Brianna Dalesandro

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Neuroprotective effects of targeting BET proteins for degradation with dBET1 in aged mice subjected to ischemic stroke.

Neurochem Int. 2019 Mar 11;:

Authors: DeMars KM, Yang C, Candelario-Jalil E

Abstract
Neuroinflammation after stroke significantly contributes to neuronal cell death. Bromodomain and Extra Terminal Domain (BET) proteins are essential to inflammatory gene transcription. BET proteins (BRD2, BRD3, BRD4, and BRDT) have varied effects including chromatin remodeling, histone acetyltransferase activity, and as scaffolds to recruit transcription factors; they couple chromatin remodeling with transcription. BRD2/4 are of particularly interest to stroke-induced neuroinflammation that contributes to delayed cell death as they are required for NF-κB-dependent gene transcription. We hypothesized that targeting BET proteins for degradation with dBET1, a proteolysis targeting chimera (PROTAC) that combines the highly selective BET inhibitor JQ1 and a ligand for cereblon E3 ubiquitin ligase, will reduce brain injury in ischemic stroke. Male aged mice (18-20 months old) were subjected to permanent occlusion of the middle cerebral artery and received either vehicle or dBET1 (10 mg/kg; i.p.) at various times after stroke. Neurobehavioral tests were performed before (baseline) and at 24 and 48 h after stroke induction. Infarct volume was quantified at 48 h. Data showed that BET degradation significantly reduced infarct volume in permanent focal cerebral ischemia in aged mice, and this was associated with reduced brain levels of pro-inflammatory mediators including TNF-α, CXCL1, CXCL10, CCL2, and matrix metalloproteinase-9. Treatment with dBET1 significantly reduced blood-brain barrier damage and infiltration of neutrophils into the ischemic brain. Importantly, treatment with the BET degrader dBET1 resulted in a significant improvement in stroke-induced neurological deficits. Collectively, these data indicate that BET proteins are a novel target for neuroprotection in ischemic stroke.

PMID: 30872008 [PubMed - as supplied by publisher]

Seeking a selectivity solution: We have worked towards an extracellular version of the antibody–drug conjugate (ADC) modality by conjugating a nonselective protease inhibitor to a high‐affinity antibody targeting a single protease. Detailed studies demonstrated that antibody binding kinetics could overcome poor small‐molecule selectivity, thus enabling simultaneous and selective binding to the target.

Abstract

Antibody–drug conjugates (ADCs) are a growing class of therapeutics that harness the specificity of antibodies and the cell‐killing potency of small‐molecule drugs. Beyond cytotoxics, there are few examples of the application of an ADC approach to difficult drug discovery targets. Here, we present the initial development of a non‐internalising ADC, with a view to selectively inhibiting an extracellular protein. Employing the wellinvestigated matrix metalloproteinase‐9 (MMP‐9) as our model, we adapted a broad‐spectrum, nonselective MMP inhibitor for conjugation and linked this to a MMP‐9‐targeting antibody. The resulting ADC fully inhibits MMP‐9, and ELISA results suggest antibody targeting can direct a nonselective inhibitor.

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Bacterial Outer Membrane Vesicles Provide Broad-Spectrum Protection against Influenza Virus Infection via Recruitment and Activation of Macrophages.

J Innate Immun. 2019 Mar 07;:1-14

Authors: Bae EH, Seo SH, Kim CU, Jang MS, Song MS, Lee TY, Jeong YJ, Lee MS, Park JH, Lee P, Kim YS, Kim SH, Kim DJ

Abstract
Influenza A virus (IAV) poses a constant worldwide threat to human health. Although conventional vaccines are available, their protective efficacy is type or strain specific, and their production is time-consuming. For the control of an influenza pandemic in particular, agents that are immediately effective against a wide range of virus variants should be developed. Although pretreatment of various Toll-like receptor (TLR) ligands have already been reported to be effective in the defense against subsequent IAV infection, the efficacy was limited to specific subtypes, and safety concerns were also raised. In this study, we investigated the protective effect of an attenuated bacterial outer membrane vesicle -harboring modified lipid A moiety of lipopolysaccharide (fmOMV) against IAV infection and the underlying mechanisms. Administration of fmOMV conferred significant protection against a lethal dose of pandemic H1N1, PR8, H5N2, and highly pathogenic H5N1 viruses; this broad antiviral activity was dependent on macrophages but independent of neutrophils. fmOMV induced recruitment and activation of macrophages and elicited type I IFNs. Intriguingly, fmOMV showed a more significant protective effect than other TLR ligands tested in previous reports, without exhibiting any adverse effect. These results show the potential of fmOMV as a prophylactic agent for the defense against influenza virus infection.

