A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.     

A nonradioactive method for isolating complementary DNAs endocing calmodulin-binding proteins

Calmodulin labeled with¹²⁵I or³⁴S has been used to screen expression libraries to isolate cDNAs encoding calmodulin-binding proteins (CBPs) from several eukaryotic systems. The use of radiolabeled calmodulin has, however, several disadvantages. We have developed a nonradiactive method to isolate cDNAs for CBPs using biotinylated calmodulin. Screening of a cDNA library in an expression vector with biotinylated calmodulin resulted in the isolation of cDNAs encoding CBPs. Avidin and biotin blocking steps, prior to incubation of the filters with biotinylated calmodulin, are found to be essential to eliminate the cDNAs that code for biotin-containing polypeptides. The cDNA clones isolated using this nonradioactive method bound calmodulin in a calcium-dependent manner. The binding of biotinylated calmodulin to these clones was completely abolished by ethylene glycolbis(\-aminoethylether)-N,N′-tetraacetic acid (EGTA), a calcium chelator. Furthermore, the isolated cDNAs were confirmed by probing the clones with³⁵S-labeled calmodulin. All the isolated clones bound to radiolabeled calmodulin in the presence of calcium but not in the presence of EGTA. The method described here is simple, fast, and does not involve preparation and handing of radiolabeled calmodulin. All the materials used in this method are commercially available; hence, this procedure should be widely applicable to isolate cDNAs encoding CBPs from any eukaryotic organism.     

Leptin modulates lymphocytes' adherence to hepatic stellate cells is associated with oxidative status alterations

We investigated leptin effects on lymphocyte interactions with hepatic-stellate-cells (HSCs). Leptin showed pro-fibrotic effects on HSCs with oxidative status imbalance.In co-cultures, leptin activates HSCs and consequently adhered HCV-lymphocytes more than healthy ones. Leptin also increased healthy and HCV lymphocyte proliferations; increased their reactive-oxygen-species; decreased antioxidants (reduced-glutathione) levels while inhibited apoptosis only of HCV-lymphocytes. The leptin-treated HCV-lymphocytes activated HSCs, increase interleukin-4 while decreased their apoptosis.Leptin-receptor-deficient (db–db)-HSCs did not adhere lymphocytes. db/db-lymphocytes however showed fewer adherences to HSCs when compared to WT-counterparts.This study presents immune and oxidative modulatory effects of leptin on lymphocytes and their consequent interaction with HSCs.     

The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells

Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.     

Initial Data on the Molecular Epidemiology of Cryptosporidiosis in Lebanon

Cryptosporidium spp. represent a major public health problem worldwide and infect the gastrointestinal tract of both immunocompetent and immunocompromised persons. The prevalence of these parasites varies by geographic region, and no data are currently available in Lebanon. To promote an understanding of the epidemiology of cryptosporidiosisin this country, the main aim of this study was to determine the prevalence Cryptosporidium in symptomatic hospitalized patients, and to analyze the genetic diversity of the corresponding isolates. Fecal specimens were collected in four hospitals in North Lebanon from 163 patients (77 males and 86 females, ranging in age from 1 to 88 years, with a mean age of 22 years) presenting gastrointestinal disorders during the period July to December 2013. The overall prevalence of Cryptosporidium spp. infection obtained by modified Ziehl-Neelsen staining and/or nested PCR was 11%, and children <5 years old showed a higher rate of Cryptosporidium spp. The PCR products of the 15 positive samples were successfully sequenced. Among them, 10 isolates (66.7%) were identified as C. hominis, while the remaining 5 (33.3%) were identified as C. parvum. After analysis of the gp60 locus, C. hominis IdA19, a rare subtype, was found to be predominant. Two C. parvum subtypes were found: IIaA15G1R1 and IIaA15G2R1. The molecular characterization of Cryptosporidium isolates is an important step in improving our understanding of the epidemiology and transmission of the infection.     

The effect of class a scavenger receptor deficiency in bone

Class A scavenger receptor (SR-A) is predominantly expressed by macrophages, and because osteoclasts are of monocyte/macrophage lineage, SR-A is of potential interest in osteoclast biology. In addition to modified low density lipoprotein uptake, SR-A is also important in cell attachment and signaling. In this study we evaluated the effect of SR-A deletion on bone. Knock-out animals have 40% greater body weight than wild type. Body composition analyses demonstrated that total lean and fat body mass were greater in knock-out animals, but there was no significant difference in percent fat and lean body mass. Bone mineral density and content were significantly greater in knock-out compared with wild type animals. Micro-computed tomography analyses confirmed that total volume, bone volume as well as trabecular number, thickness, and connectivity were significantly greater in knock-out mice. As expected, trabecular separation was greater in wild type mice. The phenotype appears to be explained by 60% fewer osteoclasts in females and 35% fewer in males compared to wild type mice with a paradoxical increase in nuclei/osteoclast in knock-out animals. Furthermore, there were no differences in adipocyte number and osteoblast number or activity. The addition of the soluble extracellular domain of SR-A to RAW264.7 cells stimulated a concentration-dependent increase in osteoclast differentiation that was receptor activator of nuclear factor-kappaB ligand (RANKL)-dependent. Soluble SR-A had no effect on cell proliferation in the presence of RANKL but stimulated a 40% increase in numbers in the absence of RANKL. We conclude that SR-A plays a role in normal osteoclast differentiation, suggesting a novel role for this receptor in bone biology.     

Solution structure of the E3 ligase HOIL-1 Ubl domain

The E3 ligases HOIL‐1 and parkin are each comprised of an N‐terminal ubiquitin‐like (Ubl) domain followed by a zinc‐binding region and C‐terminal RING–In‐between‐RING–RING domains. These two proteins, involved in the ubiquitin‐mediated degradation pathway, are the only two known E3 ligases to share this type of multidomain architecture. Further, the Ubl domain of both HOIL‐1 and parkin has been shown to interact with the S5a subunit of the 26S proteasome. The solution structure of the HOIL‐1 Ubl domain was solved using NMR spectroscopy to compare it with that of parkin to determine the structural elements responsible for S5a intermolecular interactions. The final ensemble of 20 structures had a β‐grasp Ubl‐fold with an overall backbone RMSD of 0.59 ± 0.10 Å in the structured regions between I55 and L131. HOIL‐1 had a unique extension of both β1 and β2 sheets compared to parkin and other Ubl domains, a result of a four‐residue insertion in this region. A similar 15‐residue hydrophobic core in the HOIL‐1 Ubl domain resulted in a comparable stability to the parkin Ubl, but significantly lower than that observed for ubiquitin. A comparison with parkin and other Ubl domains indicates that HOIL‐1 likely uses a conserved hydrophobic patch (W58, V102, Y127, Y129) found on the β1 face, the β3–β4 loop and β5, as well as a C‐terminal basic residue (R134) to recruit the S5a subunit as part of the ubiquitin‐mediated proteolysis pathway.