Caveolin-1 assembles type 1 inositol 1,4,5-trisphosphate receptors and canonical transient receptor potential 3 channels into a functional signaling complex in arterial smooth muscle cells

Physical coupling of sarcoplasmic reticulum (SR) type 1 inositol 1,4,5-trisphosphate receptors (IP(3)R1) to plasma membrane canonical transient receptor potential 3 (TRPC3) channels activates a cation current (I(Cat)) in arterial smooth muscle cells that induces vasoconstriction. However, structural components that enable IP(3)R1 and TRPC3 channels to communicate locally are unclear. Caveolae are plasma membrane microdomains that can compartmentalize proteins. Here, we tested the hypothesis that caveolae and specifically caveolin-1 (cav-1), a caveolae scaffolding protein, facilitate functional IP(3)R1 to TRPC3 coupling in smooth muscle cells of resistance-size cerebral arteries. Methyl-β-cyclodextrin (MβCD), which disassembles caveolae, reduced IP(3)-induced I(Cat) activation in smooth muscle cells and vasoconstriction in pressurized arteries. Cholesterol replenishment reversed these effects. Cav-1 knockdown using shRNA attenuated IP(3)-induced vasoconstriction, but did not alter TRPC3 and IP(3)R1 expression. A synthetic peptide corresponding to the cav-1 scaffolding domain (CSD) sequence (amino acids 82-101) also attenuated IP(3)-induced I(Cat) activation and vasoconstriction. A cav-1 antibody co-immunoprecipitated cav-1, TRPC3, and IP(3)R1 from cerebral artery lysate. ImmunoFRET indicated that cav-1, TRPC3 channels and IP(3)R1 are spatially co-localized in arterial smooth muscle cells. IP(3)R1 and TRPC3 channel spatial localization was disrupted by MβCD and a CSD peptide. Cholesterol replenishment re-established IP(3)R1 and TRPC3 channel close spatial proximity. Taken together, these data indicate that in arterial smooth muscle cells, cav-1 co-localizes SR IP(3)R1 and plasma membrane TRPC3 channels in close spatial proximity thereby enabling IP(3)-induced physical coupling of these proteins, leading to I(Cat) generation and vasoconstriction.     

Mechanisms of autoinhibition of IRF-7 and a probable model for inactivation of IRF-7 by Kaposi's sarcoma-associated herpesvirus protein ORF45

IRF-7 is the master regulator of type I interferon-dependent immune responses controlling both innate and adaptive immunity. Given the significance of IRF-7 in the induction of immune responses, many viruses have developed strategies to inhibit its activity to evade or antagonize host antiviral responses. We previously demonstrated that ORF45, a KSHV immediate-early protein as well as a tegument protein of virions, interacts with IRF-7 and inhibits virus-mediated type I interferon induction by blocking IRF-7 phosphorylation and nuclear translocation (Zhu, F. X., King, S. M., Smith, E. J., Levy, D. E., and Yuan, Y. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5573-5578). In this report, we sought to reveal the mechanism underlying the ORF45-mediated inactivation of IRF-7. We found that ORF45 interacts with the inhibitory domain of IRF-7. The most striking feature in the IRF-7 inhibitory domain is two α-helices H3 and H4 that contain many hydrophobic residues and two β-sheets located between the helices that are also very hydrophobic. These hydrophobic subdomains mediate intramolecular interactions that keep the molecule in a closed (inactive) form. Mutagenesis studies confirm the contribution of the hydrophobic helices and sheets to the autoinhibition of IRF-7 in the absence of viral signal. The binding of ORF45 to the critical domain of IRF-7 leads to a hypothesis that ORF45 may maintain the IRF-7 molecule in the closed form and prevent it from being activated in response to viral infection.     

