Biomedical subjects

Russell L Finley

Publications and source records attributed to Russell L Finley.

12 recordsLinked to original sources

PubMed2006

Yeast two-hybrid contributions to interactome mapping.

Interactome mapping, the systematic identification of protein interactions within an organism, promises to facilitate systems-level studies of biological processes. Using in vitro technologies that measure specific protein interactions, static maps are being generated that include many of the protein networks that occur in vivo. Most of the binary protein interaction data currently available was generated by large-scale yeast two-hybrid screens. Recent efforts to map interactions in model organisms and in humans illustrate the promise and some of the limitations of the two-hybrid approach. Although these maps are incomplete and include false positives, they are proving useful as a framework around which to elaborate and model the in vivo interactome.

Animals↗

PubMed2006

A database and tool, IM Browser, for exploring and integrating emerging gene and protein interaction data for Drosophila.

BACKGROUND: Biological processes are mediated by networks of interacting genes and proteins. Efforts to map and understand these networks are resulting in the proliferation of interaction data derived from both experimental and computational techniques for a number of organisms. The volume of this data combined with the variety of specific forms it can take has created a need for comprehensive databases that include all of the available data sets, and for exploration tools to facilitate data integration and analysis. One powerful paradigm for the navigation and analysis of interaction data is an interaction graph or map that represents proteins or genes as nodes linked by interactions. Several programs have been developed for graphical representation and analysis of interaction data, yet there remains a need for alternative programs that can provide casual users with rapid easy access to many existing and emerging data sets. DESCRIPTION: Here we describe a comprehensive database of Drosophila gene and protein interactions collected from a variety of sources, including low and high throughput screens, genetic interactions, and computational predictions. We also present a program for exploring multiple interaction data sets and for combining data from different sources. The program, referred to as the Interaction Map (IM) Browser, is a web-based application for searching and visualizing interaction data stored in a relational database system. Use of the application requires no downloads and minimal user configuration or training, thereby enabling rapid initial access to interaction data. IM Browser was designed to readily accommodate and integrate new types of interaction data as it becomes available. Moreover, all information associated with interaction measurements or predictions and the genes or proteins involved are accessible to the user. This allows combined searches and analyses based on either common or technique-specific attributes. The data can be visualized as an editable graph and all or part of the data can be downloaded for further analysis with other tools for specific applications. The database is available at http://proteome.wayne.edu/PIMdb.html CONCLUSION: The Drosophila Interactions Database described here places a variety of disparate data into one easily accessible location. The database has a simple structure that maintains all relevant information about how each interaction was determined. The IM Browser provides easy, complete access to this database and could readily be used to publish other sets of interaction data. By providing access to all of the available information from a variety of data types, the program will also facilitate advanced computational analyses.

Database Management Systems↗

PubMed2005

Tumor-suppressive maspin regulates cell response to oxidative stress by direct interaction with glutathione S-transferase.

Maspin, a novel serine protease inhibitor, suppresses tumor progression in several cancer models, including an in vivo model for prostate cancer bone metastasis. However, the molecular mechanism of maspin remains illusive, primarily because its molecular targets are unknown. To this end, we used a full-length maspin cDNA bait to screen against both a primary prostate tumor cDNA prey library and a HeLa cDNA prey library by the yeast two-hybrid method. We found that heat shock protein 90, glutathione S-transferase (GST), and heat shock protein 70 interacted with maspin with the highest frequencies. We confirmed the maspin/GST interaction using purified proteins, human epithelial cell lines, and human prostate tissues. A maspin variant that has a point mutation of Arg(340) to Ala (Mas(R340A)) showed a significantly decreased affinity for GST. Although purified maspin had no effect on the activity of purified GST in vitro, intracellular interaction between endogenous maspin and GST correlated with an elevated total GST activity in both MDA-MB-435- and DU145-derived stably transfected cells. Consistently, tumor cells treated with purified wild type maspin, but not Mas(R340A), enhanced cellular GST activity. Maspin expression in cancer cell lines also correlated with decreased basal levels of reactive oxygen species (ROS). Furthermore, H(2)O(2) treatment not only induced GST expression but also increased intracellular maspin/GST interaction, which was inversely correlated with the level of ROS generation. Conversely, maspin knockdown by small interfering RNA increased the basal, as well as H(2)O(2)-induced, ROS generation. Furthermore, the maspin effect on ROS generation was completely abolished by a GST inhibitor, indicating an essential role of GST in maspin-mediated cellular response to oxidative stress. Consistently, oxidative stress-induced vascular endothelial growth factor A expression was significantly inhibited in maspin-expressing cells. Together, our data suggest a new mechanism by which maspin, through its direct interaction with GST, may inhibit oxidative stress-induced ROS generation and vascular endothelial growth factor A induction, thus preventing further adverse effects on tumor genetics and stromal reactivity.

Alanine↗

PubMed2005

From protein networks to biological systems.

A system-level understanding of any biological process requires a map of the relationships among the various molecules involved. Technologies to detect and predict protein interactions have begun to produce very large maps of protein interactions, some including most of an organism's proteins. These maps can be used to study how proteins work together to form molecular machines and regulatory pathways. They also provide a framework for constructing predictive models of how information and energy flow through biological networks. In many respects, protein interaction maps are an entrée into systems biology.

Animals↗

PubMed2004

A Drosophila protein-interaction map centered on cell-cycle regulators.

BACKGROUND: Maps depicting binary interactions between proteins can be powerful starting points for understanding biological systems. A proven technology for generating such maps is high-throughput yeast two-hybrid screening. In the most extensive screen to date, a Gal4-based two-hybrid system was used recently to detect over 20,000 interactions among Drosophila proteins. Although these data are a valuable resource for insights into protein networks, they cover only a fraction of the expected number of interactions. RESULTS: To complement the Gal4-based interaction data, we used the same set of Drosophila open reading frames to construct arrays for a LexA-based two-hybrid system. We screened the arrays using a novel pooled mating approach, initially focusing on proteins related to cell-cycle regulators. We detected 1,814 reproducible interactions among 488 proteins. The map includes a large number of novel interactions with potential biological significance. Informative regions of the map could be highlighted by searching for paralogous interactions and by clustering proteins on the basis of their interaction profiles. Surprisingly, only 28 interactions were found in common between the LexA- and Gal4-based screens, even though they had similar rates of true positives. CONCLUSIONS: The substantial number of new interactions discovered here supports the conclusion that previous interaction mapping studies were far from complete and that many more interactions remain to be found. Our results indicate that different two-hybrid systems and screening approaches applied to the same proteome can generate more comprehensive datasets with more cross-validated interactions. The cell-cycle map provides a guide for further defining important regulatory networks in Drosophila and other organisms.

Animals↗

PubMed2003

A strategy for constructing large protein interaction maps using the yeast two-hybrid system: regulated expression arrays and two-phase mating.

Maps representing the binary interactions among proteins have become valuable tools for understanding how proteins work together to mediate biological processes. One of the most effective methods for detecting biologically important protein interactions has been the yeast two-hybrid system. Here we present an efficient two-hybrid strategy to facilitate construction of protein interaction maps on a genome-wide scale. The strategy begins with two arrays of yeast expressing known proteins fused to either a DNA binding domain (BD), or a transcription activation domain (AD). The fusion proteins are conditionally expressed using regulated promoters that can be repressed during construction and amplification of the yeast arrays. Interaction assays are conducted in two phases. In the first phase, small pools of AD strains are mated with the array of BD strains. In the second phase, individual BD strains are mated with appropriate subsets of the AD array corresponding to positive pools in the first phase. This strategy has several advantages over previously described approaches, including the ability to detect interactions with proteins that inhibit yeast growth or that activate transcription as BD fusions. Moreover, by minimizing the number of mating operations and sequencing reactions needed to test large sets of binary interactions, this strategy is more efficient than either matrix or library screening approaches. We also present a three-dimensional pooling scheme to further increase the efficiency of large-scale two-hybrid analyses.

