Intracellular bacteriolysis triggers a massive apoptotic cell death in Shigella-infected epithelial cells

Infected epithelial cells, which act as a first barrier against pathogens, seldom undergo apoptosis. Rather, infected epithelial cells undergo a slow cell death that displays hallmarks of necrosis. Here, we demonstrate that rapid intracellular lysis of Shigella flexneri, provoked by either the use of a diaminopimelic acid auxotroph mutant or treatment of infected cells with antibiotics of the beta-lactam family, resulted in a massive and rapid induction of apoptotic cell death. This intracellular bacteriolysis-mediated apoptotic death (IBAD) was characterized by the specific involvement of the mitochondrial-dependent cytochrome c/Apaf-1 axis that resulted in the activation of caspases-3, -6 and -9. Importantly, Bcl-2 family members and the NF-kappaB pathway seemed to be critical modulators of IBAD. Finally, we identified that IBAD was also triggered by Salmonella enterica serovar Typhimurium but not by the Gram-positive bacteria, Listeria monocytogenes. Together, our results demonstrate that, contrary to previous findings, epithelial cells are intrinsically able to mount an efficient apoptotic cell death response following infection. Indeed, apoptosis in normal circumstances is masked by powerful anti-apoptotic mechanisms, which are overcome in IBAD. Our results also uncover an unexpected consequence of the treatment of infected cells with certain classes of antibiotics.     

Phosphorylation of SUMO-1 occurs in vivo and is conserved through evolution

Protein dynamics is regulated by an elaborate interplay between different post-translational modifications. Ubiquitin and ubiquitin-like proteins (Ubls) are small proteins that are covalently conjugated to target proteins with important functional consequences. One such modifier is SUMO, which mainly modifies nuclear proteins. SUMO contains a unique N-terminal arm not present in ubiquitin and other Ubls, which functions in the formation of SUMO polymers. Here, we unambiguously show that serine 2 of the endogenous SUMO-1 N-terminal protrusion is phosphorylated in vivo using very high mass accuracy mass spectrometry at both the MS and the MS/MS level and complementary fragmentation techniques. Strikingly, we detected the same phosphorylation in yeast, Drosophila and human cells, suggesting an evolutionary conserved function for this modification. The nearly identical human SUMO-2 and SUMO-3 isoforms differ in serine 2; thus, only SUMO-3 could be phosphorylated at this position. Our finding that SUMO can be modified may point to an additional level of complexity through modifying a protein-modifier.     

13C NMR snapshots of the complex reaction coordinate of pyridoxal phosphate synthase

The predominant biosynthetic route to vitamin B6 is catalyzed by a single enzyme. The synthase subunit of this enzyme, Pdx1, operates in concert with the glutaminase subunit, Pdx2, to catalyze the complex condensation of ribose 5-phosphate, glutamine and glyceraldehyde 3-phosphate to form pyridoxal 5'-phosphate, the active form of vitamin B6. In previous studies it became clear that many if not all of the reaction intermediates were covalently bound to the synthase subunit, thus making them difficult to isolate and characterize. Here we show that it is possible to follow a single turnover reaction by heteronuclear NMR using (13)C- and (15)N-labeled substrates as well as (15)N-labeled synthase. By denaturing the enzyme at points along the reaction coordinate, we solved the structures of three covalently bound intermediates. This analysis revealed a new 1,5 migration of the lysine amine linking the intermediate to the enzyme during the conversion of ribose 5-phosphate to pyridoxal 5'-phosphate.     

Multi-scale simulations provide supporting evidence for the hypothesis of intramolecular protein translocation in GroEL/GroES complexes

The biological function of chaperone complexes is to assist the folding of non-native proteins. The widely studied GroEL chaperonin is a double-barreled complex that can trap non-native proteins in one of its two barrels. The ATP-driven binding of a GroES cap then results in a major structural change of the chamber where the substrate is trapped and initiates a refolding attempt. The two barrels operate anti-synchronously. The central region between the two barrels contains a high concentration of disordered protein chains, the role of which was thus far unclear. In this work we report a combination of atomistic and coarse-grained simulations that probe the structure and dynamics of the equatorial region of the GroEL/GroES chaperonin complex. Surprisingly, our simulations show that the equatorial region provides a translocation channel that will block the passage of folded proteins but allows the passage of secondary units with the diameter of an alpha-helix. We compute the free-energy barrier that has to be overcome during translocation and find that it can easily be crossed under the influence of thermal fluctuations. Hence, strongly non-native proteins can be squeezed like toothpaste from one barrel to the next where they will refold. Proteins that are already fairly close to the native state will not translocate but can refold in the chamber where they were trapped. Several experimental results are compatible with this scenario, and in the case of the experiments of Martin and Hartl, intra chaperonin translocation could explain why under physiological crowding conditions the chaperonin does not release the substrate protein.     

