New Paradigms in Ras Research Ras Is a Family of Genes Encoding Small Gtpases Involved in Cellular Signal Transduction

Consultation New paradigms in Ras research Ras is a family of genes encoding small GTPases involved in cellular signal transduction. If their signals are dysregulated, Ras proteins can cause cancer. Dr Sharon Campbell explains her lab’s research into a novel mechanism for regulation of Ras proteins by reactive free radical species Can you explain a little about the activated form and inactivated form of Ras background of your research into Ras, its don’t look all that much different from a aim and where the concept came from? structural standpoint. Although inhibitors that prevent Ras proteins from associating with We had been working on Ras for some the membrane initially appeared promising, time. In the mid-90s there was a group that it was later found that these inhibitors were published an observation that Ras could be not specifi c for Ras. More recent efforts have activated by nitric oxide (NO˙), which is a focused on how other proteins modulate Ras small, highly reactive, radical that turned and targeting those has become an area of out to be the molecule of the year a couple interest. The impact here is that now we’ve of years back because it regulates numerous found a whole host of regulatory factors that cellular processes. So, we followed up because MIKE DAVIS are distinct from protein modulatory factors they had observed this phenomenon in vitro that we can also consider as alternative as well as in cultured cells. We decided to strategies to target Ras. As the altered redox I think the complications are that these redox pursue those observations by testing whether environment in cancer cells may make Ras species are highly reactive and the chemistry we could reproduce it and if so, whether we activity particularly sensitive to redox agents, is complex. We initiated our early studies by could understand the mechanism by which approaches that target redox regulation of Ras trying to carefully manipulate the chemistry it occurred. So we did that and we were activity may provide new directions for drug so that we could really understand what was able to repeat the experiment, although the discovery efforts. In fact, fi ndings highlighted in going on with single proteins, because these mechanism that was proposed was different a recent Nature article1 indicate that a pathway reactions are fast and can generate multiple from what we found it to be. It turned out to be which links Ras to enzymes that generate nitric different species. Our longer term goal is a pretty complex electron transfer mechanism oxide, plays an important role in Ras-mediated to detect reactive GTPase intermediates involving radical chemistry rather than a cancer initiation and progession. and elucidate how redox agents regulate simpler NO-modifi cation. We then proposed GTPase-mediated processes in living cells. In that other redox agents could work by a similar So initially we started with Ras and also looked order to react in cells, reactive species need mechanism, and indeed found that Ras could at related proteins, and found that other to be generated and localized spatially with be modifi ed and regulated by a host of reactive members within this GTPase superfamily can the target GTPases. Moreover, the redox oxygen and nitrogen species. be regulated by redox agents. We have become chemistry is highly complex. As they can particularly interested in Rho GTPases, as they What in your opinion is the wider react with a multitude of biomolecules, there are disregulated in disease states such as cancer, impact of this research? may be multiple ways and types of reactive vascular disease and neurological disorders. species that regulate GTPase-mediated Once activated, Ras can regulate numerous Elucidating how reactive oxygen and nitrogen cellular processes. Furthermore, these radical and complex pathways that control cellular species regulate these Ras-related proteins may intermediates are very diffi cult to detect, so growth. However, the activation state of Ras aid in identifying novel therapeutic approaches I think where the fi eld needs to go is better was previously thought to be regulated solely that target these GTPases. While our initial detection methods of these highly reactive, by protein modulatory factors. What we funding supported our studies on Ras proteins, very fast intermediates. Elucidating how they found is that the regulation is probably more our most recent grant is focused on redox regulate GTPase activity and downsteam complicated than previously envisioned and regulation of Rho GTPases. pathways, will help us better understand how that reactive oxygen or nitrogen species can Have there been any major they modulate both physiological and patho- also regulate Ras proteins. From a therapeutic/ challenges so far? physiological processes. So those are the health standpoint, there have been numerous challenges and that’s really where we’ve put efforts to target Ras as they are the most We’ve contributed to the discovery of a new a lot of our energy, developing better tools prevalent proteins mutated in human cancer. paradigm in the fi eld and initiated research for detection and better understanding of the So far, therapeutic intervention efforts to efforts to investigate this further. It’s hard to complexities of the chemistry so that we can target Ras have not been successful. Part break through dogma and this is a new area of better interpret how these agents regulate of the problem with the protein is that the regulation so that has been a challenge. GTPase-mediated cellular processes. WWW.RESEARCHMEDIA.EU 25 ANALYSIS Alternative mechanisms for regulating Ras Activity Ras proteins have been a focus of intense investigation for close to 30 years, as they are frequently mutated in human cancer. Dr Sharon Campbell explains how the protein can be deregulated and the impact this has on pathways responsible for cell growth THEY ARE CLASSIFIED as GTPases because cultured cells, that activation of Ras can occur While a direct connection between free radical they possess high affi nity and specifi city for in the presence of nitric oxide (NO˙) radicals3. chemistry and Ras activity regulation existed at guanine diphosphate (GDP) and guanine Importantly, Lim et al. later performed studies the time, the mechanism by which activation triphosphate (GTP) and weak intrinsic GTP directly tying an enzyme that generates nitric occurred remained a mystery. Early studies by hydrolysis activity. Ras is considered to be oxide, endothelial Nitric Oxide Synthase Lander et al. indicated that redox regulation biologically active when bound to GTP and (eNOS), with Ras activation and tumor of Ras activity requires reaction of a cysteine inactive when GDP-bound. The guanine initiation, growth, and maintenance1. As tumors residue in Ras with either NO˙ gas or NO˙- nucleotide bound state and consequently the often possess an altered redox environment4, generating agents5. The Campbell laboratory activity of Ras proteins is modulated in the regulation of Ras proteins by redox agents worked with the Lander laboratory to identify cell by other proteins. Guanine Nucleotide may represent an important mechanism for the redox active cysteine in Ras as Cys-1186. Exchange Factors (GEFs) bind to Ras and regulation of Ras activity in cancer cells as well This cysteine is located in one of the conserved promote exchange of GDP for GTP, leading to as other cellular states (vascular disease, etc.) guanine nucleotide binding motifs. As cysteine an active state of the protein, whereas GTPase where the redox environment is altered. thiol-nitrosation has been shown to alter Activating Proteins (GAPs) have the opposite function and promote the inactive state by facilitating hydrolysis of GTP to GDP (Figure 1). Cycling between active GTP- and inactive Many of the mutations linking Ras to cancer alter guanine nucleotide GDP-bound states allows Ras to act as a cycling by stabilizing the active GTP bound form of Ras. This leads “molecular switch” that regulates numerous signaling pathways involved in cell growth, to chronic activation, or deregulation of the protein, which in turn differentiation, and apoptosis (programmed cell death)2. Abnormal cell growth and loss of deleteriously impacts pathways responsible for normal cell growth apoptosis are considered hallmarks of cancer development. Many of the mutations linking Ras to cancer alter guanine nucleotide cycling by stabilizing the active GTP bound form of Ras. GTP GDP This leads to chronic activation, or deregulation of the protein, which in turn deleteriously impacts pathways responsible for cell growth. While the central dogma of Ras regulation involves protein modulatory factors (e.g. .- activity regulation by GEFs and GAPs), an GEFs/NO2 /O2 alternative mechanism for regulation of Ras “Inactive” “Active” activity entails interactions between Ras Ras Ras and free radicals. Free radicals are generally short-lived, highly reactive molecules that are GDP GAPs GTP generated from both endogenous enzymes (i.e. Nitric Oxide Synthase, NADPH Oxidase) and external sources (UV radiation). Antioxidants, biomolecular scavengers, or other free radicals can typically eliminate radical species in the Pi body. However, sometimes free radicals react with DNA or proteins and alter their function. The effect of naturally occurring free radicals FIGURE 1. GUANINE NUCLEOTIDE CYCLING IN RAS IS MEDIATED BY PROTEIN MODULATORY on Ras was fi rst described by Lander et al. FACTORS (GEFS AND GAPS) AND REACTIVE OXYGEN AND NITROGEN SPECIES (NO2˙, O2˙-). when they demonstrated both in vitro and in 26 INTERNATIONAL INNOVATION ANALYSIS structure and activity of proteins, much like it combines with O2 in solution to produce Ras proteins comprise a superfamily of GTPases protein phosphorylation does, it was postulated nitrogen dioxide (NO2˙), a very powerful oxidizing that regulate a host of cellular processes that covalent modifi cation by nitric oxide may agent. NO2˙ radicals are believed to perform a including cellular growth control, motility alter Ras structure and activity.

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