Dichotomous Nitric Oxide-Dependent Post-Translational Modifications of STAT1 Are Associated with Ipilimumab Benefits in Melanoma.

Although Ipilimumab (anti-CTLA-4) is FDA-approved for stage III/IV melanoma adjuvant treatment, it is not used clinically in first-line therapy, given the superior relapse-free survival (RFS)/toxicity benefits of anti-PD-1 therapy. However, it is important to understand anti-CTLA-4's mechanistic contribution to combination anti-PD-1/CTLA-4 therapy and investigate anti-CTLA-4 therapy for BRAF-wild type melanoma cases reresected after previous adjuvant anti-PD-1 therapy. Our group published that nitric oxide (NO) increased within the immune effector cells among patients with longer RFS after adjuvant ipilimumab, whereas NO increased within the immune suppressor cells among patients with shorter RFS. Herein, we measured the post-translational modifications of STAT1 (nitration-nSTAT1 and phosphorylation-pSTAT1) that are important for regulating its activity via flow cytometry and mass spectrometry approaches. PBMCs were analyzed from 35 patients undergoing adjuvant ipilimumab treatment. Shorter RFS was associated with higher pSTAT1 levels before (p = 0.007) and after (p = 0.036) ipilimumab. Ipilimumab-treated patients with high nSTAT1 levels before and after therapy in PBMCs experienced decreased RFS, but the change in nSTAT1 levels before and after ipilimumab therapy was associated with longer RFS (p = 0.01). The measurement of post-translational modifications in STAT1 may distinguish patients with prolonged RFS from ipilimumab and provide mechanistic insight into responses to ipilimumab combination regimens.

The Genome-Wide Binding Profile for Human RE1 Silencing Transcription Factor Unveils a Unique Genetic Circuitry in Hippocampus.

Early studies in mouse neurodevelopment led to the discovery of the RE1 Silencing Transcription Factor (REST) and its role as a master repressor of neuronal gene expression. Recently, REST was reported to also repress neuronal genes in the human adult brain. These genes were found to be involved in pro-apoptotic pathways; and their repression, associated with increased REST levels during aging, were found to be neuroprotective and conserved across species. However, direct genome-wide REST binding profiles for REST in adult brain have not been identified for any species. Here, we apply this approach to mouse and human hippocampus. We find an expansion of REST binding sites in the human hippocampus that are lacking in both mouse hippocampus and other human non-neuronal cell types. The unique human REST binding sites are associated with genes involved in innate immunity processes and inflammation signaling which, on the basis of histology and recent public transcriptomic analyses, suggest that these new target genes are repressed in glia. We propose that the increases in REST expression in mid-adulthood presage the beginning of brain aging, and that human REST function has evolved to protect the longevity and function of both neurons and glia in human brain.SIGNIFICANCE STATEMENT The RE1 Silencing Transcription Factor (REST) repressor has served historically as a model for gene regulation during mouse neurogenesis. Recent studies of REST have also suggested a conserved role for REST repressor function across lower species during aging. However, direct genome-wide studies for REST have been lacking for human brain. Here, we perform the first genome-wide analysis of REST binding in both human and mouse hippocampus. The majority of REST-occupied genes in human hippocampus are distinct from those in mouse. Further, the REST-associated genes unique to human hippocampus represent a new set related to innate immunity and inflammation, where their gene dysregulation has been implicated in aging-related neuropathology, such as Alzheimer's disease.

Multi-Omics and Informatics Analysis of FFPE Tissues Derived from Melanoma Patients with Long/Short Responses to Anti-PD1 Therapy Reveals Pathways of Response.

Anti-PD-1 based immune therapies are thought to be dependent on antigen processing and presentation mechanisms. To characterize the immune-dependent mechanisms that predispose stage III/IV melanoma patients to respond to anti-PD-1 therapies, we performed a multi-omics study consisting of expression proteomics and targeted immune-oncology-based mRNA sequencing. Formalin-fixed paraffin-embedded tissue samples were obtained from stage III/IV patients with melanoma prior to anti-PD-1 therapy. The patients were first stratified into poor and good responders based on whether their tumors had or had not progressed while on anti-PD-1 therapy for 1 year. We identified 263 protein/gene candidates that displayed differential expression, of which 223 were identified via proteomics and 40 via targeted-mRNA analyses. The downstream analyses of expression profiles using MetaCore software demonstrated an enrichment of immune system pathways involved in antigen processing/presentation and cytokine production/signaling. Pathway analyses showed interferon (IFN)-γ-mediated signaling via NF-κB and JAK/STAT pathways to affect immune processes in a cell-specific manner and to interact with the inducible nitric oxide synthase. We review these findings within the context of available literature on the efficacy of anti-PD-1 therapy. The comparison of good and poor responders, using efficacy of PD-1-based therapy at 1 year, elucidated the role of antigen presentation in mediating response or resistance to anti-PD-1 blockade.

Multi-Dimensional Flow Cytometry Analyses Reveal a Dichotomous Role for Nitric Oxide in Melanoma Patients Receiving Immunotherapy.

Phenotyping of immune cell subsets in clinical trials is limited to well-defined phenotypes, due to technological limitations of reporting flow cytometry multi-dimensional phenotyping data. We developed a multi-dimensional phenotyping analysis tool and applied it to detect nitric oxide (NO) levels in peripheral blood immune cells before and after adjuvant ipilimumab co-administration with a peptide vaccine in melanoma patients. We analyzed inhibitory and stimulatory markers for immune cell phenotypes that were felt to be important in the NO analysis. The pipeline allows visualization of immune cell phenotypes without knowledge of clustering techniques and to categorize cells by association with relapse-free survival (RFS). Using this analysis, we uncovered the potential for a dichotomous role of NO as a pro- and anti-melanoma factor. NO was found in subsets of immune-suppressor cells associated with shorter-term (≤ 1 year) RFS, whereas NO was also present in immune-stimulatory effector cells obtained from patients with significant longer-term (> 1 year) RFS. These studies provide insights into the cell-specific immunomodulatory role of NO. The methods presented herein can be applied to monitor the pro- and anti-tumor effects of a variety of immune-based therapeutics in cancer patients. Clinical Trial Registration Number: NCT00084656 (https://clinicaltrials.gov/ct2/show/NCT00084656).

A high-resolution imaging approach to investigate chromatin architecture in complex tissues.

We present ChromATin, a quantitative high-resolution imaging approach for investigating chromatin organization in complex tissues. This method combines analysis of epigenetic modifications by immunostaining, localization of specific DNA sequences by FISH, and high-resolution segregation of nuclear compartments using array tomography (AT) imaging. We then apply this approach to examine how the genome is organized in the mammalian brain using female Rett syndrome mice, which are a mosaic of normal and Mecp2-null cells. Side-by-side comparisons within the same field reveal distinct heterochromatin territories in wild-type neurons that are altered in Mecp2-null nuclei. Mutant neurons exhibit increased chromatin compaction and a striking redistribution of the H4K20me3 histone modification into pericentromeric heterochromatin, a territory occupied normally by MeCP2. These events are not observed in every neuronal cell type, highlighting ChromATin as a powerful in situ method for examining cell-type-specific differences in chromatin architecture in complex tissues.

Conditional depletion of methyl-CpG-binding protein 2 in astrocytes depresses the hypercapnic ventilatory response in mice.

