Compartment-specific Synthesis of Phosphatidylethanolamine Is Required for Normal Heavy Metal Resistance

Control of lipid composition of membranes is crucial to ensure normal cellular functions. Saccharomyces cerevisiae has two different phosphatidylserine decarboxylase enzymes (Psd1 and Psd2) that catalyze formation of phosphatidylethanolamine. The mitochondrial Psd1 provides roughly 70% of the phosphatidylethanolamine (PE) biosynthesis in the cell with Psd2 carrying out the remainder. Here, we demonstrate that loss of Psd2 causes cells to acquire sensitivity to cadmium even though Psd1 remains intact. This cadmium sensitivity results from loss of normal activity of a vacuolar ATP-binding cassette transporter protein called Ycf1. Measurement of phospholipid levels indicates that loss of Psd2 causes a specific reduction in vacuolar membrane PE levels, whereas total PE levels are not significantly affected. The presence of a phosphatidylinositol transfer protein called Pdr17 is required for Psd2 function and normal cadmium tolerance. We demonstrate that Pdr17 and Psd2 form a complex in vivo that seems essential for maintenance of vacuolar PE levels. Finally, we refine the localization of Psd2 to the endosome arguing that this enzyme controls vacuolar membrane phospholipid content by regulating phospholipids in compartments that will eventually give rise to the vacuole. Disturbance of this regulation of intracellular phospholipid balance leads to selective loss of membrane protein function in the vacuole.     

Proteomics analysis of the ezrin interactome in B cells reveals a novel association with Myo18aα

The molecular regulation of recruitment and assembly of signalosomes near the B cell receptor (BCR) is poorly understood. We have previously demonstrated a role for the ERM family protein ezrin in regulating antigen-dependent lipid raft coalescence in B cells. In this study, we addressed the possibility that ezrin may collaborate with other adaptor proteins to regulate signalosome dynamics at the membrane. Using mass spectrometry-based proteomics analysis, we identified Myo18aα as a novel binding partner of ezrin. Myo18aα is an attractive candidate as it has several protein-protein interaction domains and an intrinsic motor activity. The expression of Myo18aα varied during B cell development in the bone marrow and in mature B cell subsets suggesting functional differences. Interestingly, BCR stimulation increased the association between ezrin and Myo18aα, and induced co-segregation of Myo18aα with the BCR and phosphotyrosine-containing proteins. Our data raise an intriguing possibility that the Myo18aα/ezrin complex may facilitate BCR-mediated signaling by recruiting signaling proteins that are in close proximity of the antigen receptor. Our study is not only significant with respect to understanding the molecular regulation of BCR signaling but also provides a broader basis for understanding the mechanism of action of ezrin in other cellular systems.     

Identification of B cell epitopes of alcohol dehydrogenase allergen of Curvularia lunata

Background/Objective

Epitope identification assists in developing molecules for clinical applications and is useful in defining molecular features of allergens for understanding structure/function relationship. The present study was aimed to identify the B cell epitopes of alcohol dehydrogenase (ADH) allergen from Curvularia lunata using in-silico methods and immunoassay.

Method

B cell epitopes of ADH were predicted by sequence and structure based methods and protein-protein interaction tools while T cell epitopes by inhibitory concentration and binding score methods. The epitopes were superimposed on a three dimensional model of ADH generated by homology modeling and analyzed for antigenic characteristics. Peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by ELISA using individual and pooled patients'' sera.

Result

The homology model showed GroES like catalytic domain joined to Rossmann superfamily domain by an alpha helix. Stereochemical quality was confirmed by Procheck which showed 90% residues in most favorable region of Ramachandran plot while Errat gave a quality score of 92.733%. Six B cell (P1–P6) and four T cell (P7–P10) epitopes were predicted by a combination of methods. Peptide P2 (epitope P2) showed E(X)₂GGP(X)₃KKI conserved pattern among allergens of pathogenesis related family. It was predicted as high affinity binder based on electronegativity and low hydrophobicity. The computational methods employed were validated using Bet v 1 and Der p 2 allergens where 67% and 60% of the epitope residues were predicted correctly. Among B cell epitopes, Peptide P2 showed maximum IgE binding with individual and pooled patients'' sera (mean OD 0.604±0.059 and 0.506±0.0035, respectively) followed by P1, P4 and P3 epitopes. All T cell epitopes showed lower IgE binding.

Conclusion

Four B cell epitopes of C. lunata ADH were identified. Peptide P2 can serve as a potential candidate for diagnosis of allergic diseases.     

Claudin-7 is frequently overexpressed in ovarian cancer and promotes invasion

Background

Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC.

Methodology/Principal Findings

A total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression. In real-time RT-PCR analysis, the gene for claudin-7, CLDN7, was found to be upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Small interfering RNA-mediated knockdown of claudin-7 in ovarian cancer cells led to significant changes in gene expression as measured by microarrays and validated by RT-PCR and immunoblotting. Analyses of the genes differentially expressed revealed that the genes altered in response to claudin-7 knockdown were associated with pathways implicated in various molecular and cellular functions such as cell cycle, cellular growth and proliferation, cell death, development, and cell movement. Through functional experiments in vitro, we found that both migration and invasion were altered in cells where CLDN7 had been knocked down or overexpressed. Interestingly, claudin-7 expression was associated with a net increase in invasion, but also with a decrease in migration.

Conclusion/Significance

Our work shows that claudin-7 is significantly upregulated in EOC and that it may be functionally involved in ovarian carcinoma invasion. CLDN7 may therefore represent potential marker for ovarian cancer detection and a target for therapy.     

Mismatch-repair protein MSH6 is associated with Ku70 and regulates DNA double-strand break repair

MSH6, a key component of the MSH2-MSH6 complex, plays a fundamental role in the repair of mismatched DNA bases. Herein, we report that MSH6 is a novel Ku70-interacting protein identified by yeast two-hybrid screening. Ku70 and Ku86 are two key regulatory subunits of the DNA-dependent protein kinase, which plays an essential role in repair of DNA double-strand breaks (DSBs) through the non-homologous end-joining (NEHJ) pathway. We found that association of Ku70 with MSH6 is enhanced in response to treatment with the radiomimetic drug neocarzinostatin (NCS) or ionizing radiation (IR), a potent inducer of DSBs. Furthermore, MSH6 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci after NCS or IR treatment. MSH6 colocalizes with γ-H2AX at sites of DNA damage after NCS or IR treatment. Cells depleted of MSH6 accumulate high levels of persistent DSBs, as detected by formation of γ-H2AX foci and by the comet assay. Moreover, MSH6-deficient cells were also shown to exhibit impaired NHEJ, which could be rescued by MSH6 overexpression. MSH6-deficient cells were hypersensitive to NCS- or IR-induced cell death, as revealed by a clonogenic cell-survival assay. These results suggest a potential role for MSH6 in DSB repair through upregulation of NHEJ by association with Ku70.     

The adaptor-associated kinase 1, AAK1, is a positive regulator of the Notch pathway

The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. The extracellular part of the receptor is removed by a metalloprotease of the ADAM family and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent γ-secretase activity. γ-Secretase processing of Notch has been shown to depend upon monoubiquitination as well as clathrin-mediated endocytosis (CME). We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of the γ-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization. These results suggest that after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates.     

CD40-modulated dual-specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-3 reciprocally regulate Leishmania major infection

The macrophage-expressed CD40 regulates immune responses to Leishmania major infection by reciprocal signaling through p38 MAPK and ERK1/2. CD40-induced IL-10 or IL-12 plays crucial roles in the promotion or protection from L. major infection, respectively. Because p38 MAPK and ERK1/2 are dephosphorylated by dual-specificity MAPK phosphatases (MKPs), we tested the role of CD40 in the regulation of MKPs in L. major infection. MKP-1 expression and activity increased whereas MKP-3 expression and activity decreased in virulent L. major-infected macrophages. CD40 differentially regulated the expression and activity of MKP-1 and MKP-3, which, in turn, reciprocally regulated CD40-induced p38 MAPK and ERK1/2 phosphorylation and effector functions in macrophages. Triptolide, an inhibitor of MKP-1 expression, and lentivirally expressed MKP-1 short hairpin RNA enhanced CD40-induced anti-leishmanial functions and significantly protected susceptible BALB/c mice from L. major infection. Similarly, lentivirally overexpressed MKP-3 significantly reduced disease progression and parasite burden in susceptible BALB/c mice. Thus, to our knowledge, our data show for the first time that CD40 reciprocally regulates MKP-1 and MKP-3 expression and activity while the MKPs contribute to the reciprocal CD40 signaling-regulated anti-leishmanial functions. The findings reveal a novel parasite-devised immune evasion strategy and an effective target to redirect CD40-regulated immune responses.     

Formation of amyloid fibrils via longitudinal growth of oligomers

Mature amyloid fibrils are believed to be formed by the lateral association of discrete structural units designated as protofibrils, but this lateral association of protofibrils has never been directly observed. We have recently characterized a thioesterase from Alcaligenes faecalis, which was shown to exist as homomeric oligomers with an average diameter of 21.6 nm consisting of 22 kDa subunits in predominantly beta-sheet structure. In this study, we have shown that upon incubation in a 75% ethanol solution, the oligomeric particles of protein were transformed into amyloid-like fibrils. TEM pictures obtained at various stages during fibril growth helped us to understand to a certain extent the early events in the fibrillization process. When incubated in 75% ethanol, oligomeric particles of protein grew to approximately 35-40 nm in diameter before fusion. Fusion of two oligomers of 35-40 nm resulted in the formation of a fibril. Fibril formation was accompanied by a reduction in the diameter of the particle to approximately 20-25 nm along with concomitant elongation to approximately 110 nm, indicating reorganization and strengthening of the structure. The elongation process continued by sequential addition of oligomeric units to give fibers 500-1000 nm in length with a further reduction in diameter to 17-20 nm. Further elongation resulted in the formation of fibers that were more than 4000 nm in length; the diameter, however, remained constant at 17-20 nm. These data clearly show that the mature fibrils have assembled via longitudinal growth of oligomers and not via lateral association of protofibrils.