DNA-Binding Proteins↗

PubMed2003

Active PIKfyve associates with and promotes the membrane attachment of the late endosome-to-trans-Golgi network transport factor Rab9 effector p40.

PIKfyve, a kinase that displays specificity for phosphatidylinositol (PtdIns), PtdIns 3-phosphate (3-P), and proteins, is important in multivesicular body/late endocytic function. Enzymatically inactive PIKfyve mutants elicit enormous dilation of late endocytic structures, suggesting a role for PIKfyve in endosome-to-trans-Golgi network (TGN) membrane retrieval. Here we report that p40, a Rab9 effector reported previously to bind Rab9-GTP and stimulate endosome-to-TGN transport, interacts with PIKfyve as determined by yeast two-hybrid assays, glutathione S-transferase (GST) pull-down assays, and co-immunoprecipitation in doubly transfected HEK293 cells. The interaction engages the PIKfyve chaperonin domain and four out of the six C-terminally positioned kelch repeats in p40. Differential centrifugation in a HEK293 cell line, stably expressing PIKfyveWT, showed the membrane-associated immunoreactive p40 co-sedimenting with PIKfyve in the high speed pellet (HSP) fraction. Remarkably, similar analysis in a HEK293 cell line stably expressing dominant-negative kinase-deficient PIKfyveK1831E demonstrated a marked depletion of p40 from the HSP fraction. GST-p40 failed to specifically associate with the PIKfyve lipid products PtdIns 5-P and PtdIns 3,5-P2 in a liposome binding assay but was found to be an in vitro substrate of the PIKfyve serine kinase activity. A band with the p40 electrophoretic mobility was found to react with a phosphoserine-specific antibody mainly in the PIKfyveWT-containing fractions obtained by density gradient sedimentation of total membranes from PIKfyveWT-expressing HEK293 cells. Together these results identify the Rab9 effector p40 as a PIKfyve partner and suggest that p40-PIKfyve interaction and the subsequent PIKfyve-catalyzed p40 phosphorylation anchor p40 to discrete membranes facilitating late endosome-to-TGN transport.

Binding Sites↗

PubMed2003

Simultaneous cloning of open reading frames into several different expression vectors.

Genomic sequencing has enabled the prediction of thousands of genes, most of which either cannot be assigned a function or can be only broadly categorized on the basis of sequence alone. High-throughput strategies for elucidating protein function are of high priority, and numerous approaches are being developed. Many of these approaches require the cloning of open reading frames (ORFs) into expression vectors that enable the encoded proteins to be tested for biological and biochemical activities. Typically, more than one type of vector must be employed, as different experiments require different conditions of protein production. Here we show that it is possible to simultaneously transfer a single ORF from a source vector to four target vectors using a commercially available in vitro recombination system. To test the approach, we constructed new vectors for expression of fusion proteins in yeast, including vectors for the LexA two-hybrid system. We show that individual ORFs can be efficiently transferred to four different vectors in a single in vitro reaction. The resulting expression plasmids can be separated using prototrophic markers specific to each vector. Using this system to produce multiple expression constructs simultaneously could greatly facilitate high-throughput subcloning and proteomic studies.

Bacterial Proteins↗

PubMed2002

Mediation of the DCC apoptotic signal by DIP13 alpha.

DCC (deleted in colorectal cancer) is a candidate tumor suppressor gene. However the function of DCC remains elusive. Previously, we demonstrated that forced expression of DCC induces apoptosis or cell cycle arrest (Chen, Y. Q., Hsieh, J. T., Yao, F., Fang, B., Pong, R. C., Cipriano, S. C. & Krepulat, F. (1999) Oncogene 18, 2747-2754). To delineate the DCC-induced apoptotic pathway, we have identified a protein, DIP13 alpha, which interacts with DCC. The DIP13 alpha protein has a pleckstrin homology domain and a phosphotyrosine binding domain. It interacts with a region on the DCC cytoplasmic domain that is required for the induction of apoptosis. Although ectopic expression of DIP13 alpha alone causes only a slight increase in apoptosis, co-expression of DCC and DIP13 alpha results in an approximately 5-fold increase in apoptosis. Removal of the DCC-interacting domain on DIP13 alpha abolishes its ability to enhance DCC-induced apoptosis. Inhibition of endogenous DIP13 alpha expression by small interfering RNA blocks DCC-induced apoptosis. Our data suggest that DIP13 alpha is a mediator of the DCC apoptotic pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Regulated expression of proteins in yeast using the MAL61-62 promoter and a mating scheme to increase dynamic range.