PMID: 30844806 [PubMed - as supplied by publisher]

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Profiling the susceptibility of Pseudomonas aeruginosa strains from acute and chronic infections to cell-wall-targeting immune proteins.

Sci Rep. 2019 Mar 05;9(1):3575

Authors: Torrens G, Barceló IM, Pérez-Gallego M, Escobar-Salom M, Tur-Gracia S, Munar-Bestard M, González-Nicolau MDM, Cabrera-Venegas YJ, Rigo-Rumbos EN, Cabot G, López-Causapé C, Rojo-Molinero E, Oliver A, Juan C

Abstract
In the current scenario of high antibiotic resistance, the search for therapeutic options against Pseudomonas aeruginosa must be approached from different perspectives: cell-wall biology as source of bacterial weak points and our immune system as source of weapons. Our recent study suggests that once the permeability barrier has been overcome, the activity of our cell-wall-targeting immune proteins is notably enhanced, more in mutants with impaired peptidoglycan recycling. The present work aims at analyzing the activity of these proteins [lysozyme and Peptidoglycan-Recognition-Proteins (PGLYRPs)], alone or with a permeabilizer (subinhibitory colistin) in clinical strains, along with other features related to the cell-wall. We compared the most relevant and complementary scenarios: acute (bacteremia) and chronic infections [early/late isolates from lungs of cystic fibrosis (CF) patients]. Although a low activity of lysozyme/PGLYRPs per se (except punctual highly susceptible strains) was found, the colistin addition significantly increased their activity regardless of the strains' colistin resistance levels. Our results show increased susceptibility in late CF isolates, suggesting that CF adaptation renders P. aeruginosa more vulnerable to proteins targeting the cell-wall. Thus, our work suggests that attacking some P. aeruginosa cell-wall biology-related elements to increase the activity of our innate weapons could be a promising therapeutic strategy.

PMID: 30837659 [PubMed - in process]

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Antibody neutralization of microbiota-derived circulating peptidoglycan dampens inflammation and ameliorates autoimmunity.

Nat Microbiol. 2019 Mar 04;:

Authors: Huang Z, Wang J, Xu X, Wang H, Qiao Y, Chu WC, Xu S, Chai L, Cottier F, Pavelka N, Oosting M, Joosten LAB, Netea M, Ng CYL, Leong KP, Kundu P, Lam KP, Pettersson S, Wang Y

Abstract
The human microbiota provides tonic signals that calibrate the host immune response1,2, but their identity is unknown. Bacterial peptidoglycan (PGN) subunits are likely candidates since they are well-known immunity-enhancing adjuvants, released by most bacteria during growth, and have been found in the blood of healthy people3-7. We developed a monoclonal antibody (mAb), 2E7, that targets muramyl-L-alanyl-D-isoglutamine (MDP), a conserved and minimal immunostimulatory structure of PGN. Using 2E7-based assays, we detected PGN ubiquitously in human blood at a broad range of concentrations that is relatively stable in each individual. We also detected PGN in the serum of several warm-blooded animals. However, PGN is barely detectable in the serum of germ-free mice, indicating that its origin is the host microbiota. Neutralization of circulating PGN via intraperitoneal administration of 2E7 suppressed the development of autoimmune arthritis and experimental autoimmune encephalomyelitis in mice. Arthritic NOD2-/- mice lacking the MDP sensor did not respond to 2E7, indicating that 2E7 dampens inflammation by blocking nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-mediated pathways. We propose that circulating PGN acts as a natural immune potentiator that tunes the host immune response; altering its level is a promising therapeutic strategy for immune-mediated diseases.

PMID: 30833732 [PubMed - as supplied by publisher]

Increasing resistance and a lack of new antibiotics are a serious problem for public health. Against this background, Gilles van Wezel of the Institute of Biology Leiden is looking for new medicines. Together with former Ph.D. student Changsheng Wu and colleagues he discovered the special antibiotic lugdunomycin, which they named after Leiden. The discovery was recently published in the journal Angewandte Chemie.

Indiana University researchers are advancing knowledge about how bacteria build their cell walls that could contribute to the search for new antibacterial drugs. They have created a new tool to observe living cells in real time under a microscope.

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Antibiotic resistance and host immune evasion in Staphylococcus aureus mediated by a metabolic adaptation.

Proc Natl Acad Sci U S A. 2019 Feb 26;116(9):3722-3727

Authors: Jiang JH, Bhuiyan MS, Shen HH, Cameron DR, Rupasinghe TWT, Wu CM, Le Brun AP, Kostoulias X, Domene C, Fulcher AJ, McConville MJ, Howden BP, Lieschke GJ, Peleg AY

Abstract
Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2, encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen.

PMID: 30808758 [PubMed - in process]

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Recent paradigm shift in the assembly of bacterial tripartite efflux pumps and the type I secretion system.

J Microbiol. 2019 Mar;57(3):185-194

Authors: Jo I, Kim JS, Xu Y, Hyun J, Lee K, Ha NC

Abstract
Tripartite efflux pumps and the type I secretion system of Gram-negative bacteria are large protein complexes that span the entire cell envelope. These complexes expel antibiotics and other toxic substances or transport protein toxins from bacterial cells. Elucidating the binary and ternary complex structures at an atomic resolution are crucial to understanding the assembly and working mechanism. Recent advances in cryoelectron microscopy along with the construction of chimeric proteins drastically shifted the assembly models. In this review, we describe the current assembly models from a historical perspective and emphasize the common assembly mechanism for the assembly of diverse tripartite pumps and type I secretion systems.

PMID: 30806976 [PubMed - in process]

During the co‐evolution of bacteria and phages, a mutant evolved phage gained the capacity to adhere to a different, non‐host species, thereby crossing the species barrier.

Summary

Bacteriophages (phages) are the most abundant entities in nature, yet little is known about their capacity to acquire new hosts and invade new niches. By exploiting the Gram positive soil bacterium Bacillus subtilis (B. subtilis) and its lytic phage SPO1 as a model, we followedthe co‐evolution of bacteria and phages. After infection, phageresistant bacteria were readily isolated. These bacteria were defective in production of glycosylated wall teichoic acid (WTA) polymers thatserved as SPO1 receptor. Subsequently, a SPO1mutant phage that could infect the resistant bacteriaevolved.The emerging phage contained mutations in two genes, encoding the baseplate and fibers required for host attachment. Remarkably, the mutant phage gained the capacity to infect non‐hostBacillus species that are not infected by the wild type phage. We provide evidence that the evolved phage lost its dependency on the species specificglycosylation pattern of WTA polymers. Instead, the mutant phagegained the capacity to directly adhere to the WTA backbone, conserved among different species, thereby crossing the species barrier.

This article is protected by copyright. All rights reserved.

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Development of dual and selective degraders of cyclin-dependent kinases 4 and 6.

Angew Chem Int Ed Engl. 2019 Feb 25;:

Authors: Jiang B, Wang ES, Donovan KA, Liang Y, Fischer ES, Zhang T, Gray NS

Abstract
Cyclin-dependent kinases 4 and 6 (CDK4/6) are key regulators of the cell cycle, and CDK4/6 inhibitors are FDA-approved for treating patients with metastatic breast cancer. However, due to conservation of their ATP-binding sites, development of selective agents has remained elusive. Here, we report imide-based degrader molecules capable of degrading both CDK4/6, or selectively degrading either CDK4 or CDK6. We were also able to tune the activity of these molecules against Ikaros (IKZF1) and Aiolos (IKZF3), well-established targets of imide-based degraders. We found that in mantle cell lymphoma cell lines, combined IKZF1/3 degradation with dual CDK4/6 degradation exhibited enhanced anti-proliferative effects compared to CDK4/6 inhibition, CDK4/6 degradation, or IKZF1/3 degradation. In sum, we report here the first compounds capable of inducing selective degradation of CDK4 and CDK6 as tools to pharmacologically dissect their distinct biological functions.

PMID: 30802347 [PubMed - as supplied by publisher]

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PROTAC-Mediated Degradation of Bruton's Tyrosine Kinase is Inhibited by Covalent Binding.

ACS Chem Biol. 2019 Feb 26;:

Authors: Tinworth CP, Lithgow H, Dittus L, Bassi ZI, Hughes SE, Muelbaier M, Dai H, Smith IED, Kerr WJ, Burley GA, Bantscheff M, Harling JD

Abstract
The impact of covalent binding on PROTAC-mediated degradation of BTK was investigated through the preparation of both covalent binding and reversible binding PROTACs derived from the covalent BTK inhibitor ibrutinib. It was determined that a covalent binding PROTAC inhibited BTK degradation despite evidence of target engagement, while BTK degradation was observed with a reversible binding PROTAC. These observations were consistently found when PROTACs were employed that were able to recruit either IAP or cereblon E3 ligases. Proteomics analysis determined that use of a covalently bound PROTAC did not result in the degradation of covalently bound targets, whilst degradation was observed for some reversibly bound targets. This observation highlights the importance of catalysis on successful PROTAC-mediated degradation, and highlights a potential caveat for the use of covalent target binders in PROTAC design.

PMID: 30807093 [PubMed - as supplied by publisher]

ABSTRACT

Assembly of the division machinery in Gram-negative and Gram-positive bacteria occurs in two time-dependent steps. First, the FtsZ proto-ring localizes at midcell including some FtsN molecules. Subsequently, the proteins that catalyze and regulate septal peptidoglycan (PG) synthesis are recruited including among others, the FtsBLQ-PB1B-FtsW-PBP3 complex. Further accumulation of FtsN finally allows initiation of cell division. It was known that FtsA and FtsQLB somehow prevented this initiation. Recently, A. Boes, S. Olatunji, E. Breukink, and M. Terrak (mBio 10:e01912-18, 2019, https://doi.org/10.1128/mBio.01912-18) reported that this is caused by inhibition of the activity of the PG synthases by FtsBLQ, which has to be outcompeted by accumulation of the PBP1b activating FtsN. This supports a central structural as well as regulatory role for the FtsBLQ protein complex that is conserved only in prokaryotes, making it an attractive target for antibiotic development.

ABSTRACT

Manganese (Mn) is an essential micronutrient critical for the pathogenesis of Staphylococcus aureus, a significant cause of human morbidity and mortality. Paradoxically, excess Mn is toxic; therefore, maintenance of intracellular Mn homeostasis is required for survival. Here we describe a Mn exporter in S. aureus, MntE, which is a member of the cation diffusion facilitator (CDF) protein family and conserved among Gram-positive pathogens. Upregulation of mntE transcription in response to excess Mn is dependent on the presence of MntR, a transcriptional repressor of the mntABC Mn uptake system. Inactivation of mntE or mntR leads to reduced growth in media supplemented with Mn, demonstrating MntE is required for detoxification of excess Mn. Inactivation of mntE results in elevated levels of intracellular Mn, but reduced intracellular iron (Fe) levels, supporting the hypothesis that MntE functions as a Mn efflux pump and Mn efflux influences Fe homeostasis. Strains inactivated for mntE are more sensitive to the oxidants NaOCl and paraquat, indicating Mn homeostasis is critical for resisting oxidative stress. Furthermore, mntE and mntR are required for full virulence of S. aureus during infection, suggesting S. aureus experiences Mn toxicity in vivo. Combined, these data support a model in which MntR controls Mn homeostasis by balancing transcriptional repression of mntABC and induction of mntE, both of which are critical for S. aureus pathogenesis. Thus, Mn efflux contributes to bacterial survival and virulence during infection, establishing MntE as a potential antimicrobial target and expanding our understanding of Mn homeostasis.

IMPORTANCE Manganese (Mn) is generally viewed as a critical nutrient that is beneficial to pathogenic bacteria due to its function as an enzymatic cofactor and its capability of acting as an antioxidant; yet paradoxically, high concentrations of this transition metal can be toxic. In this work, we demonstrate Staphylococcus aureus utilizes the cation diffusion facilitator (CDF) family protein MntE to alleviate Mn toxicity through efflux of excess Mn. Inactivation of mntE leads to a significant reduction in S. aureus resistance to oxidative stress and S. aureus-mediated mortality within a mouse model of systemic infection. These results highlight the importance of MntE-mediated Mn detoxification in intracellular Mn homeostasis, resistance to oxidative stress, and S. aureus virulence. Therefore, this establishes MntE as a potential target for development of anti-S. aureus therapeutics.

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Bivalent Ligands for Protein Degradation in Drug Discovery.

