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Frontiers in Basic Immunology: Celebrating the NCI Investment in Basic Immunology Research
The Center of Excellence in Immunology (CEI) is one of five Centers of Excellence in the NCI intramural research program (IRP). The mission of the CEI is to foster discovery, development, and delivery of novel immunologic approaches for the prevention and treatment of cancer and viral diseases associated with cancer. The CEI is headed by a steering committee that oversees a faculty of approximately 100 principal investigators and staff scientists. Membership spans 20 different CCR laboratories, programs, and branches and also includes representatives from the Division of Cancer Biology and the Division of Cancer Epidemiology and Genetics. CEI faculty members include a variety of accomplished researchers, including seven members of the National Academy of Sciences, five of whom are also members of the academy’s Institute of Medicine. This multidisciplinary organization represents a means to create the critical mass of basic, clinical, and translational scientists necessary to rapidly define new areas of opportunity and accelerate high-impact research in immunology and the development of immune-based approaches to preventing and treating cancer and cancer-associated viral diseases. An important goal of the CEI is to facilitate information exchange and foster collaborations among NCI investigators, as well as other NIH scientists and the extramural research community. Consequently, the CEI has initiated an annual series of meetings on cancer-related immunology research. The first of these, “Translational Immunology Related to Cancer,” was held in 2005. On September 28–29 of this year, more than 700 scientists attended the second meeting in this series, “Frontiers in Basic Immunology.” This event represented a celebration of NCI’s long-standing support for basic immunology. Al Singer, MD, headed the scientific organizing committee, which included Jon Ashwell, MD, Larry Samelson, MD, Ron Gress, MD, Joost Oppenheim, MD, Scott Durum, PhD, Bill Farrar, PhD, and John Ortaldo, PhD. All of the speakers were outstanding scientists doing innovative research in basic immunology. A complete list of the presentations at the meeting can be seen at http://web.ncifcrf.gov/events/basicimmunology/program.asp and the talks can be viewed at http://videocast.nih.gov/PastEvents.asp?c=998. Basic research in the CEI includes work being performed in several internationally renowned laboratories dedicated to understanding the development and regulation of the cellular components of immunity. Collectively, studies by CEI faculty have led to the identification of novel molecules and molecular pathways important for the normal development and function of lymphoid cells. Some of these discoveries include activation-induced T-cell apoptosis; identification of the role of DNA damage in thymocyte and B-lymphocyte survival and transformation; critical advances in protein ubiquitination and its role in inflammation; mechanisms mediating costimulatory signals between the T-cell receptor (TCR) and CD28; and the means to visualize TCR-induced signaling in single, living cells. Members of the CEI are also leaders in the field of cytokine research. These powerful molecules are critical in the development of the immune system, as well as in host defense and tumor biology. Therefore, the identification of cytokines, defining their biological activities, and understanding their mechanisms of action are important components of the efforts of the CEI to reduce the burden of cancer. CEI investigators have contributed groundbreaking work in the discovery, characterization, and immunotherapeutic potential of numerous cytokines. For example, interleukin (IL)-1, IL-2, IL-3, IL-6, IL-8, IL-15, transforming growth factor-β (TGF-β), and monocyte chemotactic protein-1 (MCP-1) were all discovered/co-discovered by CEI scientists. Cytokines with rich clinical potential that are currently distinct areas of emphasis in the research of CEI faculty include IL-2, IL-7, IL-15, TGF-β, as well as several chemokines. The bench-to-bedside progression of IL-2 is an outstanding illustration of the integration of basic, translational, and clinical research by faculty of the CEI. IL-2 and components of its receptor were discovered by NCI researchers Frank Ruscetti, PhD, and Thomas Waldmann, MD, respectively. Work from the laboratory of Steve Rosenberg, MD, PhD, established the value of this cytokine in the treatment of advanced kidney cancer and melanoma, while Dr. Waldmann’s group demonstrated that antibodies blocking the α-chain of the IL-2 receptor are useful in treating T-cell leukemia, autoimmune disease, and graft-versus-host disease (GVHD). Another bench-to-bedside project in the CEI deals with IL-15. Dr. Waldmann and co-workers in the Metabolism Branch are currently engaged in efforts to bring this promising molecule to the clinic. His group co-discovered this