Measuring Glutathione Redox Potential of HIV-1-infected Macrophages

Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E-GSH; Grx1-roGFP2) and measured subcellular changes in E-GSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E-GSH (approximately -310 mV), active viral replication induces substantial oxidative stress (E-GSH more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E-GSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about similar to 25 mV in E-GSH is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E-GSH. Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.

Activation of InsP(3) receptors is sufficient for inducing graded intrinsic plasticity in rat hippocampal pyramidal neurons

The synaptic plasticity literature has focused on establishing necessity and sufficiency as two essential and distinct features in causally relating a signaling molecule to plasticity induction, an approach that has been surprisingly lacking in the intrinsic plasticity literature. In this study, we complemented the recently established necessity of inositol trisphosphate (InsP(3)) receptors (InsP(3)R) in a form of intrinsic plasticity by asking if InsP(3)R activation was sufficient to induce intrinsic plasticity in hippocampal neurons. Specifically, incorporation of D-myo-InsP(3) in the recording pipette reduced input resistance, maximal impedance amplitude, and temporal summation but increased resonance frequency, resonance strength, sag ratio, and impedance phase lead. Strikingly, the magnitude of plasticity in all these measurements was dependent on InsP 3 concentration, emphasizing the graded dependence of such plasticity on InsP(3)R activation. Mechanistically, we found that this InsP(3)-induced plasticity depended on hyperpolarization-activated cyclic nucleotide-gated channels. Moreover, this calcium-dependent form of plasticity was critically reliant on the release of calcium through InsP(3)Rs, the influx of calcium through N-methyl-D-aspartate receptors and voltage-gated calcium channels, and on the protein kinase A pathway. Our results delineate a causal role for InsP(3)Rs in graded adaptation of neuronal response dynamics, revealing novel regulatory roles for the endoplasmic reticulum in neural coding and homeostasis.

Mycobacterium tuberculosis has diminished capacity to counteract redox stress induced by elevated levels of endogenous superoxide

Mycobacterium tuberculosis (Mtb) has evolved protective and detoxification mechanisms to maintain cytoplasmic redox balance in response to exogenous oxidative stress encountered inside host phagocytes. In contrast, little is known about the dynamic response of this pathogen to endogenous oxidative stress generated within Mtb. Using a noninvasive and specific biosensor of cytoplasmic redox state of Mtb, we for first time discovered a surprisingly high sensitivity of this pathogen to perturbation in redox homeostasis induced by elevated endogenous reactive oxygen species (ROS). We synthesized a series of hydroquinone-based small molecule ROS generators and found that ATD-3169 permeated mycobacteria to reliably enhance endogenous ROS including superoxide radicals. When Mtb strains including multidrug-resistant (MDR) and extensively drug-resistant (XDR) patient isolates were exposed to this compound, a dose-dependent, long-lasting, and irreversible oxidative shift in intramycobacterial redox potential was detected. Dynamic redox potential measurements revealed that Mtb had diminished capacity to restore cytoplasmic redox balance in comparison with Mycobacterium smegmatis (Msm), a fast growing nonpathogenic mycobacterial species. Accordingly, Mtb strains were extremely susceptible to inhibition by ATD-3169 but not Msm, suggesting a functional linkage between dynamic redox changes and survival. Microarray analysis showed major realignment of pathways involved in redox homeostasis, central metabolism, DNA repair, and cell wall lipid biosynthesis in response to ATD-3169, all consistent with enhanced endogenous ROS contributing to lethality induced by this compound. This work provides empirical evidence that the cytoplasmic redox poise of Mtb is uniquely sensitive to manipulation in steady-state endogenous ROS levels, thus revealing the importance of targeting intramycobacterial redox metabolism for controlling TB infection. (C) 2015 The Authors. Published by Elsevier Inc.

Functional characterization of heat-shock protein 90 from Oryza sativa and crystal structure of its N-terminal domain

Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 angstrom resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 from Dictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source.

