Antibiotics with new mechanisms of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. We synthesized a family of peptidomimetic antibiotics, based on the antimicrobial peptide protegrin I. Several rounds of optimization gave a lead compound that was active in the nanomolar range against gram-negative Pseudomonas sp., but was largely inactive against other Gram-negative and Gram-positive bacteria. Biochemical and genetic studies showed the peptidomimetics had a non-membrane-lytic mechanism of action and identified a homologue of the ß-barrel protein LptD (Imp/OstA), which functions in outer membrane biogenesis, as a cellular target. The peptidomimetic showed potent antimicrobial activity in a mouse septicemia infection model. Drug-resistant strains of Pseudomonas are a serious health problem, so this family of antibiotics may have important therapeutic applications. A synthesized antibiotic targets a protein involved in outer membrane biogenesis to selectively kill Pseudomonas pathogens. 1 Peptidomimetic Antibiotics Target Outer Membrane Biogenesis in Pseudomonas aeruginosa AbstractAntibiotics with new mechanisms of action are urgently required to combat the growing health

Spatial and temporal regulation of intracellular Ca 2؉signaling depends on localized Ca 2؉ microdomains containing the requisite molecular components for Ca 2؉ influx, efflux, and signal transmission. Plasma membrane Ca 2؉ -ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains. The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen. The related MAGUKs SAP90/ PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b. Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95. These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca 2؉ regulation.Calcium ion (Ca 2ϩ ) homeostasis is crucial for cell function and survival (1). A finely controlled system of Ca 2ϩ transporters, channels, and Ca 2ϩ -binding proteins allows for transient increases in the intracellular free calcium concentration ([Ca 2ϩ ] i ), 1 while over the long term maintaining a low resting [Ca 2ϩ ] i (2). The exquisite specificity of Ca 2ϩ signaling mandates that both the entry and the removal of Ca 2ϩ are under precise temporal and spatial control (3, 4). Accordingly, the molecular machinery involved in local Ca 2ϩ signaling must be assembled, maintained, and regulated with the requisite spatial and temporal resolution. Mechanisms that increase local [Ca 2ϩ ] i have been studied extensively over the last few years; consequently, significant progress has been made in understanding the regulation and targeting of calcium channels (5). By contrast, much less is known about the spatial organization of Ca 2ϩ extrusion mechanisms, specifically that provided by plasma membrane Ca 2ϩ -ATPases (PMCAs). These primary ion pumps are essential for the long term maintenance of low intracellular Ca 2ϩ but more recently have also been implicated in dynamic events such as the regulation of Ca 2ϩ spikes and local Ca 2ϩ signaling (for review, see Refs. 6 -8). A multigene family of four non-allelic members encodes four conserved mammalian PMCA isoforms (designated PMCA1-4), with additional diversity generated by alternative mRNA splicing affecting the protein at two major locations (9). The four PMCA gene products do not ...