cytokine and have made several landmark discoveries and other advances in the field. These include discovery of two of the three components of the IL-15 receptor; demonstration that IL-2 and IL-15 share receptor components; development of mice transgenic for IL-15; demonstration that IL-15 enhances effectiveness of therapeutic cancer vaccines and increases survival in some murine models of cancer; developing new treatments for graft rejection, rheumatoid arthritis, and multiple sclerosis using antibodies to a subunit of the IL-15 receptor; as well as development of inter-institute collaboration (NCI with the National Institute of Allergy and Infectious Diseases [NIAID]) for GMP (good manufacturing practices) production of IL-15 for clinical trials. To facilitate efforts to bring IL-15 to the clinic, the CEI co-sponsored a meeting with the NIH Cytokine Interest Group entitled “IL-15: Basic Research and Clinical Applications.” This meeting, chaired by Dr. Waldmann and Howard Young, PhD, was held in Lipsett Auditorium on October 30, 2006. The speakers included leading IL-15 researchers from the NIH and the extramural research community. The program can be found at http://web.ncifcrf.gov/events/IL15/program.asp. Other examples of CEI faculty translating advances in basic research into the clinic include development of a human papillomavirus (HPV) vaccine that could save up to 150,000 lives a year, a recombinant immunotoxin that has proved very effective against refractory hairy cell leukemia, and a cell-based therapy for the treatment of refractory metastatic melanoma that has resulted in improvement in 51% of patients involved in clinical trials. In addition, Dr. Rosenberg and colleagues recently demonstrated the potential for using a gene-therapy approach to re-educate a cancer patient’s immune system to treat refractory metastatic melanoma. Thus, work from scientists within the CEI has shown that strong basic research in immunology can fuel the translational and clinical advances that contribute to the NCI mission of reducing death and suffering from cancer and diseases such as AIDS. Those interested in learning more about the current activities and future plans for the CEI are encouraged to attend a steering committee meeting, held the third Monday of each month. More information on the CEI can be found at http://home.ccr.cancer.gov/coe/immunology or by contacting Diana Linnekin, PhD, at dlinnekin@ncifcrf.gov.
Integration of Cell Cycle Signals by Swe1/Wee1 in Budding Yeast
Recent studies in budding yeast revealed that these cells use the level of Swe1 (the Wee1 ortholog in this organism) to keep track of cell cycle progression and determine the timing of entry into mitosis. Swe1 is negatively regulated by a complex signaling network that relays upstream signals during assembly of the filamentous septin collar at the bud-neck, suggesting that Swe1 functions as a nodal point for integrating multiple signals that license passage into mitosis. During an unperturbed cell cycle, Swe1 localizes to the interphase nucleus and to the bud-neck at the late stages of the cell cycle. Swe1 begins to accumulate in S phase and becomes cumulatively phosphorylated as cells proceed through the cell cycle. These phosphorylation events tag Swe1 for ubiquitin-dependent degradation, which in turn obliterates Swe1’s inhibitory phosphorylation of the cyclin-dependent Cdc28 (Cdk1 homolog). Interestingly, disruption of the septin collar at the bud-neck, by using temperature-sensitive septin mutants, or loss of the septin collar–associated kinase Hsl1 (Nim1 homolog) or its adaptor Hsl7 delocalizes Swe1 from the bud-neck. The resulting hypo-phosphorylated and stabilized Swe1 imposes a G2 delay. These findings suggest that Swe1 degradation requires components associated with septin filaments and that regulation of Swe1 is critical for coordinating morphogenetic events with the commencement of mitosis. An initial search for the kinases responsible for Swe1 phosphorylation identified two bud-neck–associated kinases, Cla4 (PAK homolog) and Cdc5 (Polo kinase homolog), which appear to phosphorylate Swe1 in a temporally regulated manner (Sakchaisri K et al. Proc Natl Acad Sci U S A 101: 4124–9, 2004). In an early stage of the cell cycle, Cla4 phosphorylates Swe1 at the bud-neck (Figure 1), by associating tightly with septins, and promotes septin filament assembly. Cdc5 localizes to the bud-neck and hyper-phosphorylates Swe1 late in the cell cycle (Figure 1). Subsequent studies showed that mitotic cyclin (Clb2)–associated Cdc28 also functions in a positive feedback loop to downregulate Swe1. Further biochemical analyses revealed that Clb2-Cdc28 promotes Cdc5-dependent Swe1 phosphorylation and degradation by generating a phospho-recognition motif that enhances the interaction between Swe1 and the non-catalytic polo-box domain of Cdc5 (Asano S et al. EMBO J 24: 2194–204, 2005) (Figure 1). Septin filament assembly is dependent on Cla4 function and is required for localization of Hsl1 and Hsl7, which are, in turn, required to ensure proximity between Cdc5 and Swe1. Therefore, downregulation of Swe1 is achieved through the coordination of both septin-collar formation and stepwise phosphorylation of Swe1 by multiple kinases. Analogous interactions between the polo-box domain of mammalian polo-like kinase Plk1 and its phosphorylated substrates by Cdk1 have been previously reported (Lowery DM et al. Oncogene 24: 248–59, 2005). Thus, the concerted action of Cdc28/Cdk1 and Cdc5/Polo on their common substrates appears to be an evolutionarily conserved mechanism that is designed to effectively bring about various mitotic events. Figure 1. Model illustrating multi-kinase–dependent Swe1 phosphorylation and degradation. In an early stage of the cell cycle, Cla4 phosphorylates Swe1 at the bud-neck, but this phosphorylation is not sufficient to trigger Swe1 degradation. Later in the cell cycle, such as in G2, Swe1 is moderately phosphorylated by nascent Clb2-Cdc28 activity, as the level of Clb2 rises in the nucleus, and this promotes Cdc5-dependent Swe1 hyper-phosphorylation and, subsequently, Swe1 degradation. Once unleashed from negative regulation by Swe1, the Clb2-Cdc28 complex becomes fully active and induces mitosis. Multi-kinase phosphorylation of substrates has recently been recognized as a means of regulating diverse cellular functions. The Cla4/Cdc28/Cdc5–dependent Swe1 regulation described above highlights how yeast cells orchestrate various cellular and biochemical events to ensure completion of earlier events prior to mitotic entry. Moreover, stepwise Swe1 phosphorylation that impinges on ordered assemblies of septin filaments and the Hsl1-Hsl7 platform at the bud-neck provides a mechanism of integrating temporally distinct signals from preceding cell cycle events into one protein that plays a critical role in modulating the timing of mitosis. Whether the mechanism of Swe1 regulation is uniquely evolved in budding yeast to accurately coordinate morphogenesis throughout the budding process or if analogous mechanisms exist in other eukaryotic organisms are intriguing questions that remain to be answered.
CRACking Down on the Signals Leading to Directed Cell Migration
To establish whether CRAC is important for chemotaxis, we analyzed the chemotactic behavior of cells lacking CRAC (crac– ) and found them to be highly impaired. These results establish that CRAC has at least two functions: its previously described role in the activation of ACA and a critical role in regulating chemotaxis (Figure 1, part A). This observation raises an apparent paradox: How can a protein recruited at the front of cells to regulate chemotaxis also regulate the activity of ACA at the back of cells? To gain insight into this, we tested a series of C-terminal CRAC deletion mutants and found that expression of any of these mutants in crac– cells restores their ability to migrate directionally, but not the ability to activate ACA. These data show that distinct domains of CRAC independently regulate ACA and chemotaxis. To assess the role of PI3K signaling in CRAC function, we analyzed a series of CRAC PH domain–containing mutants. Deletion of the entire PH domain (ΔPH-CRAC) abolishes all of CRAC’s functions. A point mutant of CRAC (R42C-CRAC) that no longer binds the products of PI3K is not recruited to the leading edge of cells, fails to support chemotaxis, and confers only modest ACA activation, demonstrating that PI3K products play a critical role in all of CRAC’s functions. Overexpression of CRAC and various CRAC mutants in wild-type cells had no significant effect on chemotaxis, but they distinctively altered ACA activation, again supporting the notion that CRAC independently regulates these two processes. We conclude from these results that chemoattractant-mediated activation of PI3K is important for the CRAC-dependent regulation of both chemotaxis and ACA activation. Although we have yet to determine the exact mechanism by which CRAC controls these two processes, we envision that a CRAC-mediated activation event at the leading edge nucleates components of the chemotaxis machinery and these signals at the front are subsequently transmitted to ACA at the back to facilitate signal relay (Figure 1, part B). This relay of intracellular signals is not uncommon during chemotaxis, which requires the coordinated regulation of distinct events in the front and back of cells. Figure 1. A) Cytosolic regulator of adenylyl cyclase (CRAC) regulates chemotaxis and adenylyl cyclase (ACA) activation. Cartoon depicts the series of events leading to the chemoattractant-mediated activation of CRAC in Dictyostelium. B) Streaming during Dictyostelium chemotaxis. Left panel, a picture of Dictyostelium cells as they migrate by aligning in a head-to-tail fashion. Right panel, a model depicting the proposed localized secretion of the chemoattractant cyclic AMP (cAMP), which attracts cells to the back of the cell in front of them. The pictures represent the cellular distribution of CRAC and ACA during this process. PH, pleckstrin homology; GFP, green fluorescent protein; YFP, yellow fluorescent protein. Our study demonstrates that CRAC acts as a central regulator by integrating two interrelated aspects of chemotaxis: directed migration and signal relay. Indeed, signal relay greatly amplifies chemotaxis by enabling the recruitment of neighboring cells. As leukocytes are also known to secrete chemoattractants in response to chemoattractant stimulation, it is enticing to speculate that such a central regulator of chemotaxis also exists in higher eukaryotes.
Controlling Angiogenesis Through Thrombospondin-1 Regulation of Nitric Oxide Signaling
One of these is the potent endogenous angiogenesis inhibitor thrombospondin-1, a drug mimetic of which (ABT-510) is currently in phase II clinical trials for cancer treatment. Expression of thrombospondin-1 is commonly diminished or absent in pathology specimens from several major cancers, and studies in mice showed that approximately 0.1 nM levels of circulating thrombospondin-1 can limit tumor growth and angiogenesis. Yet, previous studies using cultured vascular endothelial cells required 1 to 10 nM concentrations of thrombospondin-1 to inhibit their growth or movement. Our collaborative studies have identified crosstalk between NO and thrombospondin-1 in endothelial cells that can explain this discrepancy. In the above, and in an accompanying paper (Ridnour LA et al. Proc Natl Acad Sci U S A 102: 13147–52, 2005), we describe novel mechanisms by which thrombospondin-1 inhibits angiogenesis stimulated by NO. In Isenberg et al., we show that low-dose NO increases the efficacy of thrombospondin-1 to inhibit endothelial cell growth, movement, and adhesion by a factor of 100 to 1000. We show that this activity is shared by antibodies that recognize the thrombospondin-1 receptor CD36 and by recombinant parts of the thrombospondin-1 molecule known to interact with this receptor on endothelial cells. This inhibition is mediated by way of thrombospondin-1 blocking the NO-mediated activation of soluble guanylyl cyclase (sGC). This enzyme mediates the synthesis of cyclic-GMP (cGMP) in cells, an important molecule in signaling pathways leading to tumor angiogenesis (Figure 1, part A). By blocking the NO-mediated activation of sGC, thrombospondin-1 also blocks the ability of an angiogenic molecule produced by many tumors, vascular endothelial growth factor, to stimulate cGMP production in endothelial cells. Finally, using transgenic mice, we show that levels of cGMP in vascular endothelial cells are elevated in the absence of endogenous thrombospondin-1 and are more sensitive to further elevation in response to NO donors. Therefore, endogenous levels of thrombospondin-1 clearly limit NO signaling through this pathway in vascular cells. In Ridnour et al., we show that NO and thrombospondin-1 form a feedback loop, whereby NO downregulates thrombospondin-1 and thrombospondin-1 inhibits NO-stimulated pathways that induce angiogenesis (Figure 1, part B). At low levels of NO (1 nM), thrombospondin-1 expression is blocked at the mRNA and protein levels, facilitating the pro-angiogenic activity of NO. This inhibition is reversed at higher levels of NO via induction of the phosphatase MKP-1, engaging inhibitory feedback to limit the angiogenic response to NO. Finally, at high NO levels such as would be produced by activated macrophages (1 μM), angiogenesis is directly inhibited by NO via phosphorylation of p53. This finely tuned feedback mechanism appears to be critical to control both wound healing and tumor angiogenesis. Figure 1. Cross talk between thrombospondin-1 and nitric oxide (NO) controls angiogenesis. A) Angiogenic signaling induced by vascular endothelial growth factor (VEGF) through its receptor activates Akt, which in turn phosphorylates and activates endothelial nitric oxide synthase (eNOS). The resulting NO binds to and activates soluble guanylyl cyclase (sGC), leading to accumulation of intracellular cyclic-GMP (cGMP). cGMP binds to and activates kinases (cGKs) and cGMP-gated channels (cNG) to stimulate endothelial cell responses required for angiogenesis. Thrombospondin-1 inhibits sGC activation and thereby prevents angiogenic signaling. B) Complementing the blocking of NO signaling by thrombospondin-1, low pro-angiogenic doses of NO suppress thrombospondin-1 expression to remove this inhibitor and facilitate angiogenesis. At higher levels of NO, this feedback is reversed by induction of additional signals that restore expression of inhibitory thrombospondin-1 as well as direct inhibition of angiogenesis by NO-derived reactive nitrogen species. Our ongoing studies suggest that both tumor growth and wound healing processes, such as those secondary to surgical treatment of solid tumors, can be controlled by peptides derived from thrombospondin-1 that target NO signaling mechanisms. Our data and those from other recent publications show that nitric oxide synthase (NOS) inhibitors can increase the efficacy of radiation and chemotherapy. Similarly, ABT-510, the drug mimetic of thrombospondin-1 mentioned earlier, binds to CD36 and enhances tumor responses to radiation and chemotherapy. The identification of this novel relationship between thrombospondin-1 and NO and the molecular mechanisms involved reveals new molecular targets for controlling angiogenic responses and could lead to novel treatment strategies combining these agents to increase cancer survival.
Protein-Protein Interactions in a Bottom-up Systems Biology Approach
Recently, we proposed that protein-protein binding sites have preferred organizations (Keskin O et al. Phys Biol 2: S24–S35, 2005; Keskin O et al. J Mol Biol 345: 1281–94, 2005). Binding sites can be divided into independent modules or “hot regions,” which consist of spatially adjacent residues that are tightly packed. They typically contain clusters of “hot spot” residues, that is, residues that have either been shown experimentally to contribute significantly (more than 2.0 Kcal/mol) to the binding free energy or found to be conserved in a multiple-structure (or sequence) alignment. Tight packing leads to high conservation because it is difficult to accommodate mutations without either steric clashes or “hole” formation. Such an organization suggests that hot spots located within a hot region contribute cooperatively to the stability of the complex. However, the contributions of separate, independent hot regions are additive. Accounting for this cooperativity has led to landmark experimental and computational investigations of the mechanisms and pathways of protein folding, seeking to answer the question of how the protein chain searches the immense number of possible nonlocal interactions to yield the hydrophobic core in the protein interior. To understand cooperativity, we need to think of the system as a cohesive unit. The overall behavior is the outcome of the properties of the entire system, rather than the sum of the properties of its components. Hence, we argue that the thermodynamic stability of the protein-protein complex is not a summation of the individual, independent contributions of the residues; rather, residues in spatial contact influence the stability of the association in a non-additive manner. When a residue is tightly packed with others, its substitution could affect the structure and interactions of its neighboring residues. If this residue and its neighbors contribute significantly to the stability of the complex, its mutation might affect that stability, not only through a change in its own interactions, but also through changes in the interactions of its neighbors. This would affect stability beyond the more direct effects of the altered interactions of the mutated residue. On the other hand, if the protein-protein interface were to consist of separate units, the impact of mutations in any of these would be independent, that is, non-cooperative. Our proposition was recently corroborated experimentally by using the TEM1–β-lactamase and β-lactamase inhibitor protein (BLIP) system (Reichmann D et al. Proc Natl Acad Sci U S A 102: 57–62, 2005). The authors have shown that within a module, mutations cause complex energetic and structural consequences; on the other hand, the structural and energetic consequences resulting from the removal of entire modules are small. A bottom-up, structure-based systems biology approach focuses on proteins. It aims to put the proteins together to create a structure-based map of interactions. The availability of maps should not be looked at only as a mere enumeration of static interactions. Rather, a structural map of the macromolecular interaction network may allow comprehension of the dynamics of the system. This is the essence of control mechanisms and of functional switches. Static maps of protein interactions tell us which proteins interact; however, they do not tell us under which conditions different paths dominate, how they dominate, or which intermolecular interactions overlap and which can coexist. To understand the dynamics of the system on the molecular level, we need to know not only which proteins interact but how they interact. This implies that we need to have at our disposal the structures of the proteins and the structures of their associations. Most highly connected proteins are among those that perform the same function for many of their partners. The kinase CDK1, the second most highly connected protein in yeast, phosphorylates more than 200 proteins in the progression of the cell cycle. Importin, the third most highly connected protein, plays a role in translocating proteins from the cytoplasm to the nucleus. These proteins have a single, promiscuous interface, creating an economical way of executing a multitude of functions through the various proteins needed for them. Interestingly, importin’s interface also contains (at least) two hot regions, as exemplified in its partners’ nuclear localization sequence.
Scientific Advisory CommitteeIf you have scientific news of interest to the CCR research community, please contact one of the scientific advisors (below) responsible for your areas of research.
CCR Frontiers in Science—StaffCenter for Cancer Research Robert H. Wiltrout, PhD, Director Deputy Directors Douglas R. Lowy, MD Editorial Staff Sue Fox, BA/BSW, Senior Editor * Palladian Partners, Inc. FOR INTERNAL USE ONLY |
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itosis comprises a series of biochemical steps and coordinated cellular events that ensure faithful partitioning of genetic and cytoplasmic components. Failure in these processes leads to genomic instability and cell death. In higher eukaryotes and fission yeast, entry into mitosis is induced by the activation of cyclin B–bound Cdk1 (Harper JW and Adams PD. Chem Rev 101: 2511–26, 2001), which is held in check by the protein kinase Wee1 (Russell P and Nurse P. Cell 49: 559–67, 1987). Wee1 is, in turn, negatively regulated via phosphorylation by Nim1/Cdr1 (Russell P and Nurse P. Cell 49: 569–76, 1987). These regulatory steps appear to have been conserved throughout evolution.
hemotaxis, the fundamental process by which cells detect and migrate up an external chemical gradient, is important in a wide range of biological processes, including wound repair, angiogenesis, and axon guidance, as well as for the survival of many lower organisms. The mechanisms that govern how cells sense and respond to chemoattractants are remarkably conserved from the social amoebae Dictyostelium discoideum to mammalian leukocytes, where chemotactic signals are transduced via G protein–coupled receptor signaling pathways. Chemotactic sensing mechanisms are extremely robust but are also highly sensitive. For example, Dictyostelium cells can accurately migrate toward chemoattractant sources that can vary by more than 4 orders of magnitude in concentration, and yet at the same time, these cells can accurately respond to very shallow gradients in which the front of the cell experiences a receptor occupancy that differs by only 1% of that at its back. This exquisite sensitivity requires cells to be able to integrate and accurately transduce signals in a spatiotemporal fashion. 
reventing angiogenesis—the recruitment of new blood vessels—has become a major focus for cancer treatment and prevention. Angiogenesis is tightly regulated by a balance between pro- and antiangiogenic factors. The gaseous redox molecule nitric oxide (NO) is known to play a crucial role in blood pressure control, but it was also recently found to promote angiogenesis at physiological levels. Although the latter activity of NO is beneficial in wound-healing responses, it may also promote angiogenesis in tumors. Several pro- and two antiangiogenic factors have been shown to modulate the endothelial form of an enzyme that generates NO. Now, ongoing studies by our group and that of David Wink, PhD (Radiation Biology Branch), reveal that additional molecular targets involved in redox signaling are convergent nodes for signaling by a variety of antiangiogenic agents. 
e propose a bottom-up, structure-based approach to systems biology. A bottom-up strategy starts from specific contacts between molecules and builds the system up to create a map. In contrast, a top-down strategy begins from the overall organization; it seeks the molecular components and the specific contacts that take place at each organizational level. A bottom-up approach, however, aims to predict which proteins will interact and how the interactions will take place. Within the systems biology framework, such predictions would assist in assigning function, providing clues to the system dynamics, and yielding information on system robustness.