Signaling networks within cells play a role in virtually every process from differentiation and proliferation to cell-cycle arrest and apoptosis. Intricate cellular communication is transduced via a complex series of protein-protein interactions, post-translational modifications, and other events. Deciphering the players and pathways is paving the way for new therapeutics, with researchers identifying novel networks from gene expression studies, developing technologies to peer inside living cells, and mining pathogens themselves for innovative strategies.
What would it be like to peer into a population of cells and see signal transduction networks in each? Scientists are now able to perform this feat by using advances in flow cytometry that allow them to simultaneously create signal transduction cellular snapshots.
“The utility of such an assay has the potential to go beyond the evaluation of biochemical coverage or pharmacodynamics, for which it is well suited, because it can identify disease-associated signaling dysregulation,” points out Gary D. Means, Ph.D., principal scientist at Amgen (www.amgen.com). “This has potential to improve our understanding of underlying pathologies.”
According to Dr. Means, “Flow cytometry has been used for decades to identify immune cell subsets with extracellular markers to profile immunophenotypes and activation states of leukocytes. Recently, we’ve learned to use flow cytometry employing intracellular markers for a variety of applications. This allows us to analyze a large number of single cells in a complex heterogeneous population or to identify relatively rare populations. We can profile a given biomarker in small constituent cell populations as well as characterize single cells and constellations of biomarkers within them, such as phosphoproteins.”
So-called phosphoprotein flow cytometry, or phosflow, represents a new approach to understanding the role of signal transduction as a contributor to disease pathology and a source of biomarkers. Measuring these markers may be used to infer patients’ response to therapeutics.
There are now a number of novel mAbs to intracellular signaling components as well as new fluorochromes that tag these antibodies, explains Dr. Means. “We can create a multicolor analysis to provide information for up to 13 parameters for each cell type identified.
“One immediate application for the new approach,” notes Dr. Means, “is quantitation of phosphoprotein biomarkers in specific cell subsets in whole blood. This provides a measure of disease states and therapeutic responses.”
Fuzzy Puzzle Networks
In signal transduction pathways, participating molecules are messengers designed to influence specific gene expression. They accomplish their goals via interactions within a complex network of components.
Gene promoters are key players in this process, according to Philip Stegmaier, research scientist and doctoral candidate at Biobase (www.biobase.de). He says that regulatory regions of genes consist of a “fuzzy puzzle” of cis-regulatory components that bind to promoters. Piecing this puzzle together may create a blueprint to identify which signal transduction networks are involved.
“The promoters of genes are the key components linking gene regulation with signal transduction networks primarily through the multiple interactions of transcription factors to their DNA binding sites,” Stegmaier explains. “What we actually observe is a fuzzy puzzle of a combination of transcription factors that form a complex near the transcription site. For example, when a ligand binds to the cell surface receptor, it may transmit a signal downstream to several intracellular transcription factors that then bind a specific gene.”
Stegmaier says that by characterizing the patterns of gene expression in diseased versus normal cells, researchers can determine which genes are up- or downregulated and map them to known pathways. “The key, however, is to obtain a causal interpretation of gene expression data in order to identify composite cis-regulatory elements in promoters of coregulated genes. This blueprint reveals not only the key transcription factors and upstream signaling molecules but also gives insights into prospective drug targets.”
Pathogenic bacteria can be major causes of death or disability. Group B Streptococcus (GBS), for example, is the foremost cause of life-threatening bacterial infections in newborns. Most bacterial pathogens possess long filamentous structures known as pili that extend from their surface. These tendrils mediate the initial contact with cells and can quickly activate host signaling cascades as the cell attempts to subvert infection.
“The potential for signal transduction becomes quite interesting,” notes John L. Telford, Ph.D., project leader, Novartis (www.novartis.com) vaccine and diagnostics division. “We performed a genomic screening of GBS and looked at every single protein that could be a candidate for inclusion in a vaccine to prevent the disease. Interestingly, we found some of these proteins were components of the pili.”
Together with Guido Grandi, Ph.D., head of the biochemistry and molecular biology unit at Novartis Vaccines & Diagnostics, Dr. Telford then studied the interactions of these pilus adhesins in a model system of bacterial interaction with human cells. “Bacteria can interact with extracellular matrix proteins that form bridges with integrins on epithelial cells,” according to Dr. Telford. “This may initiate a signaling cascade similar to that which results in polarization of the cell.
