Lipid raft-dependent and -independent signaling through HLA-DR molecules

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV(+) and EBV(-) B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the alpha and beta chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cell-cell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. MbetaCD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DR-induced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not.     

Choosing negative examples for the prediction of protein-protein interactions

The protein-protein interaction networks of even well-studied model organisms are sketchy at best, highlighting the continued need for computational methods to help direct experimentalists in the search for novel interactions. This need has prompted the development of a number of methods for predicting protein-protein interactions based on various sources of data and methodologies. The common method for choosing negative examples for training a predictor of protein-protein interactions is based on annotations of cellular localization, and the observation that pairs of proteins that have different localization patterns are unlikely to interact. While this method leads to high quality sets of non-interacting proteins, we find that this choice can lead to biased estimates of prediction accuracy, because the constraints placed on the distribution of the negative examples makes the task easier. The effects of this bias are demonstrated in the context of both sequence-based and non-sequence based features used for predicting protein-protein interactions.     

Genetic and physical mapping of a novel region close to the fragile X site on the human X chromosome

We report the isolation and characterization of a novel DNA marker (1A1) in Xqter in the region of the fragile X. Genetic studies in families segregating for the fragile X syndrome suggest that 1A1 lies between the disease mutation and the distal locus, DXS52. Studies in normal and fragile X families show that 1A1 is tightly linked to DXS52 (Zmax = 17.20; theta max = 0.03) and F8 (Zmax = 7.01; theta max = 0.08). Multipoint mapping of families supports the order Xcen-DXS105-FRAXA-1A1-DXS52-(F8, DXS115)-Xqter. Pulsed-field gel electrophoresis (PFGE) studies demonstrate that 1A1 defines a new region of at least 2 Mb of DNA not physically linked to DXS52 or F8, thus extending the physical map of Xq27-qter to over 4 Mb. Complex partial digestion PFGE patterns, probably due to differing degrees of methylation, are observed with 1A1 in unrelated normal and fragile-X-positive individuals, whereas other distal markers give uniform digestion profiles. Physical data suggest that 1A1 lies in a region less CpG rich than other distal markers in Xq27-qter.     

A defective viral superantigen-presenting phenotype in HLA-DR transfectants is corrected by CIITA

Activation of T lymphocytes by mouse mammary tumor virus superantigen (vSAg) requires binding to MHC class II molecules. The subcellular location where functional interactions occur between MHC class II molecules and vSAgs is still a matter of debate. To gain further insight into this issue, we have used human epithelial HeLa cells expressing HLA-DR1. Surprisingly, the human cells were unable to present transfected vSAg7 or vSAg9 to a series of murine T cell hybridomas. The defect is not related to a lack of vSAg processing, because these cells can indirectly activate T cells after coculture in the presence of B lymphocytes. However, after IFN-gamma treatment, the HeLa DR1(+) cells became apt at directly presenting the vSAg. Furthermore, transfection of CIITA was sufficient to restore presentation. Reconstitution experiments demonstrated the necessity of coexpressing HLA-DM and invariant chain (Ii) for efficient vSAg presentation. Interestingly, inclusion of a dileucine motif in the DRbeta cytoplasmic tail bypassed the need for HLA-DM expression and allowed the efficient presentation of vSAg7 in the presence of Ii. A similar trafficking signal was included in vSAg7 by replacing its cytoplasmic tail with the one of Ii. However, sorting of this chimeric Ii/vSAg molecule to the endocytic pathway completely abolished both its indirect and direct presentation. Together, our results suggest that functional vSAgs-DR complexes form after the very late stages of class II maturation, most probably at the cell surface.     

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface proteins are central to the cell''s ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan DIGE technology and analysis of protein expression changes using DeCyder 2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.Open in a separate windowClick here to view.^{(76M, flv)}     

Genetic tumor archeology: microdissection and genetic heterogeneity in squamous and basal cell carcinoma

Carcinogenesis is a multi-step series of somatic genetic events. The complexity of this multi-hit process makes it difficult to determine each single event and the definitive outcome of such events. To investigate the genetic alterations in cancer-related genes, sensitive and reliable detection methods are of major importance for generating relevant results. Another critical issue is the quality of starting material which largely affects the outcome of the analysis. Microdissection of cells defined under the microscope ensures a selection of representative material for subsequent genetic analysis. Skin cancer provides an advantageous model for studying the development of cancer. Detectable lesions occur early during tumor progression, facilitating molecular analysis of the cell populations from both preneoplastic and neoplastic lesions. Alterations of the p53 tumor suppressor gene are very common in non-melanoma skin cancer, and dysregulation of p53 pathways appear to be an early event in the tumor development. A high frequency of epidermal p53 clones has been detected in chronically sun-exposed skin. The abundance of clones containing p53 mutated keratinocytes adjacent to basal cell (BCC) and squamous cell carcinoma (SCC) suggests a role in human skin carcinogenesis. Studies using p53 mutations as a clonality marker have suggested a direct link between actinic keratosis, SCC in situ and invasive SCC. Microdissection-based studies have also shown that different parts of individual BCC tumors can share a common p53 mutation yet differ with respect to additional alterations within the p53 gene, consistent with subclonal development within tumors. Here, we present examples of using well-defined cell populations, including single cells, from complex tissue in combination with molecular tools to reveal features involved in skin carcinogenesis.     

Alterations in oxidative phosphorylation complex proteins in the hearts of transgenic mice that overexpress the p38 MAP kinase activator, MAP kinase kinase 6

Ischemia-reperfusion (I/R) has critical consequences in the heart. Recent studies on the functions of I/R-activated kinases, such as p38 mitogen-activated protein kinase (MAPK), showed that I/R injury is reduced in the hearts of transgenic mice that overexpress the p38 MAPK activator MAPK kinase 6 (MKK6). This protection may be fostered by changes in the levels of many proteins not currently known to be regulated by p38. To examine this possibility, we employed the multidimensional protein identification technology MudPIT to characterize changes in levels of proteins in MKK6 transgenic mouse hearts, focusing on proteins in mitochondria, which play key roles in mediating I/R injury in the heart. Of the 386 mitochondrial proteins identified, the levels of 58 were decreased, while only 2 were increased in the MKK6 transgenic mouse hearts. Among those that were decreased were 21 mitochondrial oxidative phosphorylation complex proteins, which was unexpected because p38 is not known to mediate such decreases. Immunoblotting verified that proteins in each of the five oxidative phosphorylation complexes were reduced in MKK6 mouse hearts. On assessing functional consequences of these reductions, we found that MKK6 mouse heart mitochondria exhibited 50% lower oxidative respiration and I/R-mediated reactive oxygen species (ROS) generation, both of which are predicted consequences of decreased oxidative phosphorylation complex proteins. Thus the cardioprotection observed in MKK6 transgenic mouse hearts may be partly due to decreased electron transport, which is potentially beneficial, because damaging ROS are known to be generated by mitochondrial complexes I and III during reoxygenation.     

The role of living/controlled radical polymerization in the formation of improved imprinted polymers

In this work, living/controlled radical polymerization (LRP) is compared with conventional free radical polymerization in the creation of highly and weakly cross-linked imprinted poly(methacrylic acid-co-ethylene glycol dimethacrylate) networks. It elucidates, for the first time, the effect of LRP on the chain level and begins to explain why the efficiency of the imprinting process is improved using LRP. Imprinted polymers produced via LRP exhibited significantly higher template affinity and capacity compared with polymers prepared using conventional methods. The use of LRP in the creation of highly cross-linked imprinted polymers resulted in a fourfold increase in binding capacity without a decrease in affinity; whereas weakly cross-linked gels demonstrated a nearly threefold increase in binding capacity at equivalent affinity when LRP was used. In addition, by adjusting the double bond conversion, we can choose to increase either the capacity or the affinity in highly cross-linked imprinted polymers, thus allowing the creation of imprinted polymers with tailorable binding parameters. Using free radical polymerization in the creation of polymer chains, as the template-monomer ratio increased, the average molecular weight of the polymer chains decreased despite a slight increase in the double bond conversion. Thus, the polymer chains formed were shorter but greater in number. Using LRP neutralized the effect of the template. The addition of chain transfer agent resulted in slow, uniform, simultaneous chain growth, resulting in the formation of longer more monodisperse chains. Reaction analysis revealed that propagation time was extended threefold in the formation of highly cross-linked polymers when LRP techniques were used. This delayed the transition to the diffusion-controlled stage of the reaction, which in turn led to the observed enhanced binding properties, decreased polydispersity in the chains, and a more homogeneous macromolecular architecture.     

DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation

Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive inherited PD (Bonifati et al. 2003). In a companion article (Martinat et al. 2004), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to alpha-synuclein, a protein implicated in PD pathogenesis.