Rebecca Page

A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS.

SUMMARY Among bacterial toxin/antitoxin (TA) systems, to date no antitoxin has been identified that functions by cleaving toxin mRNA. Here we demonstrate YjdO (renamed GhoT) is a membrane lytic peptide that causes ghost cell formation (lysed cells with damaged membranes) and increases persistence (persister cells are tolerant to antibiotics without undergoing genetic change). GhoT is part of a novel TA system with YjdK (renamed GhoS) since in vitro RNA degradation studies, qRT-PCR, and whole-transcriptome studies revealed GhoS masks GhoT toxicity by cleaving specifically ghoT mRNA. Alanine substitutions showed arginine 28 is important for GhoS activity, and RNA sequencing indicated the GhoS cleavage site is rich in uridine and adenosine. The NMR structure of GhoS indicates it is related to the CAS2 CRISPR RNase, and GhoS is a monomer. Hence, GhoT/GhoS is the first type V TA system where a protein antitoxin inhibits the toxin by cleaving specifically its mRNA.

Toxin-antitoxin systems in bacterial growth arrest and persistence

Bacterial persister cells constitute a subpopulation of genetically identical, metabolically slow-growing cells that are highly tolerant of antibiotics and other environmental stresses. Recent studies have demonstrated that gene loci known as toxin-antitoxin (TA) modules play a central role in the persister state. Under normal growth conditions, antitoxins potently inhibit the activities of the toxins. In contrast, under conditions of stress, the antitoxins are selectively degraded, freeing the toxins to inhibit essential cellular processes, such as DNA replication and protein translation. This inhibition results in rapid growth arrest. In this Review, we highlight recent discoveries of these multifaceted TA systems with a focus on the newly uncovered mechanisms, especially conditional cooperativity, that are used to regulate cell growth and persistence. We also discuss the potential for targeting TA systems for antimicrobial drug discovery.

Antitoxin MqsA helps mediate the bacterial general stress response

SUMMARY Although it is well-recognized that bacteria respond to environmental stress via global networks, the mechanism by which stress is relayed to the interior of the cell is poorly understood. Here we show that enigmatic toxin/antitoxin systems play a vital role in mediating the environmental stress response. Specifically, the antitoxin MqsA represses rpoS, which encodes the master regulator of stress. Repression of rpoS by MqsA reduces the concentration of the internal messenger 3,5-cyclic diguanylic acid, leading to increased motility and decreased biofilm formation. Furthermore, the repression of rpoS by MqsA decreases oxidative stress resistance via catalase activity. Upon oxidative stress, MqsA is rapidly degraded by Lon protease resulting in induction of rpoS. Hence, we show that external stress alters gene regulation controlled by toxin/antitoxin systems, such that the degradation of antitoxins during stress leads to a switch from the planktonic state (high motility) to the biofilm state (low motility).

Targeting the disordered C terminus of PTP1B with an allosteric inhibitor.

PTP1B, a validated therapeutic target for diabetes and obesity, plays a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We defined a novel mechanism of allosteric inhibition that targets the C-terminal, non-catalytic segment of PTP1B. We present the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small molecule inhibitor, MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. This new approach to inhibition of PTP1B emphasizes the potential of disordered segments of proteins as specific binding sites for therapeutic small molecules.

Three dimensional structure of the MqsR:MqsA complex: a novel TA pair comprised of a toxin homologous to RelE and an antitoxin with unique properties.

One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an α/β fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state.

Escherichia coli toxin/antitoxin pair MqsR/MqsA regulate toxin CspD

Previously we identified that the Escherichia coli protein MqsR (YgiU) functions as a toxin and that it is involved in the regulation of motility by quorum sensing signal autoinducer-2 (AI-2). Furthermore, MqsR is directly associated with biofilm development and is linked to the development of persister cells. Here we show that MqsR and MqsA (YgiT) are a toxin/antitoxin (TA) pair, which, in significant difference to other TA pairs, regulates additional loci besides its own. We have recently identified that MqsR functions as an RNase. However, using three sets of whole-transcriptome studies and two nickel-enrichment DNA binding microarrays coupled with cell survival studies in which MqsR was overproduced in isogenic mutants, we identified eight genes (cspD, clpX, clpP, lon, yfjZ, relB, relE and hokA) that are involved in a mode of MqsR toxicity in addition to its RNase activity. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) showed that (i) the MqsR/MqsA complex (and MqsA alone) represses the toxin gene cspD, (ii) MqsR overproduction induces cspD, (iii) stress induces cspD, and (iv) stress fails to induce cspD when MqsR/MqsA are overproduced or when mqsRA is deleted. Electrophoretic mobility shift assays show that the MqsA/MqsR complex binds the promoter of cspD. In addition, proteases Lon and ClpXP are necessary for MqsR toxicity. Together, these results indicate the MqsR/MqsA complex represses cspD which may be derepressed by titrating MqsA with MqsR or by degrading MqsA via stress conditions through proteases Lon and ClpXP. Hence, we demonstrate that the MqsR/MqsA TA system controls cell physiology via its own toxicity as well as through its regulation of another toxin, CspD.

Spinophilin directs protein phosphatase 1 specificity by blocking substrate binding sites.

The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets. PP1 associates with ≥200 regulatory proteins to form highly specific holoenzymes. These regulatory proteins target PP1 to its point of action within the cell and prime its enzymatic specificity for particular substrates. However, how they direct PP1s specificity is not understood. Here we show that spinophilin, a neuronal PP1 regulator, is entirely unstructured in its unbound form, and it binds PP1 through a folding-upon-binding mechanism in an elongated fashion, blocking one of PP1s three putative substrate binding sites without altering its active site. This mode of binding is sufficient for spinophilin to restrict PP1s activity toward a model substrate in vitro without affecting its ability to dephosphorylate its neuronal substrate, glutamate receptor 1 (GluR1). Thus, our work provides the molecular basis for the ability of spinophilin to dictate PP1 substrate specificity.

Structural basis for protein phosphatase 1 regulation and specificity

The ubiquitous serine/threonine protein phosphatase 1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. However, the free catalytic subunit of PP1, while an effective enzyme, lacks substrate specificity. Instead, it depends on a diverse set of regulatory proteins (≥ 200) to confer specificity towards distinct substrates. Here, we discuss recent advances in structural studies of PP1 holoenzyme complexes and summarize the new insights these studies have provided into the molecular basis of PP1 regulation and specificity.

LYP inhibits T-cell activation when dissociated from CSK

Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here, we studied the spatio-temporal dynamics of the LYP/CSK complex in T cells. We demonstrate that dissociation of this complex is necessary for recruitment of LYP to the plasma membrane, where it down-modulates TCR signaling. Development of a potent and selective chemical probe of LYP confirmed that LYP inhibits T cell activation when removed from CSK. Our findings may explain the reduced TCR-mediated signaling associated with a single nucleotide polymorphism, which confers increased risk for certain autoimmune diseases, including type 1 diabetes and rheumatoid arthritis, and results in expression of a LYP allele that is unable to bind CSK. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity.

Molecular Basis of MAP Kinase Regulation

Mitogen‐activated protein kinases (MAPKs; ERK1/2, p38, JNK, and ERK5) have evolved to transduce environmental and developmental signals (growth factors, stress) into adaptive and programmed responses (differentiation, inflammation, apoptosis). Almost 20 years ago, it was discovered that MAPKs contain a docking site in the C‐terminal lobe that binds a conserved 13‐16 amino acid sequence known as the D‐ or KIM‐motif (kinase interaction motif). Recent crystal structures of MAPK:KIM‐peptide complexes are leading to a precise understanding of how KIM sequences contribute to MAPK selectivity. In addition, new crystal and especially NMR studies are revealing how residues outside the canonical KIM motif interact with specific MAPKs and contribute further to MAPK selectivity and signaling pathway fidelity. In this review, we focus on these recent studies, with an emphasis on the use of NMR spectroscopy, isothermal titration calorimetry and small angle X‐ray scattering to investigate these processes.