Structural and functional determination of homologs of the Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA).

The Mycobacterium tuberculosis (Mtb) pathogen encodes a GlcNAc-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of GlcNAc-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential antitubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereoselective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 and 2.0 Å resolutions, respectively. The GlcNAc6P complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/ß binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria.

Unraveling Epigenetic Interplay between Inflammation, Thrombosis, and Immune-Related Disorders through a Network Meta-analysis.

Inflammation and thrombosis are two distinct yet interdependent physiological processes. The inflammation results in the activation of the coagulation system that directs the immune system and its activation, resulting in the initiation of the pathophysiology of thrombosis, a process termed immune-thrombosis. Still, the shared underlying molecular mechanism related to the immune system and coagulation has not yet been explored extensively. Inspired to answer this, we carried out a comprehensive gene expression meta-analysis using publicly available datasets of four diseases, including venous thrombosis, systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. A total of 609 differentially expressed genes (DEGs) shared by all four datasets were identified based on the combined effect size approach. The pathway enrichment analysis of the DEGs showed enrichment of various epigenetic pathways such as histone-modifying enzymes, posttranslational protein modification, chromatin organization, chromatin-modifying enzymes, HATs acetylate proteins. Network-based protein-protein interaction analysis showed epigenetic enzyme coding genes dominating among the top hub genes. The miRNA-interacting partner of the top 10 hub genes was determined. The predomination of epitranscriptomics regulation opens a layout for the meta-analysis of miRNA datasets of the same four diseases. We identified 30 DEmiRs shared by these diseases. There were 9 common DEmiRs selected from the list of miRNA-interacting partners of top 10 hub genes and shared significant DEmiRs from microRNAs dataset acquisition. These common DEmiRs were found to regulate genes involved in epigenetic modulation and indicate a promising epigenetic aspect that needs to be explored for future molecular studies in the context of immunothrombosis and inflammatory disease.

Mapping of a N-terminal α-helix domain required for human PINK1 stabilization, Serine228 autophosphorylation and activation in cells.

Autosomal recessive mutations in the PINK1 gene are causal for Parkinson's disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE : CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.

A GFP-strategy for efficient recombinant protein overexpression and purification in Mycobacterium smegmatis.

One of the major obstacles to obtaining a complete structural and functional understanding of proteins encoded by the Mycobacterium tuberculosis (Mtb) pathogen is due to significant difficulties in producing recombinant mycobacterial proteins. Recent advances that have utilised the closely related Mycobacterium smegmatis species as a native host have been effective. Here we have developed a method for the rapid screening of both protein production and purification strategies of mycobacterial proteins in whole M. smegmatis cells following green fluorescent protein (GFP) fluorescence as an indicator. We have adapted the inducible T7-promoter based pYUB1062 shuttle vector by the addition of a tobacco etch virus (TEV) cleavable C-terminal GFP enabling the target protein to be produced as a GFP-fusion with a poly-histidine tag for affinity purification. We illustrate the advantages of a fluorescent monitoring approach with the production and purification of the mycobacterial N-acetylglucosamine-6-phosphate deacetylase (NagA)-GFP fusion protein. The GFP system described here will accelerate the production of mycobacterial proteins that can be used to understand the molecular mechanisms of Mtb proteins and facilitate drug discovery efforts.

Impact of a gluten-free diet on Sseveral growth parameters in children with Type 1 Diabetes Mellitus and Celiac Disease in Western Uttar Pradesh, India / Journal of the ASEAN Federation of Endocrine Societies

Background@#Celiac disease is frequently associated with uncontrolled blood sugar and impaired linear growth in a child with type 1 diabetes mellitus. @*Objective@#To study the impact of a gluten-free diet on several growth parameters in children with type 1 diabetes mellitus and celiac disease. @*Methodology@#Two hundred and fifty six patients with Type 1 diabetes mellitus were screened (149 males and 107 females) during the study period of two years. Patients were evaluated for the clinical signs, biochemical investigations and family history of celiac disease in a tertiary care health centre in Western Uttar Pradesh, India. @*Results@#Twenty four (9.3%) patients were diagnosed to have celiac disease; the mean age at diagnosis of diabetes was 9.37±7 years. Only one out of twenty four patients with celiac disease had been diagnosed before the detection of diabetes mellitus. Weight standard deviation score (SDS) increased from -0.12±1.3 at the start of gluten free diet to 0.8±0.9 after 12 months (p<0.004). Height SDS decreased from -2.46±1.1 at the start of gluten free diet to -2.14±0.9 after 12 months later (p=0.087). Bone age SDS increased from 9.2±6.3 at the start of gluten free diet to 10.3±6.7 after 12 months later. Height velocity increased from 4.7±0.7 cm/year in the year before treatment to 5.1+1.2 cm/year during treatment (p=0.05). The increase in Haemoglobin, serum calcium, and serum iron was statistically significant (p<0.05). @*Conclusion@#Patients with celiac disease associated with type 1 diabetes mellitus frequently have poor glycemic control and impairment in several growth parameters. When these patients are put on a gluten restricted diet, they show signs of improvement in terms of weight gain, height, serum Ca, serum iron, haemoglobin, and in height velocity.

Prevalence and clinical profile of Celiac Disease in patients with Type 1 Diabetes Mellitus in Western Uttar Pradesh, India / Journal of the ASEAN Federation of Endocrine Societies

Background@#Celiac disease is frequently associated with type 1 diabetes mellitus, but is usually ill-defined and not usually suspected until the disease becomes advanced. @*Objective@#To study the prevalence and clinical profile of celiac disease among patients with type 1 diabetes mellitus in a tertiary care referral centre in north India. @*Methodology@#Two hundred and fifty six patients were screened (149 males and 107 females) during the study period of two years, patients were evaluated for the clinical signs, biochemical investigations and family history of celiac disease in tertiary care health center in western Uttar Pradesh. @*Results@#Twenty four (9.37%) patients were diagnosed to have celiac disease; the mean age at diagnosis of diabetes was 9.34 ± 7.3 years. Only 1/24 patients with celiac disease had been diagnosed before detection of diabetes mellitus. The common manifestations were normocytic normochromic anemia (66.6%) followed by diarrhoea (62.5%), abdominal pain/bloating sensation (58.3%) and short stature (58.3%). Some uncommon manifestations were also observed in small number of patients: rickets (20.8%), recurrent hypoglycemia (16.6%), carpopedal spasm (8.3%), and night blindness (8.3%). @*Conclusion@#Celiac disease was found in about 10% of patients with type 1 diabetes, almost 10-20 times higher than that observed in general pediatric population. Atypical manifestations (rickets, recurrent hypoglycemia, carpopedal spasm and night blindness) were found to be common in patients with type 1 diabetes as compared to the general population. Unexplained anemia, diarrhoea, short stature and rickets should raise suspicion for the possibility of undiagnosed celiac disease in type 1 diabetes mellitus.