A membrane leucine heptad contributes to trafficking, signaling, and transformation by latent membrane protein 1

Latent membrane protein 1 (LMP1) of Epstein Barr virus (EBV) is important for maintaining proliferation of EBV-infected B cells. LMP1, unlike its cellular counterpart, CD40, signals without a ligand and is largely internal to the plasma membrane. In order to understand how LMP1 initiates its ligand-independent signaling, we focused on a leucine heptad in LMP1's first membrane-spanning domain that was shown to be necessary for LMP1's signaling through NF-kappaB. LZ1EBV, a recombinant EBV genetically altered to express LZ1, a derivative of LMP1 in which a leucine heptad was replaced with alanines, transformed B cells with 56% of wild-type (wt) EBV's efficiency, demonstrating the importance of this heptad. To elucidate the mechanism by which this domain contributes to the functions of LMP1, the properties of the wt and LZ1 were compared in transfected cells. LZ1 failed to home to lipid rafts as efficiently as did wt LMP1. The distribution of tagged derivatives of LZ1 also differed from that of wt LMP1 in transfected cells. LZ1's defect in homing to lipid rafts and altered trafficking likely underlie the defect in transformation of LZ1EBV. While the third and fourth membrane-spanning domains of LMP1 foster its trafficking to the Golgi, the leucine heptad within the first membrane-spanning domain contributes to its trafficking, particularly to internal rafts. B cells that are successfully transformed by LZ1EBV have the same average number of viral genomes and the same fraction of cells with capped LZ1 at the cell surface but express 50% more of the LZ1 allele than wt infected cells.     

Cathelicidin LL-37 increases lung epithelial cell stiffness, decreases transepithelial permeability, and prevents epithelial invasion by Pseudomonas aeruginosa

In addition to its antibacterial activity, the cathelicidin-derived LL-37 peptide induces multiple immunomodulatory effects on host cells. Atomic force microscopy, F-actin staining with phalloidin, passage of FITC-conjugated dextran through a monolayer of lung epithelial cells, and assessment of bacterial outgrowth from cells subjected to Pseudomonas aeruginosa infection were used to determine LL-37's effect on epithelial cell mechanical properties, permeability, and bacteria uptake. A concentration-dependent increase in stiffness and F-actin content in the cortical region of A549 cells and primary human lung epithelial cells was observed after treatment with LL-37 (0.5-5 μM), sphingosine 1-phosphate (1 μM), or LPS (1 μg/ml) or infection with PAO1 bacteria. Other cationic peptides, such as RK-31, KR-20, or WLBU2, and the antibacterial cationic steroid CSA-13 did not reproduce the effect of LL-37. A549 cell pretreatment with WRW4, an antagonist of the transmembrane formyl peptide receptor-like 1 protein attenuated LL-37's ability to increase cell stiffness. The LL-37-mediated increase in cell stiffness was accompanied by a decrease in permeability and P. aeruginosa uptake by a confluent monolayer of polarized normal human bronchial epithelial cells. These results suggested that the antibacterial effect of LL-37 involves an LL-37-dependent increase in cell stiffness that prevents epithelial invasion by bacteria.     

A critical analysis of disease-associated DNA polymorphisms in the genes of cattle,goat, sheep,and pig

Genetic variations through their effects on gene expression and protein function underlie disease susceptibility in farm animal species. The variations are in the form of single nucleotide polymorphisms, deletions/insertions of nucleotides or whole genes, gene or whole chromosomal rearrangements, gene duplications, and copy number polymorphisms or variants. They exert varying degrees of effects on gene action, such as substitution of an amino acid for another, shift in reading frame and premature termination of translation, and complete deletion of entire exon(s) or gene(s) in diseased individuals. These factors influence gene function by affecting mRNA splicing pattern or by altering/eliminating protein function. Elucidating the genetic bases of diseases under the control of many genes is very challenging, and it is compounded by several factors, including host × pathogen × environment interactions. In this review, the genetic variations that underlie several diseases of livestock (under monogenic and polygenic control) are analyzed. Also, factors hampering research efforts toward identification of genetic influences on animal disease identification and control are highlighted. A better understanding of the factors analyzed could be better harnessed to effectively identify and control, genetically, livestock diseases. Finally, genetic control of animal diseases can reduce the costs associated with diseases, improve animal welfare, and provide healthy animal products to consumers, and should be given more attention.     

