Background: Methicillin-resistant Staphylococcus aureus (MRSA) PK has been recently identified as a potential novel antimicrobial drug target. Results: Screening of a marine extract library led to the identification of several bis-indole alkaloids as novel potent and selective MRSA PK inhibitors. Conclusion:These results help to understand the mechanism of the antibacterial activities of marine bis-indole alkaloids. Significance: This study provides the basis for development of potential novel antimicrobials.
Protein palmitoylation plays a critical role in sorting and targeting of several proteins to pre-and postsynaptic sites. In this study, we have analyzed the role of palmitoylation in trafficking of synaptotagmin I and its modulation by synaptic activity. We found that palmitoylation of N-terminal cysteines contributed to sorting of synaptotagmin I to an intracellular vesicular compartment at the presynaptic terminal. Presynaptic targeting is a unique feature of N-terminal sequences of synaptotagmin I because the palmitoylated N terminus of synaptotagmin VII failed to localize to presynaptic sites. We also found that palmitate was stably associated with both synaptotagmin I and SNAP-25 and that rapid neuronal depolarization did not affect palmitate turnover on these proteins. However, long-term treatment with drugs that either block synaptic activity or disrupt SNARE complex assembly modulated palmitoylation and accumulation of synaptotagmin I at presynaptic sites. We conclude that palmitoylation is involved in trafficking of specific elements involved in transmitter release and that distinct mechanisms regulate addition and removal of palmitate on select neuronal proteins.Synaptic transmission requires appropriate protein targeting and assembly of pre-and postsynaptic elements. Protein sorting to distinct domains in polarized cells appears to begin in the Golgi/trans-Golgi network, where proteins can segregate and exit in separate transport vesicles (1). One mechanism that regulates protein trafficking is palmitoylation, a post-translational modification involving the addition of palmitate, a 16-carbon fatty acid, via a labile thioester linkage (2-5). In neuronal cells, palmitoylation is critical for sorting of several synaptic proteins (6). These include the postsynaptic density protein PSD-95, the AMPA 1 receptor-binding protein, and the presynaptic proteins GAP-43 (growth-associated protein of 43 kDa) and GAD-65 (7-11). Palmitoylation of the AMPA receptor-binding protein and PSD-95 is essential for clustering at the PSD (8 -10), whereas palmitoylation of GAD-65 is important for presynaptic targeting (11,12).Acylation of several other axonal proteins as well as proteins associated with neurotransmitter release machinery has been recently reported (5, 6, 13). These include members of the synaptotagmin family that regulate synaptic vesicle trafficking and neurotransmitter release (14, 15). The synaptotagmin family includes 13 members characterized by a unique N-terminal region followed by a transmembrane domain, a cluster of cysteines (the putative palmitoylation site), a variable domain, and two C-terminal C2 domains (15-18). Synaptotagmin I, the most characterized member of the family, is proposed to act as a Ca 2ϩ sensor for regulated exocytosis (19). Other abundant members of the family include synaptotagmins III and VII (15). Interestingly, synaptotagmin I is localized to synaptic vesicles, whereas synaptotagmin VII is localized on the plasma membrane opposite synaptic vesicle docking sites (15). Despite th...
Novel classes of antimicrobials are needed to address the challenge of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Using the architecture of the MRSA interactome, we identified pyruvate kinase (PK) as a potential novel drug target based upon it being a highly connected, essential hub in the MRSA interactome. Structural modeling, including X-ray crystallography, revealed discrete features of PK in MRSA, which appeared suitable for the selective targeting of the bacterial enzyme. In silico library screening combined with functional enzymatic assays identified an acyl hydrazone-based compound (IS-130) as a potent MRSA PK inhibitor (50% inhibitory concentration [IC 50 ] of 0.1 M) with >1,000-fold selectivity over human PK isoforms. Medicinal chemistry around the IS-130 scaffold identified analogs that more potently and selectively inhibited MRSA PK enzymatic activity and S. aureus growth in vitro (MIC of 1 to 5 g/ml). These novel anti-PK compounds were found to possess antistaphylococcal activity, including both MRSA and multidrug-resistant S. aureus (MDRSA) strains. These compounds also exhibited exceptional antibacterial activities against other Gram-positive genera, including enterococci and streptococci. PK lead compounds were found to be noncompetitive inhibitors and were bactericidal. In addition, mutants with significant increases in MICs were not isolated after 25 bacterial passages in culture, indicating that resistance may be slow to emerge. These findings validate the principles of network science as a powerful approach to identify novel antibacterial drug targets. They also provide a proof of principle, based upon PK in MRSA, for a research platform aimed at discovering and optimizing selective inhibitors of novel bacterial targets where human orthologs exist, as leads for anti-infective drug development.
