The Choline-binding Protein PspC of Streptococcus pneumoniae Interacts with the C-terminal Heparin-binding Domain of Vitronectin

Adherence of Streptococcus pneumoniae is directly mediated by interactions of adhesins with eukaryotic cellular receptors or indirectly by exploiting matrix and serum proteins as molecular bridges. Pneumococci engage vitronectin, the human adhesive glycoprotein and complement inhibitor, to facilitate attachment to epithelial cells of the mucosal cavity, thereby modulating host cell signaling. In this study, we identified PspC as a vitronectin-binding protein interacting with the C-terminal heparin-binding domain of vitronectin. PspC is a multifunctional surface-exposed choline-binding protein displaying various adhesive properties. Vitronectin binding required the R domains in the mature PspC protein, which are also essential for the interaction with the ectodomain of the polymeric immunoglobulin receptor and secretory IgA. Consequently, secretory IgA competitively inhibited binding of vitronectin to purified PspC and to PspC-expressing pneumococci. In contrast, Factor H, which binds to the N-terminal part of mature PspC molecules, did not interfere with the PspC-vitronectin interaction. Using a series of vitronectin peptides, the C-terminal heparin-binding domain was shown to be essential for the interaction of soluble vitronectin with PspC. Binding experiments with immobilized vitronectin suggested a region N-terminal to the identified heparin-binding domain as an additional binding region for PspC, suggesting that soluble, immobilized, as well as cellularly bound vitronectin possesses different conformations. Finally, vitronectin bound to PspC was functionally active and inhibited the deposition of the terminal complement complex. In conclusion, this study identifies and characterizes (on the molecular level) the interaction between the pneumococcal adhesin PspC and the human glycoprotein vitronectin.     

Transmembrane and Ubiquitin-Like Domain Containing 1 (Tmub1) Regulates Locomotor Activity and Wakefulness in Mice and Interacts with CAMLG

Tmub1 (C7orf21/HOPS) encodes a protein containing a ubiquitin-like domain. Tmub1 is highly expressed in the nervous system. To study its physiological function, we generated mice with Tmub1 deleted by homologous recombination. The knockout mice were grossly normal and viable. In a comprehensive behavioral testing battery, the only knockout phenotype displayed was a strong increase in home cage locomotor activity during the dark phase (subjective day) of the light∶dark (L∶D) cycle. There were no changes in activity during the light period. There were no changes in locomotor activity observed in other assays, e.g. novel open-field. The increase in dark phase locomotor activity persisted during a seven day D∶D (complete darkness) challenge, and remained largely confined to the normally dark period. Telemetric recording in freely moving subjects for one 24 hr L∶D cycle, revealed the same increase in locomotor activity in the dark phase. In addition, EEG analysis showed that the knockout mice exhibited increased waking and decreased NREM & REM times during the dark phase, but the EEG was otherwise normal. Using lacZ as a reporter we found Tmub1 expression prominent in a few brain structures including the thalamus, a region known to drive wakefulness and arousal via its projections to the cortex. We identified calcium modulating cyclophilin ligand CAMLG/CAML as a binding partner by a yeast two-hybrid screen of a brain library. The interaction of Tmub1 and CAMLG was confirmed by co-immunoprecipitation assays in HEK cells. The two proteins were also found to be co-localized to the cytoplasm when expressed in HEK cells. Both Tmub1 and CAMLG have been recently described in the regulation of membrane trafficking of specific receptors. Taken together our results implicate Tmub1 in the regulation of locomotor activity and wakefulness and suggest that Tmub1 binds to and functions together with CAMLG.     

A Retrospective Review of the Prognostic Value of ALDH-1, Bmi-1 and Nanog Stem Cell Markers in Esophageal Squamous Cell Carcinoma

Stem cell markers are upregulated in various cancers and have potential as prognostic indicators. The objective of this study was to determine the expression of three stem cell markers, aldehyde dehydrogenase 1 (ALDH-1), B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1), and Nanog, in esophageal squamous cell carcinoma (ESCC) tissues. Immunohistochemistry was used to measure the expression of ALDH-1, Bmi-1, and Nanog in ESCC tissues from 41 patients who received pre-operative chemoradiation. We evaluated the relationship between expression of these markers, and clinicopathological features, tumor regression grade (TRG), and 5-year overall survival (OS). There were no significant associations of ALDH-1 or Bmi-1 expression with age, gender, clinical stage, and treatments (p>0.05). However, patients with Nanog-positive tumors were significantly older than those whose tumors were Nanog-negative (p = 0.033). TRG after treatment was significantly associated with expression of ALDH-1 (p = 0.001), Bmi-1 (p = 0.004), and Nanog (p<0.001). Although OS was significantly better in patients with low TRGs (p = 0.001), there were no significant correlations between ALDH-1, Bmi-1, or Nanog with OS. Expression of ALDH-1, Bmi-1, and Nanog correlated with TRG, but not OS. Further large studies are necessary to fully elucidate the prognostic value of these stem cell markers for ESCC patients.     