A human virus protein, poliovirus protein 2BC, induces membrane proliferation and blocks the exocytic pathway in the yeast Saccharomyces cerevisiae.

Inducible synthesis of poliovirus protein 2BC in Saccharomyces cerevisiae arrests cell growth in the G2 phase of the cell cycle, while no effects are observed upon expression of poliovirus genes 2B or 2C, either individually or in combination. Expression of 2BC induces a number of morphological modifications in yeast cells, one of the most striking being the proliferation of small membranous vesicles that fill most of the cytoplasm. These vesicles are morphologically similar to the cytopathic vacuoles that proliferate during the infection of human cells by poliovirus. The transport and processing of several yeast proteins, including vacuolar carboxypeptidase Y, aminopeptidase I or yeast alpha-mating factor, is hampered upon expression of poliovirus 2BC, suggesting that transport of proteins through the Golgi apparatus is impaired by this viral protein. Finally, a number of 2BC variants were generated and the effects of their expression on yeast growth, cellular morphology and protein processing were analyzed. 2BC variants defective in the NTPase activity were still able to interfere with yeast growth and the exocytic system, while deletion of 30 amino acids at the N-terminus of 2BC impairs its function. These findings lend support to the idea that 2BC, but not 2B or 2C, is the protein responsible for vesicle proliferation in poliovirus-infected cells. In addition, the activity of a human virus protein in yeast cells opens new avenues to investigate the exact location at which poliovirus 2BC interferes with the vesicular system and to test the action of other animal virus proteins potentially involved in modifying the vesicular system in mammalian cells.     

Identification of sequences in herpes simplex virus type 1 ICP22 that influence RNA polymerase II modification and viral late gene expression

Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.     

Glutamine,arginine, and leucine signaling in the intestine

Glutamine and leucine are abundant constituents of plant and animal proteins, whereas the content of arginine in foods and physiological fluids varies greatly. Besides their role in protein synthesis, these three amino acids individually activate signaling pathway to promote protein synthesis and possibly inhibit autophagy-mediated protein degradation in intestinal epithelial cells. In addition, glutamine and arginine stimulate the mitogen-activated protein kinase and mammalian target of rapamycin (mTOR)/p70 (s6) kinase pathways, respectively, to enhance mucosal cell migration and restitution. Moreover, through the nitric oxide-dependent cGMP signaling cascade, arginine regulates multiple physiological events in the intestine that are beneficial for cell homeostasis and survival. Available evidence from both in vitro and in vivo animal studies shows that glutamine and arginine promote cell proliferation and exert differential cytoprotective effects in response to nutrient deprivation, oxidative injury, stress, and immunological challenge. Additionally, when nitric oxide is available, leucine increases the migration of intestinal cells. Therefore, through cellular signaling mechanisms, arginine, glutamine, and leucine play crucial roles in intestinal growth, integrity, and function.     

Animal cloning--the route to new genomics in agriculture and medicine.

This paper reviews the origin and development of animal cloning in metazoans starting with primitive experiments performed during the late 1880's and early 1900's, followed by nuclear transplantation in amphibians in 1952, then extended to fish and insects in the 1960's, and finally to mammals in the 1980's. Emphasis is placed on the applications of mammalian cloning to agriculture, medicine, and the conservation of endangered species. In addition, the introduction of genes via random insertion or gene targeting into the genome of donor cells to be used for cloning has opened up another route for new genomics in agriculture and medicine. The production of transgenic clones starting in 1997 has indeed contributed a milestone to scientific research. Although cloning efficiency is still low, certain kinds of experiments are quite feasible, and we anticipate improvements in the future.     