Novel antimicrobial targets are urgently needed to overcome rising antibiotic resistance of important human pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Here we report the essentiality and kinetic properties of MRSA pyruvate kinase (PK). Targetron-mediated gene disruption demonstrated PK is essential for S. aureus growth and survival, suggesting that this protein may be a potential drug target. The presence of the pfk (6-phosphofructokinase)-pyk operon in MRSA252, and the nonessential nature of PFK shown by targetron, further emphasized the essential role of PK in cell viability. The importance of PK in bacterial growth was confirmed by showing that its enzymatic activity peaked during the logarithmic phase of S. aureus growth. PK from Staphylococcus and several other species of bacteria have an extra C-terminal domain (CT) containing a phosphoenolpyruvate (PEP) binding motif. To elucidate the possible structure and function of this sequence, the quaternary structures and kinetic properties of the full-length MRSA PK and truncated MRSA PK lacking the CT domain were characterized. Our results showed that (1) MRSA PK is an allosteric enzyme with homotetramer architecture activated by AMP or ribose 5-phosphate (R5P), but not by fructose 1,6-bisphosphate (FBP), which suggests a different mode of allosteric regulation when compared with human isozymes, (2) the CT domain is not required for the tetramerization of the enzyme; homotetramerization occurred in a truncated PK lacking the domain, (3) truncated enzyme exhibited high affinity toward both PEP and ADP and exhibited hyperbolic kinetics toward PEP in the presence of activators (AMP and R5P) consistent with kinetic properties of full-length enzyme, indicating that the CT domain is not required for substrate binding or allosteric regulation observed in the holoenzyme, (4) the kinetic efficiency (k(cat)/S(0.5)) of truncated enzyme was decreased by 24- and 16-fold, in ligand-free state, toward PEP and ADP, respectively, but was restored by 3-fold in AMP-bound state, suggesting that the sequence containing the CT domain (Gly(473)-Leu(585)) plays a substantial role in enzyme activity and comformational stability, and (5) full-length MRSA PK activity was stimulated at low concentrations of ATP (e.g., 1 mM) and inhibited by inorganic phosphate and high concentrations of FBP (10 mM) and ATP (e.g., >2.5 mM), whereas for truncated enzyme, stimulation at low concentrations of ATP was lost. These findings suggest that the CT domain is involved in maintaining the specificity of allosteric regulation of MRSA PK by AMP, R5P, and ATP. The CT extension also encodes a protein domain with homology to enzyme I of the Escherichia coli sugar-PTS system, suggesting that MRSA PK may also exert an important regulatory role in sugar transport metabolism. These findings yield new insights into MRSA PK function and mode of allosteric regulation which may aid in the development of clinically important drugs targeting this enzyme and further define the role of the ext...
Mortality attributable to infection with methicillin-resistant Staphylococcus aureus (MRSA) has now overtaken the death rate for AIDS in the United States, and advances in research are urgently needed to address this challenge. We report the results of the systematic identification of protein-protein interactions for the hospital-acquired strain MRSA-252. Using a high-throughput pull-down strategy combined with quantitative proteomics to distinguish specific from nonspecific interactors, we identified 13,219 interactions involving 608 MRSA proteins. Consecutive analyses revealed that this protein interaction network (PIN) exhibits scale-free organization with the characteristic presence of highly connected hub proteins. When clinical and experimental antimicrobial targets were queried in the network, they were generally found to occupy peripheral positions in the PIN with relatively few interacting partners. In contrast, the hub proteins identified in this MRSA PIN that are essential for network integrity and stability have largely been overlooked as drug targets. Thus, this empirical MRSA-252 PIN provides a rich source for identifying critical proteins essential for network stability, many of which can be considered as prospective antimicrobial drug targets.
The activation of large conductance, calcium-sensitive K ؉ (BK Ca ) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BK Ca channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BK Ca channel activity 4 -5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the ␣ isoform of type I cGMP-dependent protein kinase (cGKI). We observed that cotransfection of cells with BK Ca channels and a catalytically inactive ("dead") mutant of human cGKI␣ prevented enhancement of BK Ca channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKI␣ supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKI␣ were found to associate with BK Ca channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKI␣ was able to directly phosphorylate immunoprecipitated BK Ca channels, suggesting that cGKI␣-dependent phosphorylation of BK Ca channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKI␣ alone is sufficient to promote the enhancement of BK Ca channels in situ after activation of the NO/cGMP signaling pathway.The elevation of intracellular cGMP 1 in response to endothelium-derived nitric oxide (NO) or clinically prescribed nitrovasodilators, such as nitroglycerin and sodium nitroprusside, is known to play an important role in the hypotensive actions of these agents (1, 2). Similarly, elevation of cGMP by the phosphodiesterase inhibitor sildenafil (Viagra) (3) appears to underlie the smooth muscle-relaxing and anti-impotence effects of this drug. Although the exact mechanism(s) by which elevated cGMP causes smooth muscle relaxation has not been clearly defined, cGMP is known to influence a number of cellular processes (4), such as the levels of cytosolic free calcium, myosin light chain dephosphorylation (5), and the activity of voltagedependent, L-type calcium channels (6).In both vascular and nonvascular smooth muscle, activation of large conductance, calcium-sensitive K ϩ channels (maxi-K or BK Ca channels) is reported to occur in response to endogenous NO or exogenous NO donors (7-12). In many cases, addition of exogenous cGMP appears to mimic the effects of NO and NO donors on BK Ca channel activation (8, 10, 12-15), suggesting that cGMP acts downstream of NO. Physiologically, BK Ca channels appear to be important cellular effectors for the vasodilatory actions of the NO/cGMP signaling pathway because blockade of BK Ca channels can interfere with the relaxationpromoting effects of [16][17][18].A major intracellular target for cGMP in smooth muscle...
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