The ability to express heterologous genes in yeast has become indispensable for many biological research techniques. Expression systems that can be regulated are particularly useful because they allow an experimenter to control the timing and levels of gene expression. Despite their many advantages, however, surprisingly few conditional expression systems are available for yeast. Moreover, of those that have been described, many are not ideal either because they have high background expression levels, low induced levels, or because they require restrictive growth conditions. Here we describe a conditional expression system that takes advantage of the yeast MAL62 promoter (MAL62p), which can be controlled by adding maltose or glucose to the growth medium to induce or repress transcription, respectively. In addition, we use a mating scheme to dramatically increase the dynamic range of expression levels possible. We show that MAL62p background activity can be effectively eliminated by maintaining expression constructs in a mal(-) yeast strain. High-level expression can be induced in diploids formed by mating the mal(-) strain with a MAL(+) strain. A similar mating scheme may be useful for other conditional expression systems as well. Among other uses, this approach should aid high throughput yeast two-hybrid assays, which rely on maintaining large libraries of expression strains, which are eventually mated to conduct assays for protein interactions. We demonstrate a two-hybrid system in which MAL62p is used in conjunction with the yeast GAL1 promoter to independently regulate expression of both hybrid proteins, and to allow detection of interactions involving toxic proteins.

Amino Acid Sequence↗

PubMed2002

Galectin-3 translocates to the perinuclear membranes and inhibits cytochrome c release from the mitochondria. A role for synexin in galectin-3 translocation.

Galectin-3 is a multifunctional oncogenic protein found in the nucleus and cytoplasm and also the extracellular milieu. Although recent studies demonstrated an anti-apoptotic activity of galectin-3, neither the functional site nor the mechanism of how galectin-3 regulates apoptosis is known. In this study, we examined the subcellular localization of galectin-3 during apoptosis and investigated its anti-apoptotic actions. We report that galectin-3 translocates to the perinuclear membrane following a variety of apoptotic stimuli. Confocal microscopy and biochemical analysis revealed that galectin-3 is enriched in the mitochondria and prevents mitochondrial damage and cytochrome c release. Using a yeast two-hybrid system, we screened for galectin-3-interacting proteins that regulate galectin-3 localization and anti-apoptotic activity. Synexin, a Ca(2+)- and phospholipid-binding protein, was one of the proteins identified. We confirmed direct interaction between galectin-3 and synexin by glutathione S-transferase pull-down assay in vitro. We showed that galectin-3 failed to translocate to the perinuclear membranes when expression of synexin was down-regulated using an oligodeoxyribonucleotide complementary to the synexin mRNA, suggesting a role for synexin in galectin-3 trafficking. Furthermore, synexin down-regulation abolished anti-apoptotic activity of galectin-3. Taken together, these results suggest that synexin mediates galectin-3 translocation to the perinuclear mitochondrial membranes, where it regulates mitochondrial integrity critical for apoptosis regulation.

Annexin A7↗

PubMedDate not supplied

High-throughput cloning of Campylobacter jejuni ORfs by in vivo recombination in Escherichia coli.

A rate-limiting and costly step in many proteomics analyses is the cloning of all of the ORFs for an organism into technique-specific vectors. Here, we describe the generation of a Campylobacter jejuni expression clone set using a high-throughput cloning approach based on recombination in E. coli. The approach uses native E. coli recombination functions and requires no in vitro enzymatic steps or special strains. Our results indicate that this approach is an efficient and economical alternative for high-throughput cloning.

Campylobacter jejuni↗