Neurofibromin binds to caveolin-1 and regulates ras, FAK, and Akt

Neurofibromin (Nf1) is an approximately 280 kDa protein having tumor suppressor function, presumably by virtue of its GTPase activating domain, but little is known regarding molecular aspects of its effector pathways. Caveolin-1 (Cav-1) regulates diverse signaling molecules and has itself been implicated as a tumor suppressor. Here we demonstrate that Nf1 binds to Cav-1's scaffolding domain and co-immunoprecipitates with Cav-1. Analysis of Nf1's primary structure reveals four potential caveolin binding domains, and interestingly, in individuals with neurofibromatosis I, missense mutations occur with high frequency in 3 of the 4 putative domains. We show that Nf1 modulates ras, Akt, and focal adhesion kinase pathways, thereby affecting cytoskeletal organization; moreover, Nf1's effects on signaling are altered when lipid rafts and caveolae are disrupted by cholesterol depletion. These novel findings provide insight into possible signaling mechanisms of Nf1 and suggest that together Nf1 and Cav-1 may coordinately regulate cell growth and differentiation.     

A putative protein inhibitor of activated STAT (PIASy) interacts with p53 and inhibits p53-mediated transactivation but not apoptosis

The p53 protein has recently been reported to be capable of mediating apoptosis through a pathway that is not dependent on its transactivation function. We report here that the PIASy member of the protein inhibitor of activated STAT family inhibited p53's transactivation function without compromising its ability to induce apoptosis of the H1299 nonsmall cell lung carcinoma cell line. The p53 protein bound to PIASy in yeast two-hybrid assays and coprecipitated in complexes with p53 in immunoprecipitates from mammalian cells. PIASy inhibited the DNA-binding activity of p53 in nuclear extracts and blocked the ability of p53 to induce expression of two of its target genes, Bax and p21^Waf1/Cip1, in H1299 cells. The block in p53-mediated induction of Bax and p21 was determined to be at the level of transactivation, since PIASy inhibited p53's ability to transactivate a p21/luciferase reporter construct. PIASy did not effect the incidence of apoptosis in H1299 cells upregulated for p53. PIASy appears to regulate p53-mediated functions and may direct p53 into a transactivation-independent mode of apoptosis.     

Identifying antigenicity-associated sites in highly pathogenic H5N1 influenza virus hemagglutinin by using sparse learning

Since the isolation of A/goose/Guangdong/1/1996 (H5N1) in farmed geese in southern China, highly pathogenic H5N1 avian influenza viruses have posed a continuous threat to both public and animal health. The non-synonymous mutation of the H5 hemagglutinin (HA) gene has resulted in antigenic drift, leading to difficulties in both clinical diagnosis and vaccine strain selection. Characterizing H5N1's antigenic profiles would help resolve these problems. In this study, a novel sparse learning method was developed to identify antigenicity-associated sites in influenza A viruses on the basis of immunologic data sets (i.e., from hemagglutination inhibition and microneutralization assays) and HA protein sequences. Twenty-one potential antigenicity-associated sites were identified. A total of 17 H5N1 mutants were used to validate the effects of 11 of these predicted sites on H5N1's antigenicity, including 7 newly identified sites not located in reported antibody binding sites. The experimental data confirmed that mutations of these tested sites lead to changes in viral antigenicity, validating our method.     

Purification and structural characterization of human ERp29

ERp29 is a major resident of the endoplasmic reticulum (ER) and is postulated to play an important molecular chaperone role in most animal cells. Human ERp29 was isolated to homogeneity in high yield by using a bacterial expression system. Its secondary structure was studied by circular dichroism (CD), Fourier transformed infrared spectroscopy (FTIR) and Raman spectroscopy and it was found that human ERp29 comprises significant alpha-helical structure. The details of its temperature-induced conformational changes was studied by CD and FTIR for the first time, revealing that the protein is stable below 50 degrees C and has two distinct structural transitions between 50 degrees C and 70 degrees C. This may shed light on ERp29's inability to protect substrate proteins against thermal aggregation.     

H9724, a Monoclonal Antibody to Borrelia burgdorferi's Flagellin, Binds to Heat Shock Protein 60 (HSP60) Within Live Neuroblastoma Cells: A Potential Role for HSP60 in Peptide Hormone Signaling and in an Autoimmune Pathogenesis of the Neuropathy of Lyme Disease

Although Borrelia burgdorferi, the causative agent of Lyme disease, is found at the site of many disease manifestations, local infection may not explain all its features. B. burgdorferi's flagellin cross-reacts with a component of human peripheral nerve axon, previously identified as heat shock protein 60 (HSP60). The cross-reacting epitopes are bound by a monoclonal antibody to B. burgdorferi's flagellin, H9724. Addition of H9724 to neuroblastoma cell cultures blocks in vitro spontaneous and peptide growth-factor–stimulated neuritogenesis. Withdrawal of H9724 allows return to normal growth and differentiation. Using electron microscopy, immunoprecipitation and immunoblotting, and FACS analysis we sought to identify the site of binding of H9724, with the starting hypotheses that the binding was intracellular and not identical to the binding site of II-13, a monoclonal anti-HSP60 antibody. The current studies show that H9724 binds to an intracellular target in cultured cells with negligible, if any, surface binding. We previously showed that sera from patients with neurological manifestations of Lyme disease bound to human axons in a pattern identical to H9724's binding; these same sera also bind to an intracellular neuroblastoma cell target. II-13 binds to a different HSP60 epitope than H9724; II-13 does not modify cellular function in vitro. As predicted, II-13 bound to mitochondria, in a pattern of cellular binding very different from H9724, which bound in a scattered cytoplasmic, nonorganelle-related pattern. H9724's effect is the first evidence that HSP60 may play a role in peptide-hormone–receptor function and demonstrates the modulatory potential of a monoclonal antibody on living cells.     

Inactivation of neuregulin-1 by nitration

Nitration is a posttranslational modification that can compromise protein function. We hypothesized that nitration of growth factors secreted in the lung may alter their interaction with their respective receptors and modulate the normal growth and differentiation program induced by ligand-receptor interaction. We tested this hypothesis in vitro by nitration of neuregulin-1's (NRG-1) EGF-like domain and studying the effect on NRG-1's activity. Nitration of NRG-1's (nNRG-1) EGF-like domain resulted in an inability to activate its receptor, the human epidermal growth factor receptors 2 and 3 (HER2/HER3) heterodimer, as defined by loss of HER2 tyrosine phosphorylation induced by nNRG-1 in MCF-7 cells. Receptor activation was not restored with increasing nNRG-1 concentration or exposure times. nNRG-1 did not compete with NRG-1 for HER2/HER3 binding in competition assays. In addition, nNRG-1 no longer induced proliferation of the MCF-7 cell line, as MCF-7 cells exposed to nNRG-1 and NRG-1 concurrently had the same proliferation rate as that induced by NRG-1 alone. Thus nitration of NRG-1's EGF-like domain caused it to lose its ability to bind and activate its receptor with loss of ligand-induced proliferation. Posttranslational nitration of growth factors in states where reactive nitrogen species are increased may be an important means of regulating growth factor receptor effects in the lung.     

A novel short-chain alcohol dehydrogenase from rats with retinol dehydrogenase activity,cyclically expressed in uterine epithelium

Retinoic acid is necessary for the maintenance of many lining epithelia of the body, such as the epithelium of the luminal surface of the uterus. Administration of estrogen to prepubertal rats induces in these epithelial cells the ability to synthesize retinoic acid from retinol, coincident with the appearance of cellular retinoic acid-binding protein, type two, which is normally present in these cells only at estrus in the mature, cycling animal. Here, we report the isolation, from a cDNA library prepared from uterine mRNA collected at the estrous stage and from a rat mammary adenocarcinoma cell line, of a cDNA that encodes a novel retinol dehydrogenase. A member of the short-chain alcohol dehydrogenase family, the encoded enzyme was capable of metabolizing retinol to retinal when expressed in cells after transfection of its cDNA. When cotransfected with the cDNA of human aldehyde 6, a known retinaldehyde dehydrogenase, the transfected cells synthesized retinoic acid from retinol. Immunohistochemical analysis revealed that the protein was present in the uterine lining epithelium of the mature animal only at estrus, coincident with the presence of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two. Consequently, this novel short-chain alcohol dehydrogenase is an excellent candidate for the retinol dehydrogenase that catalyzes the first step in retinoic acid biosynthesis that occurs in uterine epithelial cells.     

