Shank2 associates with and regulates Na+/H+ exchanger 3

Na+/H+ exchanger 3 (NHE3) plays a pivotal role in transepithelial Na+ and HCO3(-) absorption across a wide range of epithelia in the digestive and renal-genitourinary systems. Accumulating evidence suggests that PDZ-based adaptor proteins play an important role in regulating the trafficking and activity of NHE3. A search for NHE3-binding modular proteins using yeast two-hybrid assays led us to the PDZ-based adaptor Shank2. The interaction between Shank2 and NHE3 was further confirmed by immunoprecipitation and surface plasmon resonance studies. When expressed in PS120/NHE3 cells, Shank2 increased the membrane expression and basal activity of NHE3 and attenuated the cAMP-dependent inhibition of NHE3 activity. Furthermore, knock-down of native Shank2 expression in Caco-2 epithelial cells by RNA interference decreased NHE3 protein expression as well as activity but amplified the inhibitory effect of cAMP on NHE3. These results indicate that Shank2 is a novel NHE3 interacting protein that is involved in the fine regulation of transepithelial salt and water transport through affecting NHE3 expression and activity.     

betaPak-interacting exchange factor-mediated Rac1 activation requires smgGDS guanine nucleotide exchange factor in basic fibroblast growth factor-induced neurite outgrowth

Neuritogenesis requires active actin cytoskeleton rearrangement in which Rho GTPases play a pivotal role. In a previous study (Shin, E. Y., Woo, K. N., Lee, C. S., Koo, S. H., Kim, Y. G., Kim, W. J., Bae, C. D., Chang, S. I., and Kim, E. G. (2004) J. Biol. Chem. 279, 1994-2004), we demonstrated that betaPak-interacting exchange factor (betaPIX) guanine nucleotide exchange factor (GEF) mediates basic fibroblast growth factor (bFGF)-stimulated Rac1 activation through phosphorylation of Ser-525 and Thr-526 at the GIT-binding domain (GBD). However, the mechanism by which this phosphorylation event regulates the Rac1-GEF activity remained elusive. We show here that betaPIX binds to Rac1 via the GBD and also activates the GTPase via an associated GEF, smgGDS, in a phosphorylation-dependent manner. Notably, the Rac1-GEF activity of betaPIX persisted for an extended period of time following bFGF stimulation, unlike other Rho GEFs containing the Dbl homology domain. We demonstrate that C-PIX, containing proline-rich, GBD, and leucine zipper domains can interact with Rac1 via the GBD in vitro and in vivo and also mediated bFGF-stimulated Rac1 activation, as determined by a modified GEF assay and fluorescence resonance energy transfer analysis. However, nonphosphorylatable C-PIX (S525A/T526A) failed to generate Rac1-GTP. Finally, betaPIX is shown to form a trimeric complex with smgGDS and Rac1; down-regulation of smgGDS expression by short interfering RNA causing significant inhibition of betaPIX-mediated Rac1 activation and neurite outgrowth. These results provide evidence for a new and unexpected mechanism whereby betaPIX can regulate Rac1 activity.     

Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X_L and Bcl-2. A structural model of the Bcl-X_L/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X_L/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X_L. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.     

Rapid estimation of triacylglycerol content of <Emphasis Type="Italic">Chlorella</Emphasis> sp. by thermogravimetric analysis

A simple and reliable method based on thermogravimetric analysis has been developed for determining triacylglycerol content in Chlorella sp. KR-1. There are two decomposing steps during pyrolysis of the microalgal cells and the second step of weight loss may be attributed to degradation and volatilization of triacylglycerols. The second peak height in the temperature derivatives of weight loss increased with the triacylglycerol content of the microalgal cells and the peak was around 390°C regardless of the triacylglycerol contents. Based on these findings, a linear equation for determining triacylglycerol content was derived. The proposed method gives satisfactory results, showing small variance and a good interpolation capability.     

Enhancement of auxin sensitivity in Ranunculus sceleratus by ethylene: A mechanism to escape from hypoxia under temporary submergence

We analyzed auxin-induced and ethylene-enhanced elongation of petiole segments in Ranunculus sceleratus L. The early time course of elongation in petiolar segments was monitored with a computer-based video digitizer system. The application of ethylene-releasing ethrel slightly increased the elongation rate in the absence of IAA. When IAA alone was applied, elongation increased after a latent period of approximately 30 min. Maximal elongation rate was attained immediately after the latent period, and then the stabilized steady rate was recorded. During this phase, addition of ethrel strongly increased the elongation rate after a period of approximately 18 min. Although ethrel could acidify the growth medium, only a small part of the enhanced elongation was due to an acid-growth effect. Most of the growth stimulation was auxin-dependent and must be ascribed to the presence of ethylene. In the presence of ethrel, the log-concentration-response curve of IAA appeared to be shifted to the left. This kinetic analysis indicates an increase, due to ethylene, in the sensitivity of the R. sceleratus petiole to auxin, which results in inducing rapid growth to escape from hypoxia under temporary submergence.     

Rescue of ΔF508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway

The most prevalent disease-causing mutation of CFTR is the deletion of Phe508 (ΔF508), which leads to defects in conventional Golgi-mediated exocytosis and cell surface expression. We report that ΔF508-CFTR surface expression can be rescued in vitro and in vivo by directing it to an unconventional GRASP-dependent secretion pathway. An integrated molecular and physiological analysis indicates that mechanisms associated with ER stress induce cell surface trafficking of the ER core-glycosylated wild-type and ΔF508-CFTR via the GRASP-dependent pathway. Phosphorylation of a specific site of GRASP and the PDZ-based interaction between GRASP and CFTR are critical for this unconventional surface trafficking. Remarkably, transgenic expression of GRASP in ΔF508-CFTR mice restores CFTR function and rescues mouse survival without apparent toxicity. These findings provide insight into how unconventional protein secretion is activated, and offer a potential therapeutic strategy for the treatment of cystic fibrosis and perhaps diseases stemming from other misfolded proteins.     

TRP14 Inhibits Osteoclast Differentiation via Its Catalytic Activity

We previously reported the inhibitory role of thioredoxin-related protein of 14 kDa (TRP14), a novel disulfide reductase, in nuclear factor-κB (NF-κB) activation, but its biological function has remained to be explored. Here, we evaluated the role of TRP14 in the differentiation and function of osteoclasts (OCs), for which NF-κB and cellular redox regulation have been known to be crucial, using RAW 264.7 macrophage cells expressing wild-type TRP14 or a catalytically inactive mutant, as well as its small interfering RNA. TRP14 depletion enhanced OC differentiation, actin ring formation, and bone resorption, as well as the accumulation of reactive oxygen species (ROS). TRP14 depletion promoted the activation of NF-κB, c-Jun NH₂-terminal kinase, and p38, the expression of c-Fos, and the consequent induction of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1), a key determinant of osteoclastogenesis. However, pretreatment with N-acetylcysteine or diphenylene iodonium significantly reduced the OC differentiation, as well as the ROS accumulation and NF-κB activation, that were enhanced by TRP14 depletion. Furthermore, receptor activator of NF-κB ligand (RANKL)-induced ROS accumulation, NF-κB activation, and OC differentiation were inhibited by the ectopic expression of wild-type TRP14 but not by its catalytically inactive mutant. These results suggest that TRP14 regulates OC differentiation and bone resorption through its catalytic activity and that enhancing TRP14 may present a new strategy for preventing bone resorption diseases.     

Genetically distinct pathways guide effector export through the type VI secretion system

Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), haemolysin co‐regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone‐like quality of Hcp. Application of this approach to the Hcp secretion island I‐encoded T6SS (H1‐T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (t ype VI s ecretion e xported 4), subsequently shown to act as a potent intra‐specific H1‐T6SS‐delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1‐T6SS effectors, Tse5 and Tse6, which differ from Hcp‐stabilized substrates by the presence of toxin‐associated PAAR‐repeat motifs and genetic linkage to members of the valine‐glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp‐stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1‐T6SS‐exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.