Phosphorylation of Enoyl-Acyl Carrier Protein Reductase InhA Impacts Mycobacterial Growth and Survival

InhA, the primary target for the first line anti-tuberculosis drug isoniazid, is a key enzyme of the fatty-acid synthase II system involved in mycolic acid biosynthesis in Mycobacterium tuberculosis. In this study, we show that InhA is a substrate for mycobacterial serine/threonine protein kinases. Using a novel approach to validate phosphorylation of a substrate by multiple kinases in a surrogate host (Escherichia coli), we have demonstrated efficient phosphorylation of InhA by PknA, PknB, and PknH, and to a lower extent by PknF. Additionally, the sites targeted by PknA/PknB have been identified and shown to be predominantly located at the C terminus of InhA. Results demonstrate in vivo phosphorylation of InhA in mycobacteria and validate Thr-266 as one of the key sites of phosphorylation. Significantly, our studies reveal that the phosphorylation of InhA by kinases modulates its biochemical activity, with phosphorylation resulting in decreased enzymatic activity. Co-expression of kinase and InhA alters the growth dynamics of Mycobacterium smegmatis, suggesting that InhA phosphorylation in vivo is an important event in regulating its activity. An InhA-T266E mutant, which mimics constitutive phosphorylation, is unable to rescue an M. smegmatis conditional inhA gene replacement mutant, emphasizing the critical role of Thr-266 in mediating post-translational regulation of InhA activity. The involvement of various serine/threonine kinases in modulating the activity of a number of enzymes of the mycolic acid synthesis pathway, including InhA, accentuates the intricacies of mycobacterial signaling networks in parallel with the changing environment.     

Regulation of sensory neuron-specific acid-sensing ion channel 3 by the adaptor protein Na+/H+ exchanger regulatory factor-1

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. The ASIC3 subunit is largely expressed in the peripheral nervous system, where it contributes to pain perception and to some aspects of mechanosensation. We report here a PDZ-dependent and protein kinase C-modulated association between ASIC3 and the Na+/H+ exchanger regulatory factor-1 (NHERF-1) adaptor protein. We show that NHERF-1 and ASIC3 are co-expressed in dorsal root ganglion neurons. NHERF-1 enhances the ASIC3 peak current in heterologous cells, including F-11 dorsal root ganglion cells, by increasing the amount of channel at the plasma membrane. Perhaps more importantly, we show that the plateau current of ASIC3 can be dramatically increased (10-30-fold) by association with NHERF-1, leading to a significant sustained current at pH 6.6. In the presence of NHERF-1, the ASIC3 subcellular localization is modified, and the channel co-localizes with ezrin, a member of the ezrin-radixin-moesin family of actin-binding proteins, providing the first direct link between ASIC3 and the cortical cytoskeleton. Given the importance of the ASIC3 sustained current in nociceptor excitability, it is likely that NHERF-1 participates in channel functions associated with nociception and mechanosensation.     

Pharmacophore modeling, virtual screening and 3D-QSAR studies of 5-tetrahydroquinolinylidine aminoguanidine derivatives as sodium hydrogen exchanger inhibitors

Sodium hydrogen exchanger (SHE) inhibitor is one of the most important targets in treatment of myocardial ischemia. In the course of our research into new types of non-acylguanidine, SHE inhibitory activities of 5-tetrahydroquinolinylidine aminoguanidine derivatives were used to build pharmacophore and 3D-QSAR models. Genetic Algorithm Similarity Program (GASP) was used to derive a 3D pharmacophore model which was used in effective alignment of data set. Eight molecules were selected on the basis of structure diversity to build 10 different pharmacophore models. Model 1 was considered as the best model as it has highest fitness score compared to other nine models. The obtained model contained two acceptor sites, two donor atoms and one hydrophobic region. Pharmacophore modeling was followed by substructure searching and virtual screening. The best CoMFA model, representing steric and electrostatic fields, obtained for 30 training set molecules was statistically significant with cross-validated coefficient (q(2)) of 0.673 and conventional coefficient (r(2)) of 0.988. In addition to steric and electrostatic fields observed in CoMFA, CoMSIA also represents hydrophobic, hydrogen bond donor and hydrogen bond acceptor fields. CoMSIA model was also significant with cross-validated coefficient (q(2)) and conventional coefficient (r(2)) of 0.636 and 0.986, respectively. Both models were validated by an external test set of eight compounds and gave satisfactory prediction (r(pred)(2)) of 0.772 and 0.701 for CoMFA and CoMSIA models, respectively. This pharmacophore based 3D-QSAR approach provides significant insights that can be used to design novel, potent and selective SHE inhibitors.     

