Bacterial AAA+ ATPase ClpB cooperates with DnaK during reactivation of aggregated proteins. The ClpB-mediated disaggregation is linked to translocation of polypeptides through the channel in the oligomeric ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and ClpB80 which does not contain the substrate-interacting N-terminal domain. The biological role of the truncated isoform ClpB80 is unknown. We found that resolubilization of aggregated proteins in E. coli after heat-shock and reactivation of aggregated proteins in vitro and in vivo occurred at higher rates in the presence of ClpB95 with ClpB80 than with ClpB95 or ClpB80 alone. Combined amounts of ClpB95 and ClpB80 bound to aggregated substrates were similar to the amounts of either ClpB95 or ClpB80 bound to the substrates in the absence of another isoform. The ATP hydrolysis rate of ClpB95 with ClpB80, which is linked to the rate of substrate translocation, was not higher than the rates measured for the isolated ClpB95 or ClpB80. We postulate that a reaction step that takes place after substrate binding to ClpB and precedes substrate translocation is rate-limiting during aggregate reactivation and its efficiency is enhanced in the presence of both ClpB isoforms. Moreover, we found that ClpB95 and ClpB80 form hetero-oligomers which are similar in size to the homo-oligomers of ClpB95 or ClpB80. Thus, the mechanism of functional cooperation of the two isoforms of ClpB may be linked to their hetero-association. Our results suggest that the functionality of other AAA+ ATPases may be also optimized by interaction and synergistic cooperation of their isoforms.

We have previously reported that protein kinase C gamma (PKCγ) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKCγ translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKCγ-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKCγ with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKCγ, the Cx43 did not disassemble out of plaques even though the PKCγ enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific "plaquetosome" arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKCγ-driven disassembly response.

Protein Kinase C (PKC) isoforms have been identified as major cellular signaling proteins that act directly in response to oxidation conditions. In retina and lens two isoforms of PKC respond to changes in oxidative stress, PKCγ and PKCε, while only PKCε is found in heart. In heart the PKCε acts on connexin 43 to protect from hypoxia. The presence of both isoforms in the lens led to this study to determine if lens PKCε had unique targets. Both lens epithelial cells in culture and whole mouse lens were examined using PKC isoform-specific enzyme activity assays, coimmunoprecipitation, confocal microscopy, immunoblots, and light and electron microscopy. PKCε was found in lens epithelium and cortex but not in the nucleus of mouse lens. The PKCε isoform was activated in both epithelium and whole lens by 5% oxygen when compared to activity at 21% oxygen. In hypoxic conditions (5% oxygen) the PKCε co-immunoprecipitated with the mitochondrial cytochrome C oxidase IV subunit (CytCOx). Concomitant with this the CytCOx enzyme activity was elevated and increased co-localization of CytCOx with PCKε was observed using immunolabeling and confocal microscopy. In contrast, no hypoxia-induced activation of CytCOx was observed in lenses from the PKCε knockout mice. Lens from 6 week old PKCε knockout mice had a disorganized bow region which was filled with vacuoles indicating a possible loss of mitochondria but the size of the lens was not altered. Electron microscopy demonstrated that the nuclei of the PCKε knockout mice were abnormal in shape. Thus, PKCε is found to be activated by hypoxia and this results in the activation of the mitochondrial protein CytCOx. This could protect the lens from mitochondrial damage under the naturally hypoxic conditions observed in this tissue. Lens oxygen levels must remain low. Elevation of oxygen which occurs during vitreal detachment or liquification is associated with cataracts. We hypothesize that elevated oxygen could cause inhibition of PKCε resulting in a loss of mitochondrial protection.

Nearly all male cystic fibrosis (CF) patients exhibit tissue abnormalities in the reproductive tract, a condition that renders them azoospermic and infertile. Two swine CF models have been reported recently that include respiratory and digestive manifestations that are comparable to human CF. The goal of this study was to determine the phenotypic changes that may be present in the vas deferens of these swine CF models. Tracts from CFTR(-/-) and CFTR(ΔF508/ΔF508) neonates revealed partial or total vas deferens and/or epididymis atresia at birth, while wild-type littermates were normal. Histopathological analysis revealed a range of tissue abnormalities and disruptions in tubular organization. Vas deferens epithelial cells were isolated and electrophysiological results support that CFTR(-/-) monolayers can exhibit Na(+) reabsorption but reveal no anion secretion following exposure to cAMP-generating compounds, suggesting that CFTR-dependent Cl(-) and/or HCO(3)(-) transport is completely impaired. SLC26A3 and SLC26A6 immunoreactivities were detected in all experimental groups, indicating that these two chloride-bicarbonate exchangers were present, but were either unable to function or their activity is electroneutral. In addition, no signs of increased mucus synthesis and/or secretion were present in the male excurrent ducts of these CF models. Results demonstrate a causal link between CFTR mutations and duct abnormalities that are manifested at birth.

The AsiA protein is a T4 bacteriophage early gene product that regulates transcription of host and viral genes. Monomeric AsiA binds tightly to the σ 70 subunit of Escherichia coli RNA polymerase, thereby inhibiting transcription from bacterial promoters and phage early promoters and co-activating transcription from phage middle promoters. Results of structural studies have identified amino acids at the protomer-protomer interface in dimeric AsiA and at the monomeric AsiA-σ 70 interface and demonstrated substantial overlap in the sets of residues that comprise each. Here we evaluate the contributions of individual interfacial amino acid side chains to protomerprotomer affinity in AsiA homodimers, to monomeric AsiA affinity for σ 70 , and to AsiA function in transcription. Sedimentation equilibrium, dynamic light scattering, electrophoretic mobility shift and transcription activity measurements were used to assess affinity and function of sitespecific AsiA mutants. Alanine substitutions for solvent-inaccessible residues positioned centrally in the protomer-protomer interface of the AsiA homodimer -V14, I17, and I40 -resulted in the largest changes in free energy of dimer association, whereas alanine substitutions at other interfacial positions had little effect. These residues also contribute significantly to AsiAdependent regulation of RNA polymerase activity, as do additional residues positioned at the periphery of the interface (K20 and F21). Notably, the relative contributions of a given amino acid side chain to RNA polymerase inhibition and activation (MotA-independent) by AsiA are very † This work was supported by the NIH (GM54998 to J.L.U. and GM59295 to K.S.) and the office of the Vice President for Research at the University of Georgia. * To whom correspondence should be addressed: The University of Georgia, Department of Chemistry, Chemistry Building, Cedar Street Building 1001, Athens, GA 30602-2556, USA. Tel.: 706-542-7922; Fax: 706-542-1738; urbauer@chem.uga.edu. # These authors contributed equally to this work SUPPORTING INFORMATION AVAILABLEOne figure detailing NMR minimal chemical shift perturbation values for all mutant AsiA proteins. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 July 27. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript similar in most cases. The mainstay for intermolecular affinity and AsiA function appears to be I17. Our results define the core interfacial residues of AsiA, establish roles for many of the interfacial amino acids, are in agreement with the tenets underlying protein-protein interactions and interfaces, and will be beneficial for a general, comprehensive understanding of the mechanistic underpinnings of bacterial RNA polymerase regulation.Following infection of Escherichia coli by bacteriophage T4, phage encoded proteins bind tightly to the host RNA polymerase to direct and redirect transcription (1). One ...