“As we begin to determine which components of the pili initiate such reactions, we become better able to design vaccines and therapeutics,” Dr. Telford continues. “We can then, by immunization, induce antibodies against pili that promote opsonization and clearance, we can interfere with the initial adhesion, and we can thus create antigens that are protective. Our approach now is to develop recombinant versions of GBS pili components that could be used as vaccines.”
Small endogenous RNAs such as miRNAs play critical regulatory roles in animal development by modulating mRNA. Research is aimed at deciphering how they exert such complex control of biological pathways. Scientists are also mining them for their enormous therapeutic potential.
Vertebrates are not the only ones to possess such molecules, however. Regulatory RNAs have been discovered in all kingdoms of life, including bacteria. “We’ve known that there are small regulatory bacterial RNAs (sRNA) for several years, but the surprise was how many have now been identified that are crucial for regulation of bacteria,” says Susan Gottesman, Ph.D., chief, biochemical genetics section, laboratory of molecular biology, NCI.
Dr. Gottesman’s work focuses on the basic science of sRNAs in bacteria. “The precise role of most of the sRNAs is still unknown. Thus far two modes of action have been described as relates to E. coli sRNAs. Some can modify protein activity, for instance, by competing with the mRNA target site for proteins that act as translational regulators. A second mechanism is by direct base-pairing with the mRNA target.”
According to Dr. Gottesman, sRNAs impact signal transduction by regulating outer membrane proteins of bacteria as well as expression of transcriptional regulators. “Additionally, sRNAs target other pathways—iron binding proteins, sugar transporters, quorum sensing, etc. As more and more are discovered and characterized, we also can use this information to examine bacterial genetics, for example, to determine natural signals that affect the sRNAs, which in turn affect the network of responses to the signals.”
Although this work is in its infancy as it relates to therapeutics, Dr. Gottesman predicts that “numerous advantages will surface as we learn more about these multitalented molecules. The identification of functional homologs present in other bacteria will provide a more global knowledge of how bacteria regulate themselves and of their role in pathogenesis.”
Master Switches of Inflammation
A family of proteases known as caspases was identified more than 15 years ago. Caspase-1 is the prototypical member of a subclass of caspases linked to cytokine maturation and has consequently been called inflammatory caspases.
New efforts to elucidate the molecular mechanisms that activate inflammatory caspases uncovered a family of intracellular adaptors, many of which are derived from the Nod-like family that mediates innate immunity. On receipt of an inflammatory stimulus, these adaptors assemble with caspase-1 into multiprotein complexes termed inflammasomes. The inflammasome triggers activation of inflammatory caspases and the generation of pro-inflammatory cytokines including interleukin-1 and -18.
Vishva Dixit, M.D., vp, Genentech (www.genentech.com), has been deciphering the workings of this system. His group identified multiple adaptor proteins that constitute the inflammasome apparatus. Cryopyrin is one such adapter and contains a so-called death domain fold motif, CARD, that interacts with a similar domain of caspase-1 and leads to the latter’s activation.
Now the plot thickens. Mutations in Cryopyrin are associated with several periodic fever syndromes such as familial cold autoinflammatory syndrome and neonatal-onset multisystem inflammatory disease, according to Dr. Dixit. He also examined Cryopyrin-deficient mice and found that Cryopryin was critical for activation of caspase-1.
“These and other studies are suggesting a link between the inflammatory and cell death signaling pathways,” notes Dr. Dixit. “Although some progress has been made in characterizing the inflammasomes, this is still an emerging field. Future studies will likely shed more light on the connection between various inflammasome pathways involved in human infection and inflammatory diseases. The ultimate goal of these studies will be to identify new functions for inflammasomes and/or inflammatory caspases that will hopefully reveal new therapeutic targets.”
Signal transduction encompasses a multitude of cross-talking networks that provide critical functions for all living cells. New components and pathways continue to be uncovered. Putting this all together, though, is no small feat. Nonetheless, many in the industry agree that for the future, signal transduction and mapping key players will continue to be a major focus aimed at better understanding disease processes and at developing targeted therapeutics.