The Centriolar Satellite Protein AZI1 Interacts with BBS4 and Regulates Ciliary Trafficking of the BBSome

Bardet-Biedl syndrome (BBS) is a well-known ciliopathy with mutations reported in 18 different genes. Most of the protein products of the BBS genes localize at or near the primary cilium and the centrosome. Near the centrosome, BBS proteins interact with centriolar satellite proteins, and the BBSome (a complex of seven BBS proteins) is believed to play a role in transporting ciliary membrane proteins. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. Here, we show that a centriolar satellite protein, AZI1 (also known as CEP131), interacts with the BBSome and regulates BBSome ciliary trafficking activity. Furthermore, we show that AZI1 interacts with the BBSome through BBS4. AZI1 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon AZI1 depletion. Under conditions in which the BBSome does not normally enter cilia, such as in BBS3 or BBS5 depleted cells, knock down of AZI1 with siRNA restores BBSome trafficking to cilia. Finally, we show that azi1 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer''s vesicle abnormalities and melanosome transport delay. These findings associate AZI1 with the BBS pathway. Our findings provide further insight into the regulation of BBSome ciliary trafficking and identify AZI1 as a novel BBS candidate gene.     

HIV-1 Tat Interacts with and Regulates the Localization and Processing of Amyloid Precursor Protein

HIV-1 Tat protein plays various roles in virus proliferation and in the regulation of numerous host cell functions. Accumulating evidence suggests that HIV-1 Tat also plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting intracellular communication. Amyloid beta (Aβ) is generated from amyloid precursor protein (APP) and accumulates in the senile plaques of Alzheimer''s disease patients. This study demonstrates that Tat interacts with APP both in vitro and in vivo, and increases the level of Aβ42 by recruiting APP into lipid rafts. Co-localization of Tat with APP in the cytosol was observed in U-87 MG cells that expressed high levels of Tat, and redistribution of APP into lipid rafts, a site of increased β- and γ-secretase activity, was demonstrated by discontinuous sucrose density gradient ultracentrifugation in the presence of Tat. Furthermore, Tat enhanced the cleavage of APP by β-secretase in vitro, resulting in 5.5-fold higher levels of Aβ42. This was consistent with increased levels of β-C-terminal fragment (β-CTF) and reduced levels of α-CTF. Moreover, stereotaxic injection of a lentiviral Tat expression construct into the hippocampus of APP/presenilin-1 (PS1) transgenic mice resulted in increased Tat-mediated production and processing of Aβ in vivo. Increased levels of Aβ42, as well as an increase in the number and size of Aβ plaques, were observed in the hippocampus following injection of Tat virus compared with mock virus. These results suggest that HIV-1 Tat may contribute to HAND by interacting with and modifying APP processing, thereby increasing Aβ production.     

The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating the intracellular trafficking and activation of matriptase. In this study, we, therefore, used genetically engineered mice to determine the effect of ablation of endogenous HAI-1 and endogenous HAI-2 on endogenous matriptase expression, subcellular localization, and activation in polarized intestinal epithelial cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase was mechanistically linked to accelerated activation and shedding of the protease caused by loss of prostasin regulation by HAI-2. Taken together, these data indicate that HAI-1 regulates the activity of activated matriptase, whereas HAI-2 has an essential role in regulating prostasin-dependent matriptase zymogen activation.     

Alternative Splicing of the RAGE Cytoplasmic Domain Regulates Cell Signaling and Function