Mice that are deficient in the transcription factor methyl-CpG-binding protein 2 (MeCP2) have a depressed hypercapnic ventilatory response (HCVR). The expression of MeCP2 can be selectively removed from astrocytes or neurons, thus offering a tool to dissect the role of this transcription factor in astrocytes from that in neurons. Studies were carried out in the progeny of mice that were a cross between those harboring a tamoxifen (TAM)-inducible Cre recombinase transgene driven by the human astrocytic glial fibrillary acidic protein (hGFAP) promoter, or Cre recombinase under control of the synapsin promoter, with mice containing a Cre-excisable exon III in the Mecp2 gene. The TAM-conditional excision of the Mecp2 exon allowed the respiratory CO2 response to be studied in the same animals before and after selective depletion of MeCP2 in astrocytes. Immunohistochemistry showed that following TAM treatment only ∼20% of GFAP-labeled cells in the retrotrapazoid nucleus and in the raphé magnus were positive for MeCP2. The slope of the relative increase in minute ventilation as a function of 1, 3, and 5% inspired CO2 was depressed in mice with depleted astrocyte MeCP2 compared with wild-type littermates. In contrast, selective depletion of MeCP2 in neurons did not significantly affect slope. While neurons which constitute the respiratory network ultimately determine the ventilatory response to CO2, this study demonstrates that loss of MeCP2 in astrocytes alone is sufficient to result in a dramatic attenuation of the HCVR. We propose that the glial contribution to HCVR is under the control of the MeCP2 gene.

Effect of Sarizotan, a 5-HT1a and D2-like receptor agonist, on respiration in three mouse models of Rett syndrome.

Disturbances in respiration are common and debilitating features of Rett syndrome (RTT). A previous study showed that the 5-HT1a receptor agonist (R)-(+)-8-hydroxy-dipropyl-2-aminotetralin hydrobromide (8-OH-DPAT) significantly reduced the incidence of apnea and the irregular breathing pattern in a mouse model of the disorder. 8-OH-DPAT, however, is not available for clinical practice. Sarizotan, a full 5-HT1a agonist and a dopamine D2-like agonist/partial agonist, has been used in clinical trials for the treatment of l-dopa-induced dyskinesia. The purpose of this study was to evaluate the effects of sarizotan on respiration and locomotion in mouse models of RTT. Studies were performed in Bird and Jaenisch strains of methyl-CpG-binding protein 2--deficient heterozygous female and Jaenisch strain Mecp2 null male mice and in knock-in heterozygous female mice of a common nonsense mutation (R168X). Respiratory pattern was determined with body plethysmography, and locomotion was determined with open-field recording. Sarizotan or vehicle was administered 20 minutes before a 30-minute recording of respiratory pattern or motor behavior. In separate studies, a crossover design was used to administer the drug for 7 and for 14 days. Sarizotan reduced the incidence of apnea in all three RTT mouse models to approximately 15% of their pretreatment levels. The irregular breathing pattern was corrected to that of wild-type littermates. When administered for 7 or 14 days, apnea decreased to 25 to 33% of the incidence seen with vehicle. This study indicates that the clinically approved drug sarizotan is an effective treatment for respiratory disorders in mouse models of RTT.

Systemic delivery of MeCP2 rescues behavioral and cellular deficits in female mouse models of Rett syndrome.

De novo mutations in the X-linked gene encoding the transcription factor methyl-CpG binding protein 2 (MECP2) are the most frequent cause of the neurological disorder Rett syndrome (RTT). Hemizygous males usually die of neonatal encephalopathy. Heterozygous females survive into adulthood but exhibit severe symptoms including microcephaly, loss of purposeful hand motions and speech, and motor abnormalities, which appear after a period of apparently normal development. Most studies have focused on male mouse models because of the shorter latency to and severity in symptoms, yet how well these mice mimic the disease in affected females is not clear. Very few therapeutic treatments have been proposed for females, the more gender-appropriate model. Here, we show that self-complementary AAV9, bearing MeCP2 cDNA under control of a fragment of its own promoter (scAAV9/MeCP2), is capable of significantly stabilizing or reversing symptoms when administered systemically into female RTT mice. To our knowledge, this is the first potential gene therapy for females afflicted with RTT.

