Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases

Covalent posttranslational protein modifications by eukaryotic transglutaminases proceed by a kinetic pathway of acylation and deacylation. Ammonia is released as the acylenzyme is formed, whereas the cross-linked product is released later in the deacylation step. Superposition of the active sites of transglutaminase type 2 (TG2) and the structurally related cysteine protease, papain, indicates that in the formation of tetrahedral intermediates, the backbone nitrogen of the catalytic Cys-277 and the N_Ɛ1 nitrogen of Trp-241 of TG2 could contribute to transition-state stabilization. The importance of this Trp-241 side chain was demonstrated by examining the kinetics of dansylcadaverine incorporation into a model peptide. Although substitution of the Trp-241 side chain with Ala or Gly had only a small effect on the Michaelis constant K_m (1.5-fold increase), it caused a >300-fold lowering of the catalytic rate constant k_cat. The wild-type and mutant TG2-catalyzed release of ammonia showed kinetics similar to the kinetics for the formation of cross-linked product, indicating that transitionstate stabilization in the acylation step was rate-limiting. In papain, a Gln residue is at the position of TG2-Trp-241. The conservation of Trp-241 in all eukaryotic transglutaminases and the finding that W241Q-TG2 had a much lower k_cat than wild-type enzyme suggest evolutionary specialization in the use of the indole group. This notion is further supported by the observation that transitionstate- stabilizing side chains of Tyr and His that operate in some serine and metalloproteases only partially substituted for Trp.

Hypoallergenic variant of the major egg white allergen Gal d 1 produced by disruption of cysteine bridges

Gal d 1 (Ovomucoid) is the most allergenic protein in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesized that disruption of selected cysteine-cysteine disulfide bridges by mutagenesis will allow us the production of a hypoallergenic variant of the protein.

Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70–86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.

N-acetyl cysteine for depressive symptoms in bipolar disorder - a double blind, randomized, placebo-controlled trial

Background: Treatment-resistant subthreshold depression is a major problem in bipolar disorder. Both depression and bipolar disorderare complicated by glutathione depletion. We hypothesized that treatment with N-acetyl cysteine (NAC), a safe, orally bioavailable precursor of glutathione, may improve the depressive component of bipolar disorder.

Methods: A randomized, double-blind, multicenter, placebo-controlled study of individuals (n 75) with bipolar disorder in the maintenance phase treated with NAC (1 g twice daily) adjunctive to usual medication over 24 weeks, with a 4-week washout. The two primary outcomes were the Montgomery Asberg Depression Rating Scale (MADRS) and time to a mood episode. Secondary outcomes included the Bipolar Depression Rating Scale and 11 other ratings of clinical status, quality of life, and functioning.

Results: NAC treatment caused a significant improvement on the MADRS (least squares mean difference [95% confidence interval]: 8.05 [13.16, 2.95], p .002) a n d most secondary scales at end point. Benefit was evident by 8 weeks on the Global Assessment of Functioning Scale and Social and Occupational Functioning Assessment Scale and at 20 weeks on the MADRS. Improvements were lost after washout. There was no effect of NAC on time to a mood episode (log-rank test: p .968) and no significant between-group differences inadverse events. Effect sizes at end point were medium to high for improvements in MADRS and 9 of the 12 secondary readouts.

Conclusions: NAC appears a safe and effective augmentation strategy for depressive symptoms in bipolar  disorder.

IgM-mediated autoimmune responses directed against anchorage epitopes are greater in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) than in major depression

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and depression are considered to be neuro-immune disorders (Maes and Twisk, BMC Medicine 8:35, 2010). There is also evidence that depression and ME/CFS are accompanied by oxidative and nitrosative stress (O&NS) and by increased autoantibodies to a number of self-epitopes some of which have become immunogenic due to damage by O&NS. The aim of this study is to examine IgM-mediated autoimmune responses to different self-epitopes in ME/CFS versus depression. We examined serum IgM antibodies to three anchorage molecules (palmitic and myristic acid and S-farnesyl-L-cysteine); acetylcholine; and conjugated NO-modified adducts in 26 patients with major depression; 16 patients with ME/CFS, 15 with chronic fatigue; and 17 normal controls. Severity of fatigue and physio-somatic (F&S) symptoms was measured with the Fibromyalgia and Chronic Fatigue Syndrome Rating Scale. Serum IgM antibodies to the three anchorage molecules and NO-phenylalanine were significantly higher in ME/CFS than in depression. The autoimmune responses to oxidatively, but not nitrosatively, modified self-epitopes were significantly higher in ME/CFS than in depression and were associated with F&S symptoms. The autoimmune activity directed against conjugated acetylcholine did not differ significantly between ME/CFS and depression, but was greater in the patients than controls. Partially overlapping pathways, i.e. increased IgM antibodies to a multitude of neo-epitopes, underpin both ME/CFS and depression, while greater autoimmune responses directed against anchorage molecules and oxidatively modified neo-epitopes discriminate patients with ME/CFS from those with depression. These autoimmune responses directed against neoantigenic determinants may play a role in the dysregulation of key cellular functions in both disorders, e.g. intracellular signal transduction, cellular differentiation and apoptosis, but their impact may be more important in ME/CFS than in depression.

Metabolite profiles in the anterior cingulate cortex of depressed patients differentiate those taking N-acetyl-cysteine versus placebo

Background: Increased oxidative stress is thought to contribute to the pathophysiology of major depressive disorder (MDD), which is in part due to diminished levels of glutathione, the primary anti-oxidant of the brain. Oral administration of N-acetyl-cysteine (NAC) replenishes glutathione and has therefore been shown to reduce depressive symptoms. Proton magnetic spectroscopy (1H-MRS) that allows quantification of brain metabolites pertinent to both MDD and oxidative biology may provide some novel insights into the neurobiological effects of NAC, and in particular metabolite concentrations within the anterior cingulate cortex (ACC) are likely to be important given the key role of this region in the regulation of affect.

Objective: The aim of this study was to determine whether the metabolite profile of the ACC in MDD patients predicts treatment with adjunctive NAC versus placebo.

Methods: This study was nested within a multicentre, randomized, double-blind, placebo-controlled study of MDD participants treated with adjunctive NAC. Participants (n = 76) from one site completed the spectroscopy component at the end of treatment (12 weeks). Spectra from a single-voxel in the ACC were acquired and absolute concentrations of glutamate (Glu), glutamate-glutamine (Glx), N-acetyl-aspartate (NAA) and myo-inositol (mI) were obtained. Binary logistic regression analysis was performed to determine whether metabolite profiles could predict NAC versus placebo group membership.

Results: When predicting group outcome (NAC or placebo), Glx, NAA and mI were a significant model, and had 75% accuracy, while controlling for depression severity and sex. However, the Glu, NAA and mI profile was only predictive at a trend level, with 68.3% accuracy. For both models, the log of the odds of a participant being in the NAC group was positively related to NAA, Glx and Glu levels and negatively related to mI levels.

Conclusion: The finding of higher Glx and NAA levels being predictive of the NAC group provides preliminary support for the putative anti-oxidative role of NAC in MDD.