Plasma membrane Ca2؉ ATPases are P-type pumps important for intracellular Ca 2؉ homeostasis. The extreme C termini of alternatively spliced "b"-type Ca 2؉ pump isoforms resemble those of K ؉ channels and Nmethyl-D-aspartate receptor subunits that interact with channel-clustering proteins of the membrane-associated guanylate kinase (MAGUK) family via PDZ domains. Yeast two-hybrid assays demonstrated strong interaction of Ca 2؉ pump 4b with the PDZ1؉2 domains of several mammalian MAGUKs. Pump 4b and PSD-95 could be co-immunoprecipitated from COS-7 cells overexpressing these proteins. Surface plasmon resonance revealed that a C-terminal pump 4b peptide interacted with the PDZ1؉2 domains of hDlg with nanomolar affinity (K D ‫؍‬ 1.6 nM), whereas binding to PDZ3 was in the micromolar range (K D ‫؍‬ 1.2 M). In contrast, the corresponding C-terminal peptide of Ca 2؉ pump 2b interacted weakly with PDZ1؉2 and not at all with PDZ3 of hDlg. Ca 2؉ pump 4b bound strongly to PDZ1؉2؉3 of hDlg on filter assays, whereas isoform 2b bound weakly, and the splice variants 2a and 4a failed to bind. Together, these data demonstrate a direct physical binding of Ca 2؉ pump isoform 4b to MAGUKs via their PDZ domains and reveal a novel role of alternative splicing within the family of plasma membrane Ca 2؉ pumps. Alternative splicing may dictate their specific interaction with PDZ domain-containing proteins, potentially influencing their localization and incorporation into functional multiprotein complexes at the plasma membrane.Temporal and spatial control of intracellular Ca 2ϩ concentrations is essential for eukaryotic cell physiology. Plasma membrane Ca 2ϩ ATPases (PMCAs) 1 represent a ubiquitous, high affinity system for the expulsion of Ca 2ϩ from the cell and are thought to be responsible for the long-term setting and maintenance of intracellular Ca 2ϩ levels (1, 2). Mammalian PMCAs are encoded by a multigene family consisting of four members termed PMCA 1-4 (3). Additional isoform diversity is generated via alternative RNA splicing (3, 4). Alternative splicing of the C-terminal tail has been shown to alter the regulatory properties of PMCA isoforms, particularly with respect to phosphorylation and calmodulin stimulation (5-10). Many PMCA isoforms and splice variants are expressed in a tissueand cell type-specific manner (11-17), and in several cell types, the PMCA has been shown to be concentrated in specific membrane domains by immunocytochemical analyses. For example, in kidney and intestinal epithelia involved in transcellular Ca 2ϩ flux, the pump is generally localized to the basolateral membrane (18). Using immunoelectron microscopy, the PMCA was recently detected at the plasma membrane surrounding the soma, as well as in the dendrites and spines of cerebellar Purkinje cells where it co-localized with P-type Ca 2ϩ channels (19). Taken together, these studies indicate that different isoforms of the PMCA may play an active role in the local control of Ca 2ϩ signaling and the dynamic regulation of Ca 2ϩ microdomains (7,20). Howev...

Modulation of Ca(2+) channels by neurotransmitters provides critical control of neuronal excitability and synaptic strength. Little is known about regulation of the Ca(2+) efflux pathways that counterbalance Ca(2+) influx in neurons. We demonstrate that bradykinin and ATP significantly facilitate removal of action potential-induced Ca(2+) loads by stimulating plasma membrane Ca(2+)-ATPases (PMCAs) in rat sensory neurons. This effect was mimicked in the soma and axonal varicosities by phorbol esters and was blocked by antagonists of protein kinase C (PKC). Reduced expression of PMCA isoform 4 abolished, and overexpression of isoform 4b enhanced, PKC-dependent facilitation of Ca(2+) efflux. This acceleration of PMCA4 underlies the shortening of the action potential afterhyperpolarization produced by activation of bradykinin and purinergic receptors. Thus, isoform-specific modulation of PMCA-mediated Ca(2+) efflux represents a novel mechanism to control excitability in sensory neurons.

Bacterial sepsis triggers robust activation of the complement system with subsequent generation of anaphylatoxins (C3a, C5a) and the terminal complement complex (TCC) that together contribute to organ failure and death. Here we tested the effect of RA101295, a 2-kDa macrocyclic peptide inhibitor of C5 cleavage, using in vitro whole-blood assays and an in vivo baboon model of Escherichia coli sepsis. RA101295 strongly inhibited E. coli-induced complement activation both in vitro and in vivo by blocking the generation of C5a and the soluble form of TCC, sC5b-9. RA101295 reduced the E. coliinduced "oxidative burst," as well as leukocyte activation, without affecting host phagocytosis of E. coli. RA101295 treatment reduced plasma LPS content in E. coli-challenged baboons, implying reduced complement-mediated bacteriolysis, whereas treated animals showed slightly improved bacterial clearance during the bacteremic stage compared with controls. Treatment with RA101295 also improved consumptive coagulopathy and preserved endothelial anticoagulant and vascular barrier functions. RA101295 abolished sepsis-induced surges in proinflammatory cytokines and attenuated systemic circulatory and febrile responses, likely reflecting decreased systemic levels of LPS and C5a. Overall, RA101295 treatment was associated with significant organ protection and markedly reduced mortality compared with nontreated controls (four of five animals survived in a 100% lethal model). We therefore conclude that inhibition of C5 cleavage during the bacteremic stage of sepsis could be an important therapeutic approach to prevent sepsis-induced inflammation, consumptive coagulopathy, and subsequent organ failure and death. complement | coagulation | sepsis | Escherichia coli | organ failure