Comput Struct Biotechnol J. 2019;17:160-176

Authors: Scheepstra M, Hekking KFW, van Hijfte L, Folmer RHA

Abstract
Targeting the "undruggable" proteome remains one of the big challenges in drug discovery. Recent innovations in the field of targeted protein degradation and manipulation of the ubiquitin-proteasome system open up new therapeutic approaches for disorders that cannot be targeted with conventional inhibitor paradigms. Proteolysis targeting chimeras (PROTACs) are bivalent ligands in which a compound that binds to the protein target of interest is connected to a second molecule that binds an E3 ligase via a linker. The E3 protein is usually either Cereblon or Von Hippel-Lindau. Several examples of selective PROTAC molecules with potent effect in cells and in vivo models have been reported. The degradation of specific proteins via these bivalent molecules is already allowing for the study of biochemical pathways and cell biology with more specificity than was possible with inhibitor compounds. In this review, we provide a comprehensive overview of recent developments in the field of small molecule mediated protein degradation, including transcription factors, kinases and nuclear receptors. We discuss the potential benefits of protein degradation over inhibition as well as the challenges that need to be overcome.

PMID: 30788082 [PubMed]

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PBP Isolation and DD-Carboxypeptidase Assay.

Methods Mol Biol. 2019;1946:207-225

Authors: Pal S, Ghosh AS

Abstract
Penicillin-binding proteins (PBPs) share the namesake because of their ability to bind penicillin or any beta-lactam antibiotic. In other words, PBPs are the targets of β-lactam antibiotics that hold nearly 60% of the global antibiotic market. These enzymes catalyze the final stages of peptidoglycan (PG) biosynthesis by acting as transglycosylases and transpeptidases. PBPs are also involved in PG remodeling by catalyzing DD-carboxypeptidase (DD-CPase) and endopeptidase reactions. Though the cross-linking abilities of PBPs are well known, the process of remodeling is still unclear, thereby drawing attention toward the DD-CPase enzymes. Here, we describe the step-by-step procedures for isolation of the bacterial cell membrane and detection of PBPs in it, followed by the purification of PBPs (DD-CPases) by both ampicillin-affinity and nickel-nitrilotriacetic acid (Ni-NTA) chromatography. The protocols to determine the enzymatic efficiency are also elucidated. The assays are aimed to determine the kinetic parameters for the interaction of the PBP with BOCILLIN, to evaluate its acylation and deacylation rates, and with its peptide substrates, to assess its DD-CPase activity.

PMID: 30798558 [PubMed - in process]

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VraSR and Virulence Trait Modulation during Daptomycin Resistance in Methicillin-Resistant Staphylococcus aureus Infection.

mSphere. 2019 Feb 13;4(1):

Authors: Taglialegna A, Varela MC, Rosato RR, Rosato AE

Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) threatens human health in hospital and community settings. The lipopeptide antibiotic daptomycin (DAP) is a frequently used treatment option for MRSA infection. DAP exposure can cause bacterial resistance because mutations are induced in genes implicated in cell membrane and cell wall metabolism. Adaptations aimed at surviving antimicrobial pressure can affect bacterial physiology and modify in vivo aptitude and pathogenesis. In this study, clinical DAP-susceptible (DAPs) and DAP-resistant (DAPr) MRSA isolates were used to investigate associations between DAP resistance and staphylococcal virulence. We previously found that VraSR is a critical sensor of cell membrane/wall homeostasis associated with DAP acquisition during MRSA infection. The present study found that DAPr CB1634 and CB5014 MRSA strains with vraSR upregulation were less virulent than their susceptible counterparts, CB1631 and CB5013. Differential gene-transcription profile analysis revealed that DAPr CB1634 had decreased agr two-component system expression, virulence factors, and highly suppressed hemolysis activity. Functional genetic analysis performed in DAPr CB1634 strains using vraSR inactivation followed by gene complementation found that vraSR acted as a transcriptional agrA regulator. These results indicated that VraSR has a broad range of regulatory functions. VraSR also appeared to affect DAPr adherence to epithelial cells, which would affect DAPr strain colonization and survival in the host. The correlation between DAP resistance and decreased virulence was also found in the CB5013 (DAPs) and CB5014 (DAPr) pair. Taken together, these findings are the first evidence that DAP resistance and MRSA virulence are tightly connected and involve compromised expression of regulatory and virulence determinants.IMPORTANCE Methicillin-resistant S. aureus continues to develop resistance to antimicrobials, including those in current clinical use as daptomycin (DAP). Resistance to DAP arises by mutations in cell membrane and cell wall genes and/or upregulation of the two-component VraSR system. However, less is known about the connection between the pathogen and virulence traits during DAP resistance development. We provide new insights into VraSR and its regulatory role for virulence factors during DAP resistance, highlighting coordinated interactions that favor the higher persistence of MRSA DAP-resistant strains in the infected host.