Assay and inhibition of diacylglycerol lipase activity

A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-(14)C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-(14)C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-(14)C]arachidonic acid.     

Discovery of benzodihydroisofurans as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase inhibitors

A series of benzodihydroisofurans were discovered as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase (PrCP) inhibitors. The structure-activity relationship (SAR) is focused on improving PrCP activity and metabolic stability, and reducing plasma protein binding. In the established diet-induced obese (eDIO) mouse model, compound ent-3a displayed target engagement both in plasma and in brain. However, this compound failed to induce significant body weight loss in eDIO mice in a five-day study.     

Interactive responses of a thalamic neuron to formalin induced lasting pain in behaving mice

Thalamocortical (TC) neurons are known to relay incoming sensory information to the cortex via firing in tonic or burst mode. However, it is still unclear how respective firing modes of a single thalamic relay neuron contribute to pain perception under consciousness. Some studies report that bursting could increase pain in hyperalgesic conditions while others suggest the contrary. However, since previous studies were done under either neuropathic pain conditions or often under anesthesia, the mechanism of thalamic pain modulation under awake conditions is not well understood. We therefore characterized the thalamic firing patterns of behaving mice in response to nociceptive pain induced by inflammation. Our results demonstrated that nociceptive pain responses were positively correlated with tonic firing and negatively correlated with burst firing of individual TC neurons. Furthermore, burst properties such as intra-burst-interval (IntraBI) also turned out to be reliably correlated with the changes of nociceptive pain responses. In addition, brain stimulation experiments revealed that only bursts with specific bursting patterns could significantly abolish behavioral nociceptive responses. The results indicate that specific patterns of bursting activity in thalamocortical relay neurons play a critical role in controlling long-lasting inflammatory pain in awake and behaving mice.     

H2O2 production downstream of FLT3 is mediated by p22phox in the endoplasmic reticulum and is required for STAT5 signalling

The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H(2)O(2)-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H(2)O(2) after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H(2)O(2) in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H(2)O(2) production in AML cells is mediated by p22phox and is critical for STAT5 signalling.     

Association of the myostatin gene with obesity, abdominal obesity and low lean body mass and in non-diabetic asian indians in north India

Background

To determine the association of the A55T and K153R polymorphisms of the Myostatin gene with obesity, abdominal obesity and lean body mass (LBM) in Asian Indians in north India.

Materials and Methods

A total of 335 subjects (238 men and 97 women) were assessed for anthropometry, % body fat (BF), LBM and biochemical parameters. Associations of Myostatin gene polymorphisms were evaluated with anthropometric, body composition and biochemical parameters. In A55T polymorphism, BMI (p = 0.04), suprailiac skinfold (p = 0.05), total skinfold (p = 0.008), %BF (p = 0.002) and total fat mass (p = 0.003) were highest and % LBM (p = 0.03) and total LBM (Kg) were lowest (p = 0.04) in subjects with Thr/Thr genotype as compared to other genotypes. Association analysis of K153R polymorphism showed that subjects with R/R genotype had significantly higher BMI (p = 0.05), waist circumference (p = 0.04), %BF (p = 0.04) and total fat mass (p = 0.03), and lower %LBM (p = 0.02) and total LBM [(Kg), (p = 0.04)] as compared to other genotypes. Using a multivariate logistic regression model after adjusting for age and sex, subjects with Thr/Thr genotype of A55T showed high risk for high %BF (OR, 3.92, 95% Cl: 2.61–12.41), truncal subcutaneous adiposity (OR, 2.9, 95% Cl: 1.57–6.60)] and low LBM (OR, 0.64, 95% CI: 0.33–0.89) whereas R/R genotype of K153R showed high risk of obesity (BMI; OR, 3.2, 95% CI: 1.2–12.9; %BF, OR, 3.6, 95% CI: 1.04–12.4), abdominal obesity (OR, 2.12, 95% CI: 2.71–14.23) and low LBM (OR, 0.61, 95% CI: 0.29–0.79).

Conclusions/Significance

We report that variants of Myostatin gene predispose to obesity, abdominal obesity and low lean body mass in Asian Indians in north India.     

Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells

Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone-responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam-mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6-associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome-mediated degradation. Furthermore, we show that Tam- and siE6AP-mediated inhibition of E6AP leads to enhanced G0-G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome-c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam-targeted E6AP inhibition is in fact required for Tam-mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer.