The structural characterization of a prophage-encoded extracellular DNase from Streptococcus pyogenes

The pathogenic bacterium Group A Streptococcus pyogenes produces several extracellular DNases that have been shown to facilitate invasive infection by evading the human host immune system. DNases degrade the chromatin in neutrophil extracellular traps, enabling the bacterium to evade neutrophil capture. Spd1 is a type I, nonspecific ββα/metal-dependent nuclease from Streptococcus pyogenes, which is encoded by the SF370.1 prophage and is likely to be expressed as a result of prophage induction. We present here the X-ray structure of this DNase in the wild-type and Asn145Ala mutant form. Through structural and sequence alignments as well as mutagenesis studies, we have identified the key residues His121, Asn145 and Glu164, which are crucial for Spd1 nucleolytic activity and shown the active site constellation. Our wild-type structure alludes to the possibility of a catalytically blocked dimeric form of the protein. We have investigated the multimeric nature of Spd1 using size-exclusion chromatography with multi-angle light scattering (SEC-MALLS) in the presence and absence of the divalent metal ion Mg2+, which suggests that Spd1 exists in a monomeric form in solution

Alterations in the amino acid profile in patients with papillary thyroid carcinoma with and without Hashimoto’s thyroiditis

PurposeAmino acids (AAs) play important physiological roles in living cells. Some amino acid changes in blood are specific for autoimmune disorders, and some are specific for thyroid cancer. The aims of this study were to profile AA metabolites in the serum of patients with papillary thyroid carcinoma (PTC0) without Hashimoto’s thyroiditis (HT) and patients with PTC with HT (PTC1) and predict whether AA metabolites are associated with thyroid disease, thyroid hormone and thyroid autoantibodies.MethodsA total of 95 serum samples were collected, including 28 healthy controls (HCs), 28 PTC0 patients and 39 PTC1 patients. Serum samples were analyzed by high-performance liquid chromatography-triple stage quadrupole-mass spectrometry (HPLC-TSQ-MS), and twenty-one amino acids (AAs) were detected.ResultsThe serum concentration of glutamic acid was significantly elevated in PTC1 patients compared with PTC0 patients. Lysine was the second amino acid that differentiated these two groups of PTC patients. In addition, the serum concentrations of glycine, alanine and tyrosine were significantly reduced in both PTC patient groups compared to the HC group. These AAs were also correlated with thyroid hormones and antibodies. Five amino acid markers, namely, glycine, tyrosine, glutamic acid, glutamine and arginine, separated/distinguished PTC0 patients from healthy subjects, and eight AA markers, the same AAs as above without arginine but with alanine, leucine, valine and histidine, separated/distinguished PTC1 patients from healthy subjects based on ROC analysis.ConclusionCompared with the HCs, changes in AAs in PTC0 and PTC1 patients showed similar patterns, suggesting the possibility of a common pathophysiological basis, which confirms preliminary research that PTC is significantly associated with pathologically confirmed HT. We found two AAs, lysine and alanine, that can perform diagnostic functions in distinguishing PTC1 from PTC0

Structure Guided Design and Synthesis of a Pyridazinone Series of Trypanosoma cruzi Proteasome Inhibitors

There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the Leishmania proteasome. A related analogue, active against Trypanosoma cruzi, showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the T. cruzi proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.</p

Tissue-specific effect of refeeding after short- and long-term caloric restriction on malic enzyme gene expression in rat tissues.

Restricting food intake to a level below that consumed voluntarily (85%, 70% and 50% of the ad libitum energy intake for 3 or 30 days) and re-feeding ad libitum for 48 h results in an increase of malic enzyme (ME) gene expression in rat white adipose tissue. The increase of ME gene expression was much more pronounced in rats maintained on restricted diet for 30 days than for 3 days. The changes in ME gene expression resembled the changes in the content of SREBP-1 in white adipose tissue. A similar increase of serum insulin concentration was observed in all groups at different degrees of caloric restriction and refed ad libitum for 48 h. Caloric restriction and refeeding caused on increase of ME activity also in brown adipose tissue (BAT) and liver. However, in liver a significant increase of ME activity was found only in rats maintained on the restricted diet for 30 days. No significant changes after caloric restriction and refeeding were found in heart, skeletal muscle, kidney cortex, and brain. These data indicate that the increase of ME gene expression after caloric restriction/refeeding occurs only in lipogenic tissues. Thus, one can conclude that caloric restriction/refeeding increases the enzymatic capacity for fatty acid biosynthesis

Association Between Cytosolic Glycerol 3-Phosphate Dehydrogenase Gene Expression in Human Subcutaneous Adipose Tissue and BMI

Background/Aims: Cytosolic glycerol 3-phosphate dehydrogenase (cGPDH) is a key enzyme providing glycerol 3-phosphate for triacylglycerol synthesis in adipose tissue and is regarded as a marker for adipocyte differentiation. The aim of this study was to test the hypothesis that an increase in cGPDH gene expression in subcutaneous adipose tissue is associated with obesity. Methods: mRNA levels in human subcutaneous adipose tissue were analysed by Real-Time PCR. Results: We found that human subcutaneous adipose tissue cGPDH activity and cGPDH mRNA level were greater in obese patients than in lean subjects and were positively correlated with BMI and fat mass. Moreover, a strong positive correlation between subcutaneous adipose tissue cGPDH mRNA level and cGPDH activity was found. The data presented here indicates also that PPAR&#947; mRNA level is positively correlated with body mass index and fat mass as well as with adipose tissue cGPDH mRNA level. Moreover, the association between subcutaneous adipose tissue cGPDH mRNA level and fatty acid translocase (FAT/CD36) mRNA level was also observed. Conclusion: The obtained results suggest that in comparison to lean subjects the increase in subcutaneous adipose tissue cGPDH gene expression in the obese, is probably the result of adipose tissue expansion during obesity

Table_1_Alterations in the amino acid profile in patients with papillary thyroid carcinoma with and without Hashimoto’s thyroiditis.doc

PurposeAmino acids (AAs) play important physiological roles in living cells. Some amino acid changes in blood are specific for autoimmune disorders, and some are specific for thyroid cancer. The aims of this study were to profile AA metabolites in the serum of patients with papillary thyroid carcinoma (PTC0) without Hashimoto’s thyroiditis (HT) and patients with PTC with HT (PTC1) and predict whether AA metabolites are associated with thyroid disease, thyroid hormone and thyroid autoantibodies.MethodsA total of 95 serum samples were collected, including 28 healthy controls (HCs), 28 PTC0 patients and 39 PTC1 patients. Serum samples were analyzed by high-performance liquid chromatography-triple stage quadrupole-mass spectrometry (HPLC-TSQ-MS), and twenty-one amino acids (AAs) were detected.ResultsThe serum concentration of glutamic acid was significantly elevated in PTC1 patients compared with PTC0 patients. Lysine was the second amino acid that differentiated these two groups of PTC patients. In addition, the serum concentrations of glycine, alanine and tyrosine were significantly reduced in both PTC patient groups compared to the HC group. These AAs were also correlated with thyroid hormones and antibodies. Five amino acid markers, namely, glycine, tyrosine, glutamic acid, glutamine and arginine, separated/distinguished PTC0 patients from healthy subjects, and eight AA markers, the same AAs as above without arginine but with alanine, leucine, valine and histidine, separated/distinguished PTC1 patients from healthy subjects based on ROC analysis.ConclusionCompared with the HCs, changes in AAs in PTC0 and PTC1 patients showed similar patterns, suggesting the possibility of a common pathophysiological basis, which confirms preliminary research that PTC is significantly associated with pathologically confirmed HT. We found two AAs, lysine and alanine, that can perform diagnostic functions in distinguishing PTC1 from PTC0.</p

The crystal structure of a family GH25 lysozyme from Aspergillus fumigatus implies a neighboring-group catalytic mechanism with retention of anomeric configuration

Lysozymes are found in many of the sequence-based families of glycoside hydrolases (www.cazy.org) where they show considerable structural and mechanistic diversity. Lysozymes from glycoside hydrolase family GH25 adopt a (a/b)5(b)3-barrel-like fold with a proposal in the literature that these enzymes act with inversion of anomeric configuration; the lack of a suitable substrate, however, means that no group has successfully demonstrated the configuration of the product. Here we report the 3-D structure of the GH25 enzyme from Bacillus anthracis at 1.4 Å resolution. We show that the active center is extremely sim- ilar to those from glycoside hydrolase families GH18, GH20, GH56, GH84, and GH85 implying that, in the absence of evidence to the contrary, GH25 enzymes also act with net retention of anomeric configuration using the neighboring-group catalytic mechanism that is common to this ‘super-family’ of enzyme

The structure of a family GH25 lysozyme from Aspergillus fumigatus

Lysins are important biomolecules which cleave the bacterial cell-wall polymer peptidoglycan. They are finding increasing commercial and medical application. In order to gain an insight into the mechanism by which these enzymes operate, the X-ray structure of a CAZy family GH25 ‘lysozyme’ from Aspergillus fumigatus was determined. This is the first fungal structure from the family and reveals a modified α/β-barrel-like fold in which an eight-stranded β-barrel is flanked by three α-helices. The active site lies toward the bottom of a negatively charged pocket and its layout has much in common with other solved members of the GH25 and related GH families. A conserved active-site DXE motif may be implicated in catalysis, lending further weight to the argument that this glycoside hydrolase family operates via a ‘substrate-assisted’ catalytic mechanism