Nucleotide sequence of the head assembly gene cluster of bacteriophage L and decoration protein characterization

The temperate Salmonella enterica bacteriophage L is a close relative of the very well studied bacteriophage P22. In this study we show that the L procapsid assembly and DNA packaging genes, which encode terminase, portal, scaffold, and coat proteins, are extremely close relatives of the homologous P22 genes (96.3 to 99.1% identity in encoded amino acid sequence). However, we also identify an L gene, dec, which is not present in the P22 genome and which encodes a protein (Dec) that is present on the surface of L virions in about 150 to 180 molecules/virion. We also show that the Dec protein is a trimer in solution and that it binds to P22 virions in numbers similar to those for L virions. Its binding dramatically stabilizes P22 virions against disruption by a magnesium ion chelating agent. Dec protein binds to P22 coat protein shells that have expanded naturally in vivo or by sodium dodecyl sulfate treatment in vitro but does not bind to unexpanded procapsid shells. Finally, analysis of phage L restriction site locations and a number of patches of nucleotide sequence suggest that phages ST64T and L are extremely close relatives, perhaps the two closest relatives that have been independently isolated to date among the lambdoid phages.     

Properties of the permeability transition pore in mitochondria devoid of Cyclophilin D

We have studied the properties of the permeability transition pore (PTP) in mitochondria from the liver of mice where the Ppif gene encoding for mitochondrial Cyclophilin D (CyP-D) had been inactivated. Mitochondria from Ppif-/- mice had no CyP-D and displayed a striking desensitization of the PTP to Ca2+, in that pore opening required about twice the Ca2+ load necessary to open the pore in strain-matched, wild-type mitochondria. Mitochondria lacking CyP-D were insensitive to Cyclosporin A (CsA), which increased the Ca2+ retention capacity only in mitochondria from wild-type mice. The PTP response to ubiquinone 0, depolarization, pH, adenine nucleotides, and thiol oxidants was similar in mitochondria from wild-type and Ppif-/- mice. These experiments demonstrate that (i) the PTP can form and open in the absence of CyP-D, (ii) that CyP-D represents the target for PTP inhibition by CsA, and (iii) that CyP-D modulates the sensitivity of the PTP to Ca2+ but not its regulation by the proton electrochemical gradient, adenine nucleotides, and oxidative stress. These results have major implications for our current understanding of the PTP and its modulation in vitro and in vivo.     

Properties of WNK1 and implications for other family members

WNKs are large serine/threonine protein kinases structurally distinct from all other members of the protein kinase superfamily. Of the four human WNK family members, WNK1 and WNK4 have been linked to a hereditary form of hypertension, pseudohypoaldosteronism type II. We characterized the biochemical properties and regulation of WNK1 that may contribute to its physiological activities and abnormal function in disease. We showed that WNK1 is activated by hypertonic stress in kidney epithelial cells and in breast and colon cancer cell lines. In addition, hypotonic stress also led to a modest increase in WNK1 activity. Gel filtration suggested that WNK1 exists as a tetramer, and yeast two-hybrid data showed that the N terminus of WNK1 (residues 1-222) interacts with residues 481-660, which includes the WNK1 autoinhibitory domain and a C-terminal coiled-coil domain. Although cell biological studies have suggested a functional interaction between WNK1 and WNK4, we found no evidence of stable interactions between these kinases. However, WNK1 phosphorylated both WNK4 and WNK2. In addition, the WNK1 autoinhibitory domain inhibited the catalytic activity of these WNKs. These findings suggest potential mechanisms for interconnected regulation of WNK family members.     

Interaction of the mammalian endosomal sorting complex required for transport (ESCRT) III protein hSnf7-1 with itself, membranes, and the AAA+ ATPase SKD1

SKD1/VPS4B is an AAA+ (ATPase associated with a variety of cellular activities) protein involved in multivesicular body (MVB) biogenesis. In this study, we show that the impairment in MVB biogenesis caused by the ATP hydrolysis-deficient mutant SKD1(E235Q) is accompanied by assembly of a large detergent-insoluble protein complex that includes normally soluble endogenous components of mammalian endosomal sorting complex required for transport (ESCRT) I and ESCRT-III complexes. Membrane-bound ESCRT-III complex has been proposed to be the substrate that recruits SKD1 to nascent MVBs. To explore this relationship, we studied interactions among the human ESCRT-III components hSnf7-1 and hVps24, membranes, and SKD1. We found that a significant portion of overexpressed hSnf7-1 associated with membranes where it formed a large protein complex that recruited SKD1 and perturbed normal MVB biogenesis. Overexpressed hVps24 also associated with membranes and perturbed endosome structure but only when fused to green fluorescent protein. Domain analysis revealed that the basic N-terminal half of hSnf7-1 localized to membranes and formed detergent-resistant polymers, some of which looked like filopodia extending into the lumen of swollen endosomes or out from the plasma membrane. The C-terminal acidic half of hSnf7-1 did not associate with membranes and was required for interaction of hSnf7-1 with SKD1. Together with earlier studies, our work suggests that a variety of ESCRT-III-containing polymers can assemble on membranes and recruit SKD1 during formation of the MVB.     

Bacteriophage P22 tail accessory factor GP26 is a long triple-stranded coiled-coil

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.     

Chronic airway infection/inflammation induces a Ca2+i-dependent hyperinflammatory response in human cystic fibrosis airway epithelia

Hyperinflammatory responses to infection have been postulated as a component of cystic fibrosis (CF) lung disease. Studies have linked intracellular calcium (Ca(2+)(i)) mobilization with inflammatory responses in several systems. We have reported that the pro-inflammatory mediator bradykinin (BK) promotes larger Ca(2+)(i) signals in CF compared with normal bronchial epithelia, a response that reflects endoplasmic reticulum (ER)/Ca(2+) store expansion induced by chronic luminal airway infection/inflammation. The present study investigated whether CF airway epithelia were hyperinflammatory and, if so, whether the hyperinflammatory CF phenotype was linked to larger Ca(2+) stores in the ER. We found that DeltaF508 CF bronchial epithelia were hyperinflammatory as defined by an increased basal and mucosal BK-induced interleukin (IL)-8 secretion. However, the CF hyperinflammation expressed in short-term (6-11-day-old) primary cultures of DeltaF508 bronchial epithelia was lost in long-term (30-40-day-old) primary cultures of DeltaF508 bronchial epithelia, indicating this response was independent of mutant cystic fibrosis transmembrane conductance regulator. Exposure of 30-40-day-old cultures of normal airway epithelia to supernatant from mucopurulent material (SMM) from CF airways reproduced the increased basal and mucosal BK-stimulated IL-8 secretion of short-term CF cultures. The BK-triggered increased IL-8 secretion in SMM-treated cultures was mediated by an increased Ca(2+)(i) mobilization consequent to an ER expansion associated with increases in protein synthesis (total, cytokines, and antimicrobial factors). The increased ER-dependent, Ca(2+)(i)-mediated hyperinflammatory epithelial response may represent a general beneficial airway epithelial adaptation to transient luminal infection. However, in CF airways, the Ca(2+)(i)-mediated hyperinflammation may be ineffective in promoting the eradication of infection in thickened mucus and, consequently, may have adverse effects in the lung.     

In vivo and in vitro analysis of cardiac troponin I phosphorylation

Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during beta-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser(23)/Ser(24)) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation.