Expression profiles of two thaumatin‐like protein (TmTLP) genes in responses to various micro‐organisms from Tenebrio molitor

It is well known that anti‐fungal thaumatin‐like proteins (TLPs) play important roles in plants. Here, we investigated the expression analysis of thaumatin‐like protein genes TmTLPs in response to various pathogens in Tenebrio molitor. Developmental expression patterns of TmTLPs show that TmTLPs are highly expressed in the early pupal and adult stages. Furthermore, tissue‐specific expression patterns of TmTLPs indicate that TmTLP1 is highly expressed in the integument and gut of last instar larvae and the integument and Malpighian tubules of 5‐day old adults. In contrast, TmTLP2 is highly expressed in the gut of both last instar larvae and 5‐day old adults. We hypothesize that the expression of TmTLP genes in developmental stages may be related to molting and body remodeling stresses. In addition, the induction patterns of TmTLP genes indicate that TmTLPs were slightly induced by Escherichia coli, Staphylococcus aureus, Candida albicans and Listeria monocytogenes. Furthermore, TmTLP1 and TmTLP2 were strongly induced in response to E. coli at 9 h post‐injection and L. monocytogenes at 3 h post‐injection. Our results suggest that TmTLPs may possess antimicrobial functions in T. molitor.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Maize ZmVPP5 is a truncated Vacuole H+‐PPase that confers hypersensitivity to salt stress

In plants, Vacuole H⁺‐PPases (VPPs) are important proton pumps and encoded by multiple genes. In addition to full‐length VPPs, several truncated forms are expressed, but their biological functions are unknown. In this study, we functionally characterized maize vacuole H⁺‐PPase 5 (ZmVPP5), a truncated VPP in the maize genome. Although ZmVPP5 shares high sequence similarity with ZmVPP1, ZmVPP5 lacks the complete structure of the conserved proton transport and the inorganic pyrophosphatase‐related domain. Phylogenetic analysis suggests that ZmVPP5 might be derived from an incomplete gene duplication event. ZmVPP5 is expressed in multiple tissues, and ZmVPP5 was detected in the plasma membrane, vacuole membrane and nuclei of maize cells. The overexpression of ZmVPP5 in yeast cells caused a hypersensitivity to salt stress. Transgenic maize lines with overexpressed ZmVPP5 also exhibited the salt hypersensitivity phenotype. A yeast two‐hybrid analysis identified the ZmBag6‐like protein as a putative ZmVPP5‐interacting protein. The results of bimolecular luminescence complementation (BiLC) assay suggest an interaction between ZmBag6‐like protein and ZmVPP5 in vivo. Overall, this study suggests that ZmVPP5 might act as a VPP antagonist and participate in the cellular response to salt stress. Our study of ZmVPP5 has expanded the understanding of the origin and functions of truncated forms of plant VPPs.     

Iron‐regulated surface determinant B (IsdB) promotes Staphylococcus aureus adherence to and internalization by non‐phagocytic human cells

Staphylococcus aureus is a human pathogen that causes invasive and recurring infections. The ability to internalize into and persist within host cells is thought to contribute to infection. Here we report a novel role for the well‐characterized iron‐regulated surface determinant B (IsdB) protein which we have shown can promote adhesion of 293T, HeLa cells and platelets to immobilized bacteria independently of its ability to bind haemoglobin. IsdB bound to the active form of the platelet integrin α_IIbβ₃, both on platelets and when the integrin was expressed ectopically in CHO cells. IsdB also promoted bacterial invasion into human cells. This was clearly demonstrated with bacteria lacking fibronectin‐binding proteins (FnBPs), which are known to promote invasion in the presence of fibronectin. However, IsdB also contributed significantly to invasion by cells expressing FnBPs in the presence of serum. Thus IsdB appears to be able to interact with the broader family of integrins that bind ligands with the RGD motif and to act as a back up mechanism to promote interactions with mammalian cells.     

Visualization of the post‐Golgi vesicle‐mediated transportation of TGF‐β receptor II by quasi‐TIRFM

Transforming growth factor β receptor II (Tβ RII) is synthesized in the cytoplasm and then transported to the plasma membrane of cells to fulfil its signalling duty. Here, we applied live‐cell fluorescence imaging techniques, in particular quasi‐total internal reflection fluorescence microscopy, to imaging fluorescent protein‐tagged Tβ RII and monitoring its secretion process. We observed punctuate‐like Tβ RII‐containing post‐Golgi vesicles formed in MCF7 cells. Single‐particle tracking showed that these vesicles travelled along the microtubules at an average speed of 0.51 μm/s. When stimulated by TGF‐β ligand, these receptor‐containing vesicles intended to move towards the plasma membrane. We also identified several factors that could inhibit the formation of such post‐Golgi vesicles. Although the inhibitory mechanisms still remain unknown, the observed characteristics of Tβ RII‐containing vesicles provide new information on intracellular Tβ RII transportation. It also renders Tβ RII a good model system for studying post‐Golgi vesicle‐trafficking and protein transportation. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Fibronectin-binding protein A of Staphylococcus aureus has multiple, substituting, binding regions that mediate adherence to fibronectin and invasion of endothelial cells

Invasive Staphylococcus aureus infection frequently involves bacterial seeding from the bloodstream to other body tissues, a process necessarily involving interactions between circulating bacteria and vascular endothelial cells. Staphylococcus aureus fibronectin‐binding protein is central to the invasion of endothelium, fibronectin forming a bridge between bacterial fibronectin‐binding proteins and host cell receptors. To dissect further the mechanisms of invasion of endothelial cells by S. aureus, a series of truncated FnBPA proteins that lacked one or more of the A, B, C or D regions were expressed on the surface of S. aureus and tested in fibronectin adhesion, endothelial cell adhesion and invasion assays. We found that this protein has multiple, substituting, fibronectin‐binding regions, each capable of conferring both adherence to fibronectin and endothelial cells, and endothelial cell invasion. By expressing S. aureus FnBPA on the surface of the non‐invasive Gram‐positive organism Lactococcus lactis, we have found that no other bacterial factor is required for invasion. Furthermore, we have demonstrated that, as with other cell types, invasion of endothelial cells is mediated by integrin α₅β₁. These findings may be of relevance to the development of preventive measures against systemic infection, and bacterial spread in the bacteraemic patient.     

LIPH promotes metastasis by enriching stem‐like cells in triple‐negative breast cancer

Lipase member H (LIPH), a novel member of the triglyceride lipase family. The clinical implications of its expression in breast cancer are still unclear. Therefore, in this study, we investigated the associations between LIPH and the tumorigenic behaviours of 144 triple‐negative breast cancer (TNBC) patients. The ratio and mammosphere‐forming ability of CD44+/CD24? stem‐like cells were tested. The role of LIPH in breast cancer cell migration and invasion was also evaluated. In addition, the effect of LIPH silencing on mitochondrial respiration was determined using the Seahorse assay. Finally, the effect of LIPH silencing on protein expression was determined via tandem mass tag‐based spectrometry and Western blotting. We found that LIPH expression was associated with metastasis in lymph nodes and distant organs (P = 0.025), resulting in poor survival among breast cancer patients (P = 0.027). LIPH knockdown significantly decreased both the ratio of CD44⁺/CD24^? stem‐like cells and their mammosphere‐forming ability. LIPH silencing promoted apoptosis, arrested cell cycle in the G2/M phase, mitigated the oxidation‐related oxygen consumption rate in the mitochondria, and reduced metabolism. LIPH inhibited adhesion between tumour cells and enhanced the epithelial‐mesenchymal transition. Tandem mass spectrometric analysis presented 68 proteins were differentially expressed in LIPH‐silenced cells and LIPH‐mediated modulation of tumour cell adhesion depended on integrin‐related CAPN2 and paxillin signalling. Overall, our findings provided strong evidence that LIPH up‐regulation promoted metastasis and the stemness of TNBC cells. Therefore, targeting LIPH is a potentially viable strategy for preventing metastasis in TNBC.     

Mussel and dogwhelk distribution along the north‐west Atlantic coast: testing predictions derived from the abundant‐centre model

Aim We performed the first test of predictions from the abundant‐centre model using north‐west Atlantic coastal organisms. We tested the hypotheses that the density of intertidal mussels (Mytilus edulis and M. trossulus) and dogwhelks (Nucella lapillus) and mussel age and size would peak at an intermediate location along their distribution range. We also assessed the latitudinal variation in critical aerial exposure time. Location North‐west Atlantic coast between Newfoundland (Canada) and New York (USA), covering 1800 km of shoreline. Methods Using a nested design, we measured mussel density, age and size and dogwhelk density in 60 wave‐exposed rocky intertidal sites spread evenly in six regions. Critical aerial exposure times were determined using online data. Results Mytilus edulis peaked in abundance in Maine and was much less abundant in the other regions. Mytilus trossulus peaked in abundance in southern Nova Scotia and Maine, was less abundant in the other regions to the north, and was absent in the southernmost region (New York). Both mussel species were least abundant in a northern region (Cape Breton), although not in the northernmost region (Newfoundland). Critical aerial exposure times were negatively correlated with overall mussel density. Mussel age and size were similar among regions. Dogwhelks peaked in abundance in Maine and were much less abundant in the other regions, being positively correlated with overall mussel density across regions. Main conclusions Density data for M. edulis and N. lapillus provide limited support for an abundant‐centre pattern, while M. trossulus shows a clear ramped‐south distribution. Critical aerial exposure times suggest that physiological stress during summer and winter low tides may be lowest in Maine and southern Nova Scotia, which might partially explain mussel predominance in those regions. Winter ice scour in Cape Breton may explain the abundance trough observed there. Mussel size and age may be more limited by wave exposure at our sites (as they all face open waters) than by regional differences in environmental stress. Dogwhelks, which prey on mussels, seem to respond positively to prey density at the regional scale. Our study supports the notion that, while the abundant‐centre model is a useful starting point for research, it often represents an oversimplification of reality.     

Outer membrane protein OmpV mediates Salmonella enterica serovar typhimurium adhesion to intestinal epithelial cells via fibronectin and α1β1 integrin

Salmonella typhimurium is an invasive Gram‐negative enteric bacterium, which causes salmonellosis, a type of gastroenteritis in humans and typhoid‐like symptoms in mice. Upon entering through the contaminated food and water, S. typhimurium adheres, colonises, and invades intestinal epithelial cells (IECs) of the small intestine. In this study, we have shown that upon deletion of the outer membrane protein OmpV, there is a significant decrease in adherence of S. typhimurium to the IECs, indicating that OmpV is an important adhesin of S. typhimurium. Further, our study showed that OmpV binds to the extracellular matrix component fibronectin and signals through α1β1 integrin receptor on the IECs and OmpV‐mediated activation of α1β1, resulting in the activation of focal adhesion kinase and F‐actin modulation. Actin modulation is crucial for bacterial invasion. To the best of our knowledge, this is the first report of an adhesin mediated its effect through integrin in S. typhimurium. Further, we have observed a decrease in pathogenicity in terms of increased LD₅₀ dose, lesser bacterial numbers in stool, and less colonisation of bacteria in different organs of mice infected with Δompv mutant compared with the wild‐type bacteria, thus confirming the crucial role of OmpV in the pathogenesis of S. typhimurium.     