The Receptor for Advanced Glycation End-products (RAGE) is a multi-ligand receptor present on most cell types. Upregulation of RAGE is seen in a number of pathological states including, inflammatory and vascular disease, dementia, diabetes and various cancers. We previously demonstrated that alternative splicing of the RAGE gene is an important mechanism which regulates RAGE signaling through the production of soluble ligand decoy isoforms. However, no studies have identified any alternative splice variants within the intracellular region of RAGE, a region critical for RAGE signaling. Herein, we have cloned and characterized a novel splice variant of RAGE that has a truncated intracellular domain (RAGEΔICD). RAGEΔICD is prevalent in both human and mouse tissues including lung, brain, heart and kidney. Expression of RAGEΔICD in C6 glioma cells impaired RAGE-ligand induced signaling through various MAP kinase pathways including ERK1/2, p38 and SAPK/JNK. Moreover, RAGEΔICD significantly affected tumor cell properties through altering cell migration, invasion, adhesion and viability in C6 glioma cells. Furthermore, C6 glioma cells expressing RAGEΔICD exhibited drastic inhibition on tumorigenesis in soft agar assays. Taken together, these data indicate that RAGEΔICD represents a novel endogenous mechanism to regulate RAGE signaling. Significantly, RAGEΔICD could play an important role in RAGE related disease states through down regulation of RAGE signaling.     

Characterization of the Mitochondrial Inner Membrane Translocase Complex: the Tim23p Hydrophobic Domain Interacts with Tim17p but Not with Other Tim23p Molecules

Tim23p is a mitochondrial inner membrane protein essential for the import of proteins from the cytosol. Tim23p contains an amino-terminal hydrophilic segment and a carboxyl-terminal hydrophobic domain (Tim23Cp). To study the functions and interactions of the two parts of Tim23p separately, we constructed tim23N, encoding only the hydrophilic region of Tim23p, and tim23C, encoding only the hydrophobic domain of Tim23p. Only the Tim23C protein is imported into mitochondria, indicating that the mitochondrial targeting information in Tim23p resides in its membrane spans or intervening loops. Tim23Cp, however, cannot substitute for full-length Tim23p, suggesting that the hydrophilic portion of Tim23p also performs an essential function in mitochondrial protein import. We found that overexpression of Tim23Cp is toxic to yeast cells that carry the tim23-1 mutation. Excess Tim23Cp causes Tim23-1p to disappear, leaving tim23-1 cells without a full-length version of the Tim23 protein. If Tim17p, another inner membrane import component, is overexpressed along with Tim23Cp, the toxicity of Tim23Cp is largely reversed and the Tim23-1 protein no longer disappears. In coimmunoprecipitations from solubilized mitochondria, Tim17p associates with the Tim23C protein. In addition, we show that Tim23p and Tim17p can be chemically cross-linked to each other in intact mitochondria. We conclude that the hydrophobic domain encoded by tim23C targets Tim23p to the mitochondria and mediates the direct interaction between Tim23p and Tim17p. In contrast, Tim23Cp cannot be coimmunoprecipitated with Tim23p, raising the possibility that the hydrophobic domain of Tim23p does not interact with other Tim23 molecules.     

Knock-in Mutation Reveals an Essential Role for Focal Adhesion Kinase Activity in Blood Vessel Morphogenesis and Cell Motility-Polarity but Not Cell Proliferation

Focal adhesion kinase (FAK) associates with both integrins and growth factor receptors in the control of cell motility and survival. Loss of FAK during mouse development results in lethality at embryonic day 8.5 (E8.5) and a block in cell proliferation. Because FAK serves as both a scaffold and signaling protein, gene knock-outs do not provide mechanistic insights in distinguishing between these modes of FAK function. To determine the role of FAK activity during development, a knock-in point mutation (lysine 454 to arginine (R454)) within the catalytic domain was introduced by homologous recombination. Homozygous FAK^R454/R454 mutation was lethal at E9.5 with defects in blood vessel formation as determined by lack of yolk sac primary capillary plexus formation and disorganized endothelial cell patterning in FAK^R454/R454 embryos. In contrast to the inability of embryonic FAK^−/− cells to proliferate ex vivo, primary FAK^R454/R454 mouse embryo fibroblasts (MEFs) were established from E8.5 embryos. R454 MEFs exhibited no difference in cell growth compared with normal MEFs, and R454 FAK localized to focal adhesions but was not phosphorylated at Tyr-397. In E8.5 embryos and primary MEFs, FAK R454 mutation resulted in decreased c-Src Tyr-416 phosphorylation. R454 MEFs exhibited enhanced focal adhesion formation, decreased migration, and defects in cell polarity. Within immortalized MEFs, FAK activity was required for fibronectin-stimulated FAK-p190RhoGAP association and p190RhoGAP tyrosine phosphorylation linked to decreased RhoA GTPase activity, focal adhesion turnover, and directional motility. Our results establish that intrinsic FAK activity is essential for developmental processes controlling blood vessel formation and cell motility-polarity but not cell proliferation. This work supports the use of FAK inhibitors to disrupt neovascularization.