Interaction of Mycobacterium tuberculosis RshA and SigH is mediated by salt bridges.

The alternate sigma factor sigH of Mycobacterium tuberculosis is expressed under stress and acts as a major regulator of several genes, including some other sigma factors and redox systems. While it is auto-regulated by its own promoter at the transcriptional level, its regulation at the post-translational level is through its cognate protein, an anti-sigma factor, RshA. Hither before RshA was believed to be a zinc-associated anti-sigma factor (ZAS) and the binding of RshA to SigH is redox dependent. Here, we show that RshA coordinates a [2Fe-2S] cluster using cysteines as ligands and native RshA has more affinity to [2Fe-2S] cluster than to zinc. Furthermore, we used amide hydrogen deuterium exchange mass spectrometry (HDX-MS), followed by site-directed mutagenesis in SigH and RshA, to elucidate the interaction mechanism of RshA and SigH and the potential role of metal ion clustering in SigH regulation. Three regions in SigH, comprising of residues 1-25, 58-69, 90-111, 115-132 and 157-196 and residues 35-57 of RshA show decreased deuterium exchange and reflect decreased solvent accessibility upon complexation with SigH. Of the three RshA mutants, created based on the HDX results, the RsHA E37A mutant shows stronger interaction with SigH, relative to WT RshA, while the H49A mutant abolishes interactions and the C(53)XXC(56)AXXA mutant has no effect on complexation with SigH. The D22A, D160A and E162 SigH mutants show significantly decreased binding to RshA and the E168A mutant completely abolished interactions with RshA, indicating that the SigH-RshA interaction is mediated by salt bridges. In addition, SigH-RshA interaction does not require clustering of metal ions. Based on our results, we propose a molecular model of the SigH-RshA interaction.

A role for glia in the progression of Rett's syndrome.

Rett's syndrome (RTT) is an X-chromosome-linked autism spectrum disorder caused by loss of function of the transcription factor methyl-CpG-binding protein 2 (MeCP2). Although MeCP2 is expressed in most tissues, loss of MeCP2 expression results primarily in neurological symptoms. Earlier studies suggested the idea that RTT is due exclusively to loss of MeCP2 function in neurons. Although defective neurons clearly underlie the aberrant behaviours, we and others showed recently that the loss of MECP2 from glia negatively influences neurons in a non-cell-autonomous fashion. Here we show that in globally MeCP2-deficient mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern, and greatly prolonged lifespan compared to globally null mice. Furthermore, restoration of MeCP2 in the mutant astrocytes exerted a non-cell-autonomous positive effect on mutant neurons in vivo, restoring normal dendritic morphology and increasing levels of the excitatory glutamate transporter VGLUT1. Our study shows that glia, like neurons, are integral components of the neuropathology of RTT, and supports the targeting of glia as a strategy for improving the associated symptoms.

YjbH-enhanced proteolysis of Spx by ClpXP in Bacillus subtilis is inhibited by the small protein YirB (YuzO).

The Spx protein of Bacillus subtilis is a global regulator of the oxidative stress response. Spx concentration is controlled at the level of proteolysis by the ATP-dependent protease ClpXP and a substrate-binding protein, YjbH, which interacts with Spx. A yeast two-hybrid screen was carried out using yjbH as bait to uncover additional substrates or regulators of YjbH activity. Of the several genes identified in the screen, one encoded a small protein, YirB (YuzO), which elevated Spx concentration and activity in vivo when overproduced from an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible yirB construct. Pulldown experiments using extracts of B. subtilis cells producing a His-tagged YirB showed that native YjbH interacts with YirB in B. subtilis. Pulldown experiments using affinity-tagged Spx showed that YirB inhibited YjbH interaction with Spx. In vitro, YjbH-mediated proteolysis of Spx by ClpXP was inhibited by YirB. The activity of YirB is similar to that of the antiadaptor proteins that were previously shown to reduce proteolysis of a specific ClpXP substrate by interacting with a substrate-binding protein.