In silico analysis of expression pattern of a Wnt/β-catenin responsive gene ANLN in gastric cancer

Actin-binding protein anillin (ANLN) is primarily involved in the cytokinesis and known to be dysregulated in many cancers including gastric cancer (GC). However, the regulation and clinical significance of ANLN in GC are far less clear. In the present study, we aimed to investigate the clinical significance and possible regulators of ANLN in GC. We have identified the Wnt/β-catenin associated regulation of ANLN by analyzing the in vitro perturbed β-catenin mRNA expression profiles. Investigating the gastric tumors from publicly available genome-wide mRNA expression profiles, we have identified the over expression of ANLN in gastric tumors. Association between ANLN expression and clinical characteristics of GC showed elevated expression in intestinal type GC. Performing a single sample prediction method across GC mRNA expression profiles, we have identified the over expression of ANLN in proliferative type gastric tumors compared to the invasive and metabolic type gastric tumors. In silico pathway prediction analysis revealed the association between Wnt/β-catenin signaling and ANLN expression in gastric tumors. Our results highlight that expression of a Wnt/β-catenin responsive gene ANLN in GC is a molecular predictor of intestinal and proliferative type gastric tumors.     

The role of bronchial epithelial cells in the pathogenesis of COPD in Z-alpha-1 antitrypsin deficiency

Background

Alpha-1 antitrypsin is the main inhibitor of neutrophil elastase in the lung. Although it is principally synthesized by hepatocytes, alpha-1 antitrypsin is also secreted by bronchial epithelial cells. Gene mutations can lead to alpha-1 antitrypsin deficiency, with the Z variant being the most clinically relevant due to its propensity to polymerize. The ability of bronchial epithelial cells to produce Z-variant protein and its polymers is unknown.We investigated the expression, accumulation, and secretion of Z-alpha-1 antitrypsin and its polymers in cultures of transfected cells and in cells originating from alpha-1 antitrypsin-deficient patients.

Methods

Experiments using a conformation-specific antibody were carried out on M- and Z-variant–transfected 16HBE cells and on bronchial biopsies and ex vivo bronchial epithelial cells from Z and M homozygous patients. In addition, the effect of an inflammatory stimulus on Z-variant polymer formation, elicited by Oncostatin M, was investigated. Comparisons of groups were performed using t-test or ANOVA. Non-normally distributed data were assessed by Mann–Whitney U test or the Kruskal-Wallis test, where appropriate. A P value of < 0.05 was considered to be significant.

Results

Alpha-1 antitrypsin polymers were found at a higher concentration in the culture medium of ex vivo bronchial epithelial cells from Z-variant homozygotes, compared with M-variant homozygotes (P < 0.01), and detected in the bronchial epithelial cells and submucosa of patient biopsies. Oncostatin M significantly increased the expression of alpha-1 antitrypsin mRNA and protein (P < 0.05), and the presence of Z-variant polymers in ex vivo cells (P < 0.01).

Conclusions

Polymers of Z-alpha-1 antitrypsin form in bronchial epithelial cells, suggesting that these cells may be involved in the pathogenesis of lung emphysema and in bronchial epithelial cell dysfunction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0112-3) contains supplementary material, which is available to authorized users.     

NMR-Based Detection of Hydrogen/Deuterium Exchange in Liposome-Embedded Membrane Proteins

Membrane proteins play key roles in biology. Determination of their structure in a membrane environment, however, is highly challenging. To address this challenge, we developed an approach that couples hydrogen/deuterium exchange of membrane proteins to rapid unfolding and detection by solution-state NMR spectroscopy. We show that the method allows analysis of the solvent protection of single residues in liposome-embedded proteins such as the 349-residue Tom40, the major protein translocation pore in the outer mitochondrial membrane, which has resisted structural analysis for many years.     

Are Articulatory Settings Mechanically Advantageous for Speech Motor Control?

We address the hypothesis that postures adopted during grammatical pauses in speech production are more “mechanically advantageous” than absolute rest positions for facilitating efficient postural motor control of vocal tract articulators. We quantify vocal tract posture corresponding to inter-speech pauses, absolute rest intervals as well as vowel and consonant intervals using automated analysis of video captured with real-time magnetic resonance imaging during production of read and spontaneous speech by 5 healthy speakers of American English. We then use locally-weighted linear regression to estimate the articulatory forward map from low-level articulator variables to high-level task/goal variables for these postures. We quantify the overall magnitude of the first derivative of the forward map as a measure of mechanical advantage. We find that postures assumed during grammatical pauses in speech as well as speech-ready postures are significantly more mechanically advantageous than postures assumed during absolute rest. Further, these postures represent empirical extremes of mechanical advantage, between which lie the postures assumed during various vowels and consonants. Relative mechanical advantage of different postures might be an important physical constraint influencing planning and control of speech production.