Novel mutations in TLR genes cause hyporesponsiveness to Mycobacterium avium subsp. paratuberculosis infection

<p>Abstract</p> <p>Background</p> <p>Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.</p> <p>Results</p> <p>The study presents association between TLR gene mutations and increased susceptibility to <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further <it>in silico </it>analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.</p> <p>Conclusion</p> <p>The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^thamino acid position in LRR motif (TLR1–LRR10) and 4^thresidue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.</p

Arginine residues as stabilizing elements in proteins

Site-specific substitutions of arginine for lysine in the thermostable D-xylose isomerase (XI) from Actinoplanes missouriensis are shown to impart significant heat stability enhancement in the presence of sugar substrates most probably by interfering with nonenzymatic glycation. The same substitutions are also found to increase beat stability in the absence of any sugar derivatives, where a mechanism based on prevention of glycation can no longer be invoked. This rather conservative substitution is moreover shown to improve thermostability in two other structurally unrelated proteins, human copper, zinc-superoxide dismutase (CuZnSOD) and D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus subtilis. The stabilizing effect of Lys --> Arg substitutions is rationalized on the basis of a detailed analysis of the crystal structures of wild-type XI and of engineered variants with Lys --> Arg substitution at four distinct locations, residues 253, 309, 319, and 323. Molecular model building analysis of the structures of wild-type and mutant CuZnSOD (K9R) and GAPDH (G281K and G281R) is used to explain the observed stability enhancement in these proteins. In addition to demonstrating that even thermostable proteins can lend themselves to further stability improvement, our findings provide direct evidence that arginine residues are important stabilizing elements in proteins. Moreover, the stabilizing role of electrostatic interactions, particularly between subunits in oligomeric proteins, is documented

The Role of Alpha-Hemoglobin Stabilizing Protein in Redox Chemistry, Denaturation, and Hemoglobin Assembly

Hemoglobin biosynthesis in erythrocyte precursors involves several steps. The correct ratios and concentrations of normal alpha (α) and beta (β) globin proteins must be expressed; apoproteins must be folded correctly; heme must be synthesized and incorporated into these globins rapidly; and the individual α and β subunits must be rapidly and correctly assembled into heterotetramers. These events occur on a large scale in vivo, and dysregulation causes serious clinical disorders such as thalassemia syndromes. Recent work has implicated a conserved erythroid protein known as Alpha-Hemoglobin Stabilizing Protein (AHSP) as a participant in these events. Current evidence suggests that AHSP enhances α subunit stability and diminishes its participation in harmful redox chemistry. There is also evidence that AHSP facilitates one or more early-stage post-translational hemoglobin biosynthetic events. In this review, recent experimental results are discussed in light of several current models describing globin subunit folding, heme uptake, assembly, and denaturation during hemoglobin synthesis. Particular attention is devoted to molecular interactions with AHSP that relate to α chain oxidation and the ability of α chains to associate with partner β chains. Antioxid. Redox Signal. 12, 219–232