Dysregulated haemolysin promotes bacterial outer membrane vesicles‐induced pyroptotic‐like cell death in zebrafish

Inflammasomes are important innate immune components in mammals. However, the bacterial factors modulating inflammasome activation in fish, and the mechanisms by which they alter fish immune defences, remain to be investigated. In this work, a mutant of the fish pathogen Edwardsiella piscicida (E. piscicida), called 0909I, was shown to overexpress haemolysin, which could induce a robust pyroptotic‐like cell death dependent on caspase‐5‐like activity during infection in fish nonphagocyte cells. E. piscicida haemolysin was found to mainly associate with bacterial outer membrane vesicles (OMVs), which were internalised into the fish cells via a dynamin‐dependent endocytosis and induced pyroptotic‐like cell death. Importantly, bacterial immersion infection of both larvae and adult zebrafish suggested that dysregulated expression of haemolysin alerts the innate immune system and induces intestinal inflammation to restrict bacterial colonisation in vivo. Taken together, these results suggest a critical role of zebrafish innate immunity in monitoring invaded pathogens via detecting the bacterial haemolysin‐associated OMVs and initiating pyroptotic‐like cell death. These new additions to the understanding of haemolysin‐mediated pathogenesis in vivo provide evidence for the existence of noncanonical inflammasome signalling in lower vertebrates.     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

Site‐specific inhibition of integrin αvβ3‐vitronectin association by a ser‐asp‐val sequence through an Arg‐Gly‐Asp‐binding site of the integrin

A functional proteomic technology using protein chip and molecular simulation was used to demonstrate a novel biomolecular interaction between P11, a peptide containing the Ser‐Asp‐Val (SDV) sequence and integrin αvβ3. P11 (HSDVHK) is a novel antagonistic peptide of integrin αvβ3 screened from hexapeptide library through protein chip system. An in silico docking study and competitive protein chip assay revealed that the SDV sequence of P11 is able to create a stable inhibitory complex onto the vitronectin‐binding site of integrin αvβ3. The Arg‐Gly‐Asp (RGD)‐binding site recognition by P11 was site specific because the P11 was inactive for the complex formation of a denatured form of integrin–vitronectin. P11 showed a strong antagonism against αvβ3‐GRGDSP interaction with an IC₅₀ value of 25.72±3.34 nM, whereas the value of GRGDSP peptide was 1968.73±444.32 nM. The binding‐free energies calculated from the docking simulations for each P11 and RGD peptide were ?3.99 and ?3.10 kcal/mol, respectively. The free energy difference between P11 and RGD corresponds to approximately a 4.5‐fold lower K_i value for the P11 than the RGD peptide. The binding orientation of the docked P11 was similar to the crystal structure of the RGD in αvβ3. The analyzed docked poses suggest that a divalent metal–ion coordination was a common driving force for the formation of both SDV/αvβ3 and RGD/αvβ3 complexes. This is the first report on the specific recognition of the RGD‐binding site of αvβ3 by a non‐RGD containing peptide using a computer‐assisted proteomic approach.     

Development of the muscle system and contractile activity in the mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> (Mollusca,Bivalvia)

The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and fluorescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 μm in both mussel larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing video workstation. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult molluscs.     

Solution structure of a bacterial immunoglobulin‐like domain of the outer membrane protein (LigB) from Leptospira

Leptospiral immunoglobulin‐like (Lig) proteins are surface proteins expressed in pathogenic strains of Leptospira. LigB, an outer membrane protein containing tandem repeats of bacterial Ig‐like (Big) domains and a no‐repeat tail, has been identified as a virulence factor involved in adhesion of pathogenic Leptospira interrogans to host cells. A Big domain of LigB, LigBCen2R, was reported previously to bind the GBD domain of fibronectin, suggesting its important role in leptospiral infections. In this study, we determined the solution structure of LigBCen2R by nuclear magnetic resonance (NMR) spectroscopy. LigBCen2R adopts a canonical immunoglobulin‐like fold which is comprised of a beta‐sandwich of ten strands in three sheets. We indicated that LigBCen2R is able to bind to Ca²⁺ with a high affinity by isothermal titration calorimetry assay. NMR perturbation experiment identified a number of residues responsible for Ca²⁺ binding. Structural comparison of it with other Big domains demonstrates that they share a similar fold pattern, but vary in some structural characters. Since Lig proteins play a vital role in the infection to host cells, our study will contribute a structural basis to understand the interactions between Leptospira and host cells. Proteins 2015; 83:195–200. © 2014 Wiley Periodicals, Inc.