/biossíntese , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidadeRESUMO
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.
RESUMO
Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in the United States. PqsE, a thioesterase enzyme, is vital for virulence of P. aeruginosa, making PqsE an attractive target for inhibition. Neither the substrate nor the product of PqsE catalysis has been identified. A library of 550 million DNA-encoded drug-like small molecules was screened for those that bind to the purified PqsE protein. The structures of the bound molecules were identified by high throughput sequencing of the attached DNA barcodes. Putative PqsE binders with the strongest affinity features were examined for inhibition of PqsE thioesterase activity in vitro. The most potent inhibitors were resynthesized off DNA and examined for the ability to alter PqsE thermal melting and for PqsE thioesterase inhibition. Here, we report the synthesis, biological activity, mechanism of action, and early structure-activity relationships of a series of 2-(phenylcarbamoyl)benzoic acids that noncompetitively inhibit PqsE. A small set of analogs designed to probe initial structure-activity relationships showed increases in potency relative to the original hits, the best of which has an IC50 = 5 µM. Compound refinement is required to assess their in vivo activities as the current compounds do not accumulate in the P. aeruginosa cytosol. Our strategy validates DNA-encoded compound library screening as a rapid and effective method to identify catalytic inhibitors of the PqsE protein, and more generally, for discovering binders to bacterial proteins revealed by genetic screening to have crucial in vivo activities but whose biological functions have not been well-defined.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , DNA/química , Inibidores Enzimáticos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Tioléster Hidrolases/antagonistas & inibidores , Benzamidas/síntese química , Benzamidas/farmacologia , Inibidores Enzimáticos/síntese química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ácidos Ftálicos/síntese química , Ácidos Ftálicos/farmacologia , Pseudomonas aeruginosa/enzimologia , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-AtividadeRESUMO
Legionella pneumophila (L.p.), the microbe responsible for Legionnaires' disease, secretes â¼300 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.
Assuntos
Células Eucarióticas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Interações Hospedeiro-Patógeno , Legionella pneumophila/enzimologia , Fosfotransferases/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Células Eucarióticas/microbiologia , Doença dos Legionários/microbiologia , Fosforilação , Fatores de Virulência/metabolismoRESUMO
Allosteric inhibitors can be more difficult to optimize without an understanding of how their binding influences the conformational motions of the target. Here, we used an integrated computational and experimental approach to probe the molecular mechanism of an allosteric inhibitor of heat shock protein 70 (Hsp70). The anticancer compound, MKT-077, is known to bind a conserved site in members of the Hsp70 family, which favors the ADP-bound state and interferes with a protein-protein interaction (PPI) at long range. However, the binding site does not overlap with either the nucleotide-binding cleft or the PPI contact surface, so its mechanism is unclear. To this end, we modeled Hsp70's internal dynamics and studied how MKT-077 alters local sampling of its allosteric states. The results pointed to a set of concerted motions between five loops in Hsp70's nucleotide-binding domain (NBD), surrounding the MKT-077 binding site. To test this prediction, we mutated key residues and monitored chaperone activities in vitro. Together, the results indicate that MKT-077 interacts with loop222 to favor a pseudo-ADP bound conformer of Hsp70's NBD, even when ATP is present. We used this knowledge to synthesize an analog of MKT-077 that would better prevent motions of loop222 and confirmed that it had improved antiproliferative activity in breast cancer cells. These results provide an example of how to unlock and leverage the complex mechanisms of allosteric inhibitors.
Assuntos
Antineoplásicos/química , Proteínas de Choque Térmico HSC70/química , Piridinas/química , Tiazóis/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Regulação Alostérica , Sítios de Ligação , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
Heat shock protein 70 (Hsp70) is a molecular chaperone that plays critical roles in protein homeostasis. Hsp70's chaperone activity is coordinated by intra-molecular interactions between its two domains, as well as inter-molecular interactions between Hsp70 and its co-chaperones. Each of these contacts represents a potential opportunity for the development of chemical inhibitors. To illustrate this concept, we review three classes of recently identified molecules that bind distinct pockets on Hsp70. Although all three compounds share the ability to interrupt core biochemical functions of Hsp70, they stabilize different conformers. Accordingly, each compound appears to interrupt a specific subset of inter- and intra-molecular interactions. Thus, an accurate definition of an Hsp70 inhibitor may require a particularly detailed understanding of the molecule's binding site and its effects on protein-protein interactions.
Assuntos
Proteínas de Choque Térmico HSP70/fisiologia , Regulação Alostérica , Sítios de Ligação , Proteínas de Choque Térmico HSP70/antagonistas & inibidoresRESUMO
AH197, a trinuclear Ru(III)/Pt(II) metal complex, is strikingly more effective than the hallmark anticancer drug cisplatin and the Ru(III) clinical candidate NAMI-A in its binding to RNA and inhibition of primer DNA synthesis. Heteromultinuclear complexes could potentially serve as far better chemotherapeutics than mononuclear complexes.