A novel src homology 3 domain-containing adaptor protein, HIP-55, that interacts with hematopoietic progenitor kinase 1

Hematopoietic progenitor kinase 1 (HPK1) is a member of the mitogen-activated protein kinase kinase kinase kinase (MAP4K) family and an upstream activator of the c-Jun N-terminal kinase (JNK) signaling cascade. HPK1 interacts, through its proline-rich domains, with growth factor receptor-bound 2 (Grb2), CT10-regulated kinase (Crk), and Crk-like (CrkL) adaptor proteins. We identified a novel HPK1-interacting protein of 55 kDa (HIP-55), similar to the mouse SH3P7 protein, containing an N-terminal actin-binding domain and a C-terminal Src homology 3 domain. We found that HPK1 bound to HIP-55 both in vitro and in vivo. When co-transfected, HIP-55 increased HPK1's kinase activity as well as JNK1's kinase activity. A dominant-negative HPK1 mutant blocked activation of JNK1 by HIP-55 showing that HIP-55 activates the JNK1 signaling pathway via HPK1. Our results identify a novel protein, HIP-55, that binds to HPK1 and regulates the JNK1 signaling cascade.     

An early look at macromolecular crowding

Interest in the interaction between proteins and polysaccharides in semidilute systems developed in the late 1950's and early 1960's both in the field of matrix biology and in the construction of new separation techniques. The author gives an account of how his work in the 50's on the characterization of a connective tissue polysaccharide, hyaluronan, led him into studies on polysaccharide networks, sieve effects, molecular exclusion, a theory of gel filtration, solubility of proteins and chemical equilibria in polymer solutions, water and protein homeostatis in vivo, and ordered convectional flow in concentrated polymer systems.     

Ro60 and Y RNAs: structure,functions, and roles in autoimmunity

Ro60, also known as SS-A or TROVE2, is an evolutionarily conserved RNA-binding protein that is found in most animal cells, approximately 5% of sequenced prokaryotic genomes and some archaea. Ro60 is present in cells as both a free protein and as a component of a ribonucleoprotein complex, where its best-known partners are members of a class of noncoding RNAs called Y RNAs. Structural and biochemical analyses have revealed that Ro60 is a ring-shaped protein that binds Y RNAs on its outer surface. In addition to Y RNAs, Ro60 binds misfolded and aberrant noncoding RNAs in some animal cell nuclei. Although the fate of these defective Ro60-bound noncoding RNAs in animal cells is not well-defined, a bacterial Ro60 ortholog functions with 3′ to 5′ exoribonucleases to assist structured RNA degradation. Studies of Y RNAs have revealed that these RNAs regulate the subcellular localization of Ro60, tether Ro60 to effector proteins and regulate the access of other RNAs to its central cavity. As both mammalian cells and bacteria lacking Ro60 are sensitized to ultraviolet irradiation, Ro60 function may be important during exposure to some environmental stressors. Here we summarize the current knowledge regarding the functions of Ro60 and Y RNAs in animal cells and bacteria. Because the Ro60 RNP is a clinically important target of autoantibodies in patients with rheumatic diseases such as Sjogren’s syndrome, systemic lupus erythematosus, and neonatal lupus, we also discuss potential roles for Ro60 RNPs in the initiation and pathogenesis of systemic autoimmune rheumatic disease.     

Characterization of LMP-1's association with TRAF1, TRAF2, and TRAF3.

The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the immortalizing activity of EBV in primary, human B lymphocytes. LMP-1 is targeted to the plasma membrane, where it influences signaling pathways of infected cells. LMP-1 has been found to associate with members of the tumor necrosis factor receptor-associated factor (TRAF) family of proteins. As with LMP-1, the TRAF molecules have been shown to participate in cell signaling pathways. We have characterized and mapped in detail a region of LMP-1 that associates with TRAF1, TRAF2, and TRAF3. TRAF3 alone associates with LMP-1 in a yeast two-hybrid assay, whereas all three TRAF molecules associate with LMP-1 under various conditions when they are assayed in extracts of human cells. TRAF1, TRAF2, and TRAF3 appear to associate independently with LMP-1 but bind an overlapping target site. TRAF3 associates with LMP-1 most avidly and can compete with TRAF1 and TRAF2 for binding to LMP-1. TRAF2 associates with truncated derivatives of the carboxy terminus of LMP-1 more efficiently than with the intact terminus, indicating that LMP-1's conformation may regulate its association with TRAF2. Finally, point mutations that decrease LMP-1's association with the three TRAF molecules to 3 to 20% of wild-type levels do not detectably affect otherwise intact LMP-1's induction of NF-kappaB activity. Therefore, these associations are not necessary for the majority of intact LMP-1's induction of this signaling pathway.