Evolutionary analysis of the TPP-dependent enzyme family

The evolutionary relationships of the thiamine pyrophosphate (TPP)-dependent family of enzymes was investigated by generation of a neighbor joining phylogenetic tree using sequences from the conserved pyrophosphate (PP) and pyrimidine (Pyr) binding domains of 17 TPP-dependent enzymes. This represents the most comprehensive analysis of TPP-dependent enzyme evolution to date. The phylogeny was shown to be robust by comparison with maximum likelihood trees generated for each individual enzyme and also broadly confirms the evolutionary history proposed recently from structural comparisons alone (Duggleby 2006). The phylogeny is most parsimonious with the TPP enzymes having arisen from a homotetramer which subsequently diverged into an α2β2 heterotetramer. The relationship between the PP- and Pyr-domains and the recruitment of additional protein domains was examined using the transketolase C-terminal (TKC)-domain as an example. This domain has been recruited by several members of the family and yet forms no part of the active site and has unknown function. Removal of the TKC-domain was found to increase activity toward β-hydroxypyruvate and glycolaldehyde. Further truncations of the Pyr-domain yielded several variants with retained activity. This suggests that the influence of TKC-domain recruitment on the evolution of the mechanism and specificity of transketolase (TK) has been minor, and that the smallest functioning unit of TK comprises the PP- and Pyr-domains, whose evolutionary histories extend to all TPP-dependent enzymes

Localization of post -golgi trafficking of tumor necrosis factor-a in macrophages.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine secreted by activated macrophages, In this study, we examined the intracellular distribution and trafficking of TNF-alpha, Immunofluorescence and immunogold localization demonstrated that in lipopolysaccharide (LPS)-stimulated RAW264 macrophages, the greatest concentration of TNF-alpha is found in the perinuclear Golgi complex, Staining of the Golgi complex appeared 20 min after activation of cells and persisted for 2-12 h, and TNF-cu appeared on the cell surface only transiently during this time. The rate of disappearance of Golgi staining correlated with the release of the cleaved, mature TNP-alpha into the medium, Pulse chase labeling and subcellular fractionation studies indicated that both 26-kDa and 17-kDa forms of TNF-alpha may be present at the level of the Golgi complex, Post-Golgi trafficking of TNF-alpha was modulated by agents that disrupt the cytoskeleton, Interferon-gamma (IFN-gamma), which primes macrophages for TNF-alpha-dependent cellular cytotoxicity, potentiated the effect of LPS by sustaining enhanced intracellular pools of TNF-alpha and also promoted redistribution of TNF-alpha into post-Golgi vesicular compartments. We propose that the primary pool of biologically active TNF-alpha in activated macrophages is held in the Golgi complex and that the cytokine is recruited directly from this intracellular pool for release in response to tumor cells or pathogens