PMID: 30760612 [PubMed - in process]

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The cyclic peptide labaditin does not alter the outer membrane integrity of Salmonella enterica serovar Typhimurium.

Sci Rep. 2019 Feb 13;9(1):1993

Authors: Barbosa SC, Nobre TM, Volpati D, Cilli EM, Correa DS, Oliveira ON

Abstract
Antimicrobial peptides are a promising class of new antibiotics with the ability to kill bacteria by disrupting their cell membrane, which is especially difficult for Gram-negative bacteria whose cell wall contains an outer layer of lipopolysaccharides (LPS). Here we show that the cyclic decapeptide Labaditin (Lo), with proven activity against the Gram-positive Staphylococcus aureus and Streptococcus mutans, is not able to kill the Gram-negative Salmonella enterica serovar Typhimurium (S.e.s. Typhimurium). We found that Lo induced significant changes in the surface pressure isotherms of Langmuir monolayers representing the Salmonella enterica serovar Typhimurium inner membrane (S.e.s. Typhimurium IM), and caused leakage in large unilamellar vesicles made with this IM lipid composition. On the basis of these results one should expect bactericidal activity against S.e.s. Typhimurium. However, Lo could not interact with a monolayer of LPS, causing no significant changes in either the surface pressure isotherms or in the polarization-modulated infrared reflection absorption spectra (PM-IRRAS). Therefore, the failure of Lo to kill S.e.s. Typhimurium is associated with the lack of interaction with LPS from the outer bacteria membrane. Our approach with distinct monolayer compositions and combined techniques to investigate molecular-level interactions is useful for drug design to fight antibiotic-resistant bacteria.

PMID: 30760803 [PubMed - in process]

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Peptidoglycan Remodeling Enables Escherichia coli To Survive Severe Outer Membrane Assembly Defect.

MBio. 2019 Feb 05;10(1):

Authors: Morè N, Martorana AM, Biboy J, Otten C, Winkle M, Serrano CKG, Montón Silva A, Atkinson L, Yau H, Breukink E, den Blaauwen T, Vollmer W, Polissi A

Abstract
Gram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show that Escherichia coli cells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.IMPORTANCE In Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show that Escherichia coli cells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane.

PMID: 30723128 [PubMed - in process]

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SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate.

ACS Chem Biol. 2019 Feb 05;:

Authors: Roy M, Winkler S, Hughes SJ, Whitworth C, Galant M, Farnaby W, Rumpel K, Ciulli A

Abstract
Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target:PROTAC:ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of BET proteins Brd2, Brd3 and Brd4 to the E3 ligase VHL. We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4BD2 or Brd2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilised due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.

PMID: 30721025 [PubMed - as supplied by publisher]

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Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae.

Proc Natl Acad Sci U S A. 2019 Feb 04;:

Authors: Perez AJ, Cesbron Y, Shaw SL, Bazan Villicana J, Tsui HT, Boersma MJ, Ye ZA, Tovpeko Y, Dekker C, Holden S, Winkler ME

Abstract
Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae.

PMID: 30718427 [PubMed - as supplied by publisher]

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In silico Modeling of PROTAC-Mediated Ternary Complexes: Validation and Application.

J Chem Inf Model. 2019 Feb 04;:

Authors: Drummond ML, Willaims C

Abstract
In this work, four methods are described and validated for generating in silico ensembles of PROTAC-mediated ternary complexes. Filters based on characteristics of the proposed ternary complexes are developed to identify those that resemble known crystal structures. We then show how to use these modeling techniques a priori to discriminate the PROTAC-mediated degradation behavior of a mutant protein vs. its wild type, of three closely related targets, and among three different PROTAC molecules.

PMID: 30714732 [PubMed - as supplied by publisher]

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Binding from Both Sides: TolR and Full-Length OmpA Bind and Maintain the Local Structure of the E. coli Cell Wall.

Structure. 2019 Jan 17;:

Authors: Boags AT, Samsudin F, Khalid S

Abstract
We present a molecular modeling and simulation study of the E. coli cell envelope, with a particular focus on the role of TolR, a native protein of the E. coli inner membrane, in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed binds peptidoglycan largely through electrostatic interactions. Non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides, respectively, maintain the position of the cell wall even in the absence of Braun's lipoprotein. The charged residues that mediate the cell-wall interactions of TolR in our simulations are conserved across a number of species of gram-negative bacteria.

PMID: 30713026 [PubMed - as supplied by publisher]