Studies on structural and functional divergence among seven WhiB proteins of Mycobacterium tuberculosis H37Rv.

The whiB-like genes (1-7) of Mycobacterium tuberculosis are involved in cell division, nutrient starvation, pathogenesis, antibiotic resistance and stress sensing. Although the biochemical properties of WhiB1, WhiB3 and WhiB4 are known, there is no information about the other proteins. Here, we elucidate in detail the biochemical and biophysical properties of WhiB2, WhiB5, WhiB6 and WhiB7 of M. tuberculosis and present a comprehensive comparative study on the molecular properties of all WhiB proteins. UV-Vis spectroscopy has suggested the presence of a redox-sensitive [2Fe-2S] cluster in each of the WhiB proteins, which remains stably bound to the proteins in the presence of 8 m urea. The [2Fe-2S] cluster of each protein was oxidation labile but the rate of cluster loss decreased under reducing environments. The [2Fe-2S] cluster of each WhiB protein responded differently to the oxidative effect of air and oxidized glutathione. In all cases, disassembly of the [2Fe-2S] cluster was coupled with the oxidation of cysteine-thiols and the formation of two intramolecular disulfide bonds. Both CD and fluorescence spectroscopy revealed that WhiB proteins are structurally divergent members of the same family. Similar to WhiB1, WhiB3 and WhiB4, apo WhiB5, WhiB6 and WhiB7 also reduced the disulfide of insulin, a model substrate. However, the reduction efficiency varied significantly. Surprisingly, WhiB2 did not reduce the insulin disulfide, even though its basic properties were similar to those of others. The structural and functional divergence among WhiB proteins indicated that each WhiB protein is a distinguished member of the same family and together they may represent a novel redox system for M. tuberculosis.

The YjbH protein of Bacillus subtilis enhances ClpXP-catalyzed proteolysis of Spx.

The global transcriptional regulator Spx of Bacillus subtilis is controlled at several levels of the gene expression process. It is maintained at low concentrations during unperturbed growth by the ATP-dependent protease ClpXP. Under disulfide stress, Spx concentration increases due in part to a reduction in ClpXP-catalyzed proteolysis. Recent studies of Larsson and coworkers (Mol. Microbiol. 66:669-684, 2007) implicated the product of the yjbH gene as being necessary for the proteolytic control of Spx. In the present study, yeast two-hybrid analysis and protein-protein cross-linking showed that Spx interacts with YjbH. YjbH protein was shown to enhance the proteolysis of Spx in reaction mixtures containing ClpXP protease but not ClpCP protease. An N-terminal truncated form of YjbH with a deletion of residues 1 to 24 (YjbH(Delta1-24)) showed no proteolysis enhancement activity. YjbH is specific for Spx as it did not accelerate proteolysis of the ClpXP substrate green fluorescent protein (GFP)-SsrA, a GFP derivative with a C-terminal SsrA tag that is recognized by ClpXP. Using inductively coupled plasma atomic emission spectroscopy and 4-(2-pyridylazo) resorcinol release experiments, YjbH was found to contain zinc atoms. Zinc analysis of YjbH(Delta1-24) revealed that the N-terminal histidine-rich region is indispensable for the coordination of at least one Zn atom. A Zn atom coordinated by the N-terminal region was rapidly released from the protein upon treatment with a strong oxidant. In conclusion, YjbH is proposed to be an adaptor for ClpXP-catalyzed Spx degradation, and a model of YjbH redox control involving Zn dissociation is presented.

Molecular function of WhiB4/Rv3681c of Mycobacterium tuberculosis H37Rv: a [4Fe-4S] cluster co-ordinating protein disulphide reductase.

The genome sequence of Mycobacterium tuberculosis H37Rv revealed the presence of seven whiB-like open reading frames. In spite of several genetic studies on whiB genes, the biochemical properties of WhiB proteins are poorly understood. All WhiB-like proteins have four conserved cysteine residues, out of which two are present in a CXXC motif. We report for the first time the detailed biochemical and biophysical properties of M. tuberculosis WhiB4/Rv3681c and demonstrate the functional relevance of four conserved cysteines and the CXXC motif. UV-visible absorption spectra of freshly purified mWhiB4 showed the presence of a [2Fe-2S] cluster, whereas the electron paramagnetic resonance (EPR) spectra of reconstituted protein showed the presence of a [4Fe-4S] cluster. The iron-sulphur cluster was redox sensitive but stably co-ordinated to the protein even in the presence of high concentration of chaotropic agents. Despite primary sequence divergence from thioredoxin family proteins, the apo mWhiB4 has properties similar to thioredoxins and functions as a protein disulphide reductase, whereas holo mWhiB4 is enzymatically inactive. Apart from the cysteine thiol of CXXC motif the distantly placed thiol pair also contributes equally to the enzymatic activity of mWhiB4. A functional model of mWhiB4 in redox signaling during oxidative stress in M. tuberculosis has been presented.

Expression and characterization of alpha-(1,4)-glucan branching enzyme Rv1326c of Mycobacterium tuberculosis H37Rv.

Glycogen branching enzyme (GlgB, EC 2.4.1.18) catalyzes the third step of glycogen biosynthesis by the cleavage of an alpha-(1,4)-glucosidic linkage and subsequent transfer of cleaved oligosaccharide to form a new alpha-(1,6)-branch. A single glgB gene Rv1326c is present in Mycobacterium tuberculosis. The predicted amino acid sequence of GlgB of M. tuberculosis has all the conserved regions of alpha-amylase family proteins. The overall amino acid identity to other GlgBs ranges from 48.5 to 99%. The glgB gene of M. tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity using metal affinity and ion exchange chromatography. The recombinant protein is a monomer as evidenced by gel filtration chromatography, is active as an enzyme, and uses amylose as the substrate. Enzyme activity was optimal at pH 7.0, 30 degrees C and divalent cations such as Zn2+ and Cu2+ inhibited activity. CD spectroscopy, proteolytic cleavage and mass spectroscopy analyses revealed that cysteine residues of GlgB form structural disulfide bond(s), which allow the protein to exist in two different redox-dependent conformational states. These conformations have different surface hydrophobicities as evidenced by ANS-fluorescence of oxidized and reduced GlgB. Although the conformational change did not affect the branching enzyme activity, the change in surface hydrophobicity could influence the interaction or dissociation of different cellular proteins with GlgB in response to different physiological states.

Characterization of Mycobacterium tuberculosis WhiB1/Rv3219 as a protein disulfide reductase.

WhiB family of protein is emerging as one of the most fascinating group and is implicated in stress response as well as pathogenesis via their involvement in diverse cellular processes. Surprisingly, available in vivo data indicate an organism specific physiological role for each of these proteins. The WhiB proteins have four conserved cysteine residues where two of them are present in a C-X-X-C motif. In thioredoxins and similar proteins, this motif works as an active site and confers thiol-disulfide oxidoreductase activity to the protein. The recombinant WhiB1/Rv3219 was purified in a single step from Escherichia coli using Ni(2+)-NTA affinity chromatography and was found to exist as a homodimer. Mass spectrometry of WhiB1 shows that the four cysteine residues form two intramolecular disulfide bonds. Using intrinsic tryptophan fluorescence as a measure of redox state, the redox potential of WhiB1 was calculated as -236+/-2mV, which corresponds to the redox potential of many cytoplasmic thioredoxin-like proteins. WhiB1 catalyzed the reduction of insulin disulfide thus clearly demonstrating that it functions as a protein disulfide reductase. Present study for the first time suggests that WhiB1 may be a part of the redox network of Mycobacterium tuberculosis through its involvement in thiol-disulfide exchange with other cellular proteins.