Covalent binding of uracil DNA glycosylase UdgX to abasic DNA upon uracil excision.

Uracil DNA glycosylases (UDGs) are important DNA repair enzymes that excise uracil from DNA, yielding an abasic site. Recently, UdgX, an unconventional UDG with extremely tight binding to DNA containing uracil, was discovered. The structure of UdgX from Mycobacterium smegmatis in complex with DNA shows an overall similarity to that of family 4 UDGs except for a protruding loop at the entrance of the uracil-binding pocket. Surprisingly, H109 in the loop was found to make a covalent bond to the abasic site to form a stable intermediate, while the excised uracil remained in the pocket of the active site. H109 functions as a nucleophile to attack the oxocarbenium ion, substituting for the catalytic water molecule found in other UDGs. To our knowledge, this change from a catalytic water attack to a direct nucleophilic attack by the histidine residue is unprecedented. UdgX utilizes a unique mechanism of protecting cytotoxic abasic sites from exposure to the cellular environment.

Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci.

Precise enumeration of living intracellular bacteria is the key step to estimate the invasion potential of pathogens and host immune responses to understand the mechanism and kinetics of bacterial pathogenesis. Therefore, quantitative assessment of host-pathogen interactions is essential for development of novel antibacterial therapeutics for infectious disease. The gentamicin protection assay (GPA) is the most widely used method for these estimations by counting the CFU of intracellular living pathogens. Here, we assess the longstanding drawbacks of the GPA by employing an antistaphylococcal endopeptidase as a bactericidal agent to kill extracellular Staphylococcus aureus We found that the difference between the two methods for the recovery of intracellular CFU of S. aureus was about 5 times. We prove that the accurate number of intracellular CFU could not be precisely determined by the GPA due to the internalization of gentamicin into host cells during extracellular bacterial killing. We further demonstrate that lysostaphin-mediated extracellular bacterial clearance has advantages for measuring the kinetics of bacterial internalization on a minute time scale due to the fast and tunable activity and the inability of protein to permeate the host cell membrane. From these results, we propose that accurate quantification of intracellular bacteria and measurement of internalization kinetics can be achieved by employing enzyme-mediated killing of extracellular bacteria (enzyme protection assay [EPA]) rather than the host-permeative drug gentamicin, which is known to alter host physiology.

Coupling of radiofrequency with magnetic nanoparticles treatment as an alternative physical antibacterial strategy against multiple drug resistant bacteria.

Antibiotic resistant bacteria not only affect human health and but also threatens the safety in hospitals and among communities. However, the emergence of drug resistant bacteria is inevitable due to evolutionary selection as a consequence of indiscriminate antibiotic usage. Therefore, it is necessary to develop a novel strategy by which pathogenic bacteria can be eliminated without triggering resistance. We propose a novel magnetic nanoparticle-based physical treatment against pathogenic bacteria, which blocks biofilm formation and kills bacteria. In this approach, multiple drug resistant Staphylococcus aureus USA300 and uropathogenic Escherichia coli CFT073 are trapped to the positively charged magnetic core-shell nanoparticles (MCSNPs) by electrostatic interaction. All the trapped bacteria can be completely killed within 30 min owing to the loss of membrane potential and dysfunction of membrane-associated complexes when exposed to the radiofrequency current. These results indicate that MCSNP-based physical treatment can be an alternative antibacterial strategy without leading to antibiotic resistance, and can be used for many purposes including environmental and therapeutic applications.

Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa.

The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co(2+) rather than Zn(2+): the kcat (s(-1)) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co(2+), and Zn(2+)conditions, respectively. Consistently, addition of low concentrations of Co(2+) to PaAP previously saturated with Zn(2+) greatly enhanced the enzymatic activity, suggesting that Co(2+)may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co(2+) or Zn(2+) commonly showed two metal ions in the active site coordinated with three conserved residues and a bicarbonate ion in a tetragonal geometry. However, Co(2+)- and Zn(2+)-bound structures showed no noticeable alterations relevant to differential effects of metal species, except the relative orientation of Glu-265, a general base in the active site. The characterization of mutant PaAP revealed that the first metal binding site is primarily responsible for metal preference. Similar to PaAP, Streptococcus pneumonia glutamyl aminopeptidase (SpGP), belonging to aminopeptidase family M42 in clan MH, also showed requirement for Co(2+) for maximum activity. These results proposed that clan MH peptidases might be a cocatalytic cobalt peptidase rather than a zinc-dependent peptidase.

Crystal structure of the LG3 domain of endorepellin, an angiogenesis inhibitor.

Endorepellin, the C-terminal region of perlecan, inhibits angiogenesis by disrupting actin cytoskeleton and focal adhesions. The C-terminal laminin-like globular domain (LG3) of endorepellin directs most of this antiangiogenic activity. To investigate the angiostatic mechanism and to identify structural determinants, we have solved crystal structures of the LG3 domain in both apo- and calcium-bound forms at resolutions of 1.5 Å and 2.8 Å, respectively. The conserved core has the jellyroll fold characteristic of LG domains. The calcium-induced structural changes seem very restricted, and the calcium binding site appears to be preformed, suggesting that the bound calcium ion, rather than structural rearrangements, contributes to antiangiogenesis. We have identified H4268 on the EF loop as a key residue for the biochemical function of LG3, since its mutation abolishes antiangiogenic activity, and mutant LG3 can no longer form a direct interaction with integrin. Taken together, we propose that these two distinct structural elements contribute to the angiostatic effect of endorepellin.

Systematic evaluation of transcellular activities of secretory phospholipases A2. High activity of group V phospholipases A2 to induce eicosanoid biosynthesis in neighboring inflammatory cells.

The mechanisms by which secretory phospholipase A2 (PLA2) exerts cellular effects are not fully understood. To elucidate these mechanisms, we systematically and quantitatively assessed the activities of human group IIA, V, and X PLA2s on originating and neighboring cells using orthogonal fluorogenic substrates in various mixed cell systems. When HEK293 cells stably expressing each of these PLA2s were mixed with non-transfected HEK293 cells, group V and X PLA2s showed strong transcellular lipolytic activity, whereas group IIA PLA2 exhibited much lower transcellular activity. The transcellular activity of group V PLA2 was highly dependent on the presence of cell surface heparan sulfate proteoglycans of acceptor cells. Activation of RBL-2H3 and DLD-1 cells that express endogenous group V PLA2 led to the secretion of group V PLA2 and its transcellular action on neighboring human neutrophils and eosinophils, respectively. Similarly, activation of human bronchial epithelial cells, BEAS-2B, caused large increases in arachidonic acid and leukotriene C4 release from neighboring human eosinophils. Collectively, these studies show that group V and X PLA2s can act transcellularly on mammalian cells and suggest that group V PLA2 released from neighboring cells may function in triggering the activation of inflammatory cells under physiological conditions.

The mechanism of membrane targeting of human sphingosine kinase 1.

Sphingosine 1-phosphate is a bioactive sphingolipid that regulates cell growth and suppresses programmed cell death. The biosynthesis of sphingosine 1-phosphate is catalyzed by sphingosine kinase (SK) but the mechanism by which the subcellular localization and activity of SK is regulated in response to various stimuli is not fully understood. To elucidate the origin and structural determinant of the specific subcellular localization of SK, we performed biophysical and cell studies of human SK1 (hSK1) and selected mutants. In vitro measurements showed that hSK1 selectively bound phosphatidylserine over other anionic phospholipids and strongly preferred the plasma membrane-mimicking membrane to other cellular membrane mimetics. Mutational analysis indicates that conserved Thr54 and Asn89 in the putative membrane-binding surface are essential for lipid selectivity and membrane targeting both in vitro and in the cell. Also, phosphorylation of Ser225 enhances the membrane affinity and plasma membrane selectivity of hSK1, presumably by modulating the interaction of Thr54 and Asn89 with the membrane. Collectively, these studies suggest that the specific plasma membrane localization and activation of SK1 is mediated largely by specific lipid-protein interactions.