Kinetic modeling of the assembly, dynamic steady state, and contraction of the FtsZ ring in prokaryotic cytokinesis

Cytokinesis in prokaryotes involves the assembly of a polymeric ring composed of FtsZ protein monomeric units. The Z ring forms at the division plane and is attached to the membrane. After assembly, it maintains a stable yet dynamic steady state. Once induced, the ring contracts and the membrane constricts. In this work, we present a computational deterministic biochemical model exhibiting this behavior. The model is based on biochemical features of FtsZ known from in vitro studies, and it quantitatively reproduces relevant in vitro data. An essential part of the model is a consideration of interfacial reactions involving the cytosol volume, where monomeric FtsZ is dispersed, and the membrane surface in the cell's mid-zone where the ring is assembled. This approach allows the same chemical model to simulate either in vitro or in vivo conditions by adjusting only two geometrical parameters. The model includes minimal reactions, components, and assumptions, yet is able to reproduce sought-after in vivo behavior, including the rapid assembly of the ring via FtsZ-polymerization, the formation of a dynamic steady state in which GTP hydrolysis leads to the exchange of monomeric subunits between cytoplasm and the ring, and finally the induced contraction of the ring. The model gives a quantitative estimate for coupling between the rate of GTP hydrolysis and of FtsZ subunit turnover between the assembled ring and the cytoplasmic pool as observed. Membrane constriction is chemically driven by the strong tendency of GTP-bound FtsZ to self-assembly. The model suggests a possible mechanism of membrane contraction without a motor protein. The portion of the free energy of GTP hydrolysis released in cyclization is indirectly used in this energetically unfavorable process. The model provides a limit to the mechanistic complexity required to mimic ring behavior, and it highlights the importance of parallel in vitro and in vivo modeling.     

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

Release of hemoglobin from the erythrocyte during intravascular hemolysis contributes to the pathology of a variety of diseased states. This effect is partially due to the enhanced ability of cell-free plasma hemoglobin, which is primarily found in the ferrous, oxygenated state, to scavenge nitric oxide. Oxidation of the cell-free hemoglobin to methemoglobin, which does not effectively scavenge nitric oxide, using inhaled nitric oxide has been shown to be effective in limiting pulmonary and systemic vasoconstriction. However, the ferric heme species may be reduced back to ferrous hemoglobin in plasma and has the potential to drive injurious redox chemistry. We propose that compounds that selectively convert cell-free hemoglobin to ferric, and ideally iron-nitrosylated heme species that do not actively scavenge nitric oxide, would effectively treat intravascular hemolysis. We show here that nitroxyl generated by Angeli's salt (sodium alpha-oxyhyponitrite, Na2N2O3) preferentially reacts with cell-free hemoglobin compared to that encapsulated in the red blood cell under physiologically relevant conditions. Nitroxyl oxidizes oxygenated ferrous hemoglobin to methemoglobin and can convert the methemoglobin to a more stable, less toxic species, iron-nitrosyl hemoglobin. These results support the notion that Angeli's salt or a similar compound could be used to effectively treat conditions associated with intravascular hemolysis.     

Relevance of the capacity of phosphorylated fructose to scavenge the hydroxyl radical

The hydroxyl radical (OH) has detrimental biological activity due to its very high reactivity. Our experiments were designed to determine the effects of equimolar concentrations of glucose, fructose and mannitol and three phosphorylated forms of fructose (fructose-1-phosphate (F1P); fructose-6-phosphate (F6P); and fructose-1,6-bis(phosphate) (F16BP)) on OH radical production via the Fenton reaction. EPR spectroscopy using spin-trap DEPMPO was applied to detect radical production. We found that the percentage inhibition of OH radical formation decreased in the order F16BP > F1P > F6P > fructose > mannitol = glucose. As ketoses can sequester redox-active iron thus preventing the Fenton reaction, the Haber-Weiss-like system was also employed to generate OH, so that the effect of iron sequestration could be distinguished from direct OH radical scavenging. In the latter system, the rank order of OH scavenging activity was F16BP > F1P > F6P > fructose = mannitol = glucose. Our results clearly demonstrate that intracellular phosphorylated forms of fructose have more scavenging properties than fructose or glucose, leading us to conclude that the acute administration of fructose could overcome the body’s reaction to exogenous antioxidants during appropriate therapy in certain pathophysiological conditions related to oxidative stress, such as sepsis, neurodegenerative diseases, atherosclerosis, malignancy, and some complications of pregnancy.