Remarkable Effect of Chalcogen Substitution on an Enzyme Mimetic for Deiodination of Thyroid Hormones

Iodothyronine deiodinases are selenoenzymes which regulate the thyroid hormone homeostasis by catalyzing the regioselective deiodination of thyroxine (T4). Synthetic deiodinase mimetics are important not only to understand the mechanism of enzyme catalysis, but also to develop therapeutic agents as abnormal thyroid hormone levels have implications in different diseases, such as hypoxia, myocardial infarction, critical illness, neuronal ischemia, tissue injury, and cancer. Described herein is that the replacement of sulfur/selenium atoms in a series of deiodinase mimetics by tellurium remarkably alters the reactivity as well as regioselectivity toward T4. The tellurium compounds reported in this paper represent the first examples of deiodinase mimetics which mediate sequential deiodination of T4 to produce all the hormone derivatives including T0 under physiologically relevant conditions.

Robust Glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI Family Member Protein, Is Critical for Oxidative Stress Resistance in Saccharomyces cerevisiae

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. The accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging, and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, their functions are largely dependent on the glutathione cofactor, the availability of which is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein, a member of the ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae, plays an indispensable role in regulating redox homeostasis. Our results show that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared with another paralog, Hsp34. On the other hand, glyoxalase-defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. Additionally, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress, and Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1, which is implicated in the familial form of Parkinson disease, complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying the importance of these proteins in the maintenance of ROS homeostasis across phylogeny.

Chemical Shifts to Metabolic Pathways: Identifying Metabolic Pathways Directly from a Single 2D NMR Spectrum

Identifying cellular processes in terms of metabolic pathways is one of the avowed goals of metabolomics studies. Currently, this is done after relevant metabolites are identified to allow their mapping onto specific pathways. This task is daunting due to the complex nature of cellular processes and the difficulty in establishing the identity of individual metabolites. We propose here a new method: ChemSMP (Chemical Shifts to Metabolic Pathways), which facilitates rapid analysis by identifying the active metabolic pathways directly from chemical shifts obtained from a single two-dimensional (2D) C-13-H-1] correlation NMR spectrum without the need for identification and assignment of individual metabolites. ChemSMP uses a novel indexing and scoring system comprised of a ``uniqueness score'' and a ``coverage score''. Our method is demonstrated on metabolic pathways data from the Small Molecule Pathway Database (SMPDB) and chemical shifts from the Human Metabolome Database (HMDB). Benchmarks show that ChemSMP has a positive prediction rate of >90% in the presence of deduttered data and can sustain the same at 60-70% even in the presence of noise, such as deletions of peaks and chemical shift deviations. The method tested on NMR data acquired for a mixture of 20 amino acids shows a success rate of 93% in correct recovery of pathways. When used on data obtained from the cell lysate of an unexplored oncogenic cell line, it revealed active metabolic pathways responsible for regulating energy homeostasis of cancer cells. Our unique tool is thus expected to significantly enhance analysis of NMIR-based metabolomics data by reducing existing impediments.

First-in-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating M-tuberculosis

Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38?350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type Mtb as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional Delta APSR mutant, which could be restored by complementation with Mtb APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live Mtb, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between Mtb persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value.

The mycobacterial iron-dependent regulator IdeR induces ferritin (bfrB) by alleviating Lsr2 repression

Emerging evidence indicates that precise regulation of iron (Fe) metabolism and maintenance of Fe homeostasis in Mycobacterium tuberculosis (Mtb) are essential for its survival and proliferation in the host. IdeR is a central transcriptional regulator of Mtb genes involved in Fe metabolism. While it is well understood how IdeR functions as a repressor, how it induces transcription of a subset of its targets is still unclear. We investigated the molecular mechanism of IdeR-mediated positive regulation of bfrB, the gene encoding the major Fe-storage protein of Mtb. We found that bfrB induction by Fe required direct interaction of IdeR with a DNA sequence containing four tandem IdeR-binding boxes located upstream of the bfrB promoter. Results of in vivo and in vitro transcription assays identified a direct repressor of bfrB, the histone-like protein Lsr2. IdeR counteracted Lsr2-mediated repression in vitro, suggesting that IdeR induces bfrB transcription by antagonizing the repressor activity of Lsr2. Together, these results elucidate the main mechanism of bfrB positive regulation by IdeR and identify Lsr2 as a new factor contributing to Fe homeostasis in mycobacteria.

