Mechanistic heterogeneity in contractile properties of α-tropomyosin (TPM1) mutants associated with inherited cardiomyopathies.

The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are 30 single-residue mutations in TPM1, the gene encoding α-tropomyosin. We examined seven mutant tropomyosins, E62Q, D84N, I172T, L185R, S215L, D230N, and M281T, that were chosen based on their clinical severity and locations along the molecule. The goal of our study was to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn could provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca(2+) sensitivity of human ß-cardiac myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca(2+) activation, and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca(2+) concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that different substitutions perturb the structure of the regulatory complex in different ways. Moreover, we observed changes in protein stability and protein-protein interactions in these mutants. Our results suggest multiple mechanistic pathways to hypertrophic and dilated cardiomyopathies. Finally, we examined a computationally designed mutant, E181K, that is hypersensitive, confirming predictions derived from in silico structural analysis.

A glutamine synthetase inhibitor increases survival and decreases cytokine response in a mouse model of acute liver failure.

BACKGROUND: Acute liver failure (ALF) can be induced in mice by administering Escherichia coli lipopolysaccharide (LPS) and D-galactosamine (D-GalN), which induce an inflammatory response involving tumour necrosis factor (TNF)-α production and a hepatocyte-specific transcriptional block. Under these conditions, binding of TNF-α to its cognate receptor on hepatocytes eventually leads to their apoptosis. AIMS: As part of an effort to identify drugs to treat this disease model, we have investigated whether the glutamine synthetase inhibitor methionine sulfoximine (MSO) could play a protective role, given its effectiveness in the inhibition of brain swelling associated with hyperammonaemia. METHODS: Mouse survival, glutamine synthetase activity, hepatocyte apoptosis and induction of inflammatory cytokines were measured in mice treated with MSO before an intraperitoneal injection of LPS/D-GalN. The effect of MSO on viability and on TNF-α release was also assessed on inflammatory and liver cells. RESULTS: We have found that, in mice treated with LPS/D-GalN, MSO (i) drastically increases animal survival; (ii) sharply reduces glutamine synthetase activity, without inhibiting its other target, γ-glutamyl cysteine synthetase; (iii) inhibits death receptor-mediated apoptosis in hepatocytes upstream to cytokine binding; (iv) strongly reduces the overall inflammatory cytokine response, including a significant decrease in TNF-α induction in vivo and ex vivo, and in the interferon-γ level and signalling. CONCLUSIONS: These results demonstrate that the MSO target glutamine synthetase is required for the early steps of the cytokine response to endotoxins, and that its pharmacological inhibition may be exploited to treat inflammation.

In vitro suppression of inflammatory cytokine response by methionine sulfoximine.

BACKGROUND: The glutamine synthetase inhibitor methionine sulfoximine (MSO), shown previously to prevent death caused by an inflammatory liver response in mice, was tested on in vitro production of cytokines by mouse peritoneal macrophages triggered with lipopolysaccharide (LPS). RESULTS: MSO significantly reduced the production of Interleukin 6 (IL-6) and Tumor Necrosis Factor Alpha (TNFα) at 4 and 6 h after LPS-treatment. This reduction did not result from decreased transcription of IL-6 and TNFα genes, and therefore appeared to result from post-transcriptional inhibition of synthesis of these cytokines. MSO treatment did not inhibit total protein synthesis and did not reduce the production of a third LPS-triggered cytokine CXCL1, so the effect was not a toxic or global downregulation of the LPS response. The anti-inflammatory effects of a glutamine synthetase inhibitor were seen even though the medium contained abundant (2 mM) glutamine, suggesting that the target for this activity was not glutamine synthetase. In agreement with this hypothesis, the L,R isomer of MSO, which does not inhibit glutamine synthetase and was previously thought to be inert, both significantly reduced IL-6 secretion in isolated macrophages and increased survival in a mouse model for inflammatory liver failure. CONCLUSIONS: Our findings provide evidence for a novel target of MSO. Future attempts to identify the additional target would therefore also provide a target for therapies to treat diseases involving damaging cytokine responses.

Methionine sulfoximine, an inhibitor of glutamine synthetase, lowers brain glutamine and glutamate in a mouse model of ALS.

In an effort to alter the levels of neurochemicals involved in excitotoxicity, we treated mice with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. Since glutamate toxicity has been proposed as a mechanism for the degeneration of motor neurons in a variety of neurodegenerative diseases, we tested the effects of MSO on the transgenic mouse that overexpresses the mutant human SOD1(G93A) gene, an animal model for the primary inherited form of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS). This treatment in vivo reduced glutamine synthetase activity measured in vitro by 85%. Proton magnetic resonance spectroscopy, with magic angle spinning of intact samples of brain tissue, showed that MSO treatment reduced brain levels of glutamine by 60% and of glutamate by 30% in both the motor cortex and the anterior striatum, while also affecting levels of GABA and glutathione. Kaplan-Meyer survival analysis revealed that MSO treatment significantly extended the lifespan of these mice by 8% (p<0.01). These results show that in the SOD1(G93A) model of neurodegenerative diseases, the concentration of brain glutamate (determined with (1)H-MRS) can be lowered by inhibiting in vivo the synthesis of glutamine with non-toxic doses of MSO.