Crystal structure of the hypoxia-inducible form of 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase (PFKFB3): A possible new target for cancer therapy

The hypoxia-inducible form of 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase (PFKFB3) plays a crucial role in the progression of cancerous cells by enabling their glycolytic pathways even under severe hypoxic conditions. To understand its structural architecture and to provide a molecular scaffold for the design of new cancer therapeutics, the crystal structure of the human form was determined. The structure at 2.1 Å resolution shows that the overall folding and functional dimerization are very similar to those of the liver (PFKFB1) and testis (PFKFB4) forms, as expected from sequence homology. However, in this structure, the N-terminal regulatory domain is revealed for the first time among the PFKFB isoforms. With a β-hairpin structure, the N terminus interacts with the 2-Pase domain to secure binding of fructose-6-phosphate to the active pocket, slowing down the release of fructose-6-phosphate from the phosphoenzyme intermediate product complex. The C-terminal regulatory domain is mostly disordered, leaving the active pocket of the fructose-2,6-bisphosphatase domain wide open. The active pocket of the 6-phosphofructo-2-kinase domain has a more rigid conformation, allowing independent bindings of substrates, fructose-6-phosphate and ATP, with higher affinities than other isoforms. Intriguingly, the structure shows an EDTA molecule bound to the fructose-6-phosphate site of the 6-phosphofructo-2-kinase active pocket despite its unfavorable liganding concentration, suggesting a high affinity. EDTA is not removable from the site with fructose-6-P alone but is with both ATP and fructose-6-P or with fructose-2,6-bisphosphate. This finding suggests that a molecule in which EDTA is covalently linked to ADP is a good starting molecule for the development of new cancer-therapeutic molecules. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc

Proteomic Analysis of Salmonella enterica Serovar Typhimurium Isolated from RAW 264.7 Macrophages

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LPS Tolerance Inhibits Cellular Respiration and Induces Global Changes in the Macrophage Secretome

Inflammatory response plays an essential role in the resolution of infections. However, inflammation can be detrimental to an organism and cause irreparable damage. For example, during sepsis, a cytokine storm can lead to multiple organ failures and often results in death. One of the strongest triggers of the inflammatory response is bacterial lipopolysaccharides (LPS), acting mostly through Toll-like receptor 4 (TLR4). Paradoxically, while exposure to LPS triggers a robust inflammatory response, repeated or prolonged exposure to LPS can induce a state of endotoxin tolerance, a phenomenon where macrophages and monocytes do not respond to new endotoxin challenges, and it is often associated with secondary infections and negative outcomes. The cellular mechanisms regulating this phenomenon remain elusive. We used metabolic measurements to confirm differences in the cellular metabolism of naïve macrophages and that of macrophages responding to LPS stimulation or those in the LPS-tolerant state. In parallel, we performed an unbiased secretome survey using quantitative mass spectrometry during the induction of LPS tolerance, creating the first comprehensive secretome profile of endotoxin-tolerant cells. The secretome changes confirmed that LPS-tolerant macrophages have significantly decreased cellular metabolism and that the proteins secreted by LPS-tolerant macrophages have a strong association with cell survival, protein metabolism, and the metabolism of reactive oxygen species

Bacillus anthracis Interacts with Plasmin(ogen) to Evade C3b-Dependent Innate Immunity

The causative agent of anthrax, Bacillus anthracis, is capable of circumventing the humoral and innate immune defense of the host and modulating the blood chemistry in circulation to initiate a productive infection. It has been shown that the pathogen employs a number of strategies against immune cells using secreted pathogenic factors such as toxins. However, interference of B. anthracis with the innate immune system through specific interaction of the spore surface with host proteins such as the complement system has heretofore attracted little attention. In order to assess the mechanisms by which B. anthracis evades the defense system, we employed a proteomic analysis to identify human serum proteins interacting with B. anthracis spores, and found that plasminogen (PLG) is a major surface-bound protein. PLG efficiently bound to spores in a lysine- and exosporium-dependent manner. We identified a-enolase and elongation factor tu as PLG receptors. PLG-bound spores were capable of exhibiting anti-opsonic properties by cleaving C3b molecules in vitro and in rabbit bronchoalveolar lavage fluid, resulting in a decrease in macrophage phagocytosis. Our findings represent a step forward in understanding the mechanisms involved in the evasion of innate immunity by B. anthracis through recruitmen

Correction to Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

Correction to Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virion