SYNGAP1: Mind the Gap

A cardinal feature of early stages of human brain development centers on the sensory, cognitive, and emotional experiences that shape neuronal-circuit formation and refinement. Consequently, alterations in these processes account for many psychiatric and neurodevelopmental disorders. Neurodevelopment disorders affect 3-4% of the world population. The impact of these disorders presents a major challenge to clinicians, geneticists, and neuroscientists. Mutations that cause neurodevelopmental disorders are commonly found in genes encoding proteins that regulate synaptic function. Investigation of the underlying mechanisms using gain or loss of function approaches has revealed alterations in dendritic spine structure, function, and plasticity, consequently modulating the neuronal circuit formation and thereby raising the possibility of neurodevelopmental disorders resulting from synaptopathies. One such gene, SYNGAP1 (Synaptic Ras-GTPase-activating protein) has been shown to cause Intellectual Disability (ID) with comorbid Autism Spectrum Disorder (ASD) and epilepsy in children. SYNGAP1 is a negative regulator of Ras, Rap and of AMPA receptor trafficking to the postsynaptic membrane, thereby regulating not only synaptic plasticity, but also neuronal homeostasis. Recent studies on the neurophysiology of SYNGAP1, using Syngapl mouse models, have provided deeper insights into how downstream signaling proteins and synaptic plasticity are regulated by SYNGAP1. This knowledge has led to a better understanding of the function of SYNGAP1 and suggests a potential target during critical period of development when the brain is more susceptible to therapeutic intervention.

SYNGAP1: Mind the Gap

A cardinal feature of early stages of human brain development centers on the sensory, cognitive, and emotional experiences that shape neuronal-circuit formation and refinement. Consequently, alterations in these processes account for many psychiatric and neurodevelopmental disorders. Neurodevelopment disorders affect 3-4% of the world population. The impact of these disorders presents a major challenge to clinicians, geneticists, and neuroscientists. Mutations that cause neurodevelopmental disorders are commonly found in genes encoding proteins that regulate synaptic function. Investigation of the underlying mechanisms using gain or loss of function approaches has revealed alterations in dendritic spine structure, function, and plasticity, consequently modulating the neuronal circuit formation and thereby raising the possibility of neurodevelopmental disorders resulting from synaptopathies. One such gene, SYNGAP1 (Synaptic Ras-GTPase-activating protein) has been shown to cause Intellectual Disability (ID) with comorbid Autism Spectrum Disorder (ASD) and epilepsy in children. SYNGAP1 is a negative regulator of Ras, Rap and of AMPA receptor trafficking to the postsynaptic membrane, thereby regulating not only synaptic plasticity, but also neuronal homeostasis. Recent studies on the neurophysiology of SYNGAP1, using Syngapl mouse models, have provided deeper insights into how downstream signaling proteins and synaptic plasticity are regulated by SYNGAP1. This knowledge has led to a better understanding of the function of SYNGAP1 and suggests a potential target during critical period of development when the brain is more susceptible to therapeutic intervention.

Physiological role of FolD (methylenetetrahydrofolate dehydrogenase), FchA (methenyltetrahydrofolate cyclohydrolase) and Fhs (formyltetrahydrofolate synthetase) from Clostridium perfringens in a heterologous model of Escherichia coli

Most organisms possess bifunctional FolD 5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrandrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (Eco FolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (Cpe FoID) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of Cpe FoID is about five times more efficient than that of Eco FolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose cyclohydrolase activity is similar to 10 times more efficient than that of Eco FolD. Kinetic parameters for Cpe Fhs were also determined for utilization of all of its substrates. Both Cpe FoID and Cpe FchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of Cpe FoID and Cpe FchA is also necessary to rescue an E coli folD deletion strain (harbouring Cpe Fhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell.