Hot off the presses! Jun 29 Immunol Rev

The Jun 29 issue of the Immunol Rev is now up on Pubget (About Immunol Rev): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Glycoimmunology: ignore at your peril!
  - Immunol Rev 230(1):5-8 (2009)
  
• Mannose-binding lectin and innate immunity
  - Immunol Rev 230(1):9-21 (2009)
  Summary: Innate immunity is the earliest response to invading microbes and acts to contain infection in the first minutes to hours of challenge. Unlike adaptive immunity that relies upon clonal expansion of cells that emerge days after antigenic challenge, the innate immune response is immediate. Soluble mediators, including complement components and the mannose binding lectin (MBL) make an important contribution to innate immune protection and work along with epithelial barriers, cellular defenses such as phagocytosis, and pattern-recognition receptors that trigger pro-inflammatory signaling cascades. These four aspects of the innate immune system act in concert to protect from pathogen invasion. Our work has focused on understanding the protection provided by this complex defense system and, as discussed in this review, the particular contribution of soluble mediators such as MBL and phagocytic cells. Over the past two decades both human epidemiological data and mous! e models have indicated that MBL plays a critical role in innate immune protection against a number of pathogens. As demonstrated by our recent in vitro work, we show that MBL and the innate immune signaling triggered by the canonical pattern-recognition receptors (PRRs), the Toll-like receptors (TLRs), are linked by their spatial localization to the phagosome. These observations demonstrated a novel role for MBL as a TLR co-receptor and establishes a new paradigm for the role of opsonins, which we propose to function not only to increase microbial uptake but also to spatially coordinate, amplify, and synchronize innate immune defenses mechanism. In this review we discuss both the attributes of MBL that make it a unique soluble pattern recognition molecule and also highlight its broader role in coordinating innate immune activation.
• Endogenous ligands for C-type lectin receptors: the true regulators of immune homeostasis
  - Immunol Rev 230(1):22-37 (2009)
  Summary: C-type lectin receptors (CLRs) have long been known as pattern-recognition receptors implicated in the recognition of pathogens by the innate immune system. However, evidence is accumulating that many CLRs are also able to recognize endogenous 'self' ligands and that this recognition event often plays an important role in immune homeostasis. In the present review, we focus on the human and mouse CLRs for which endogenous ligands have been described. Special attention is given to the signaling events initiated upon recognition of the self ligand and the regulation of glycosylation as a switch modulating CLR recognition, and therefore, immune homeostasis.
• -glucan recognition by the innate immune system
  - Immunol Rev 230(1):38-50 (2009)
  Summary: β-glucans are recognized by the innate immune system. This recognition plays important roles in host defense and presents specific opportunities for clinical modulation of the host immune response. Neutrophils, macrophages, and dendritic cells among others express several receptors capable of recognizing β-glucan in its various forms. This review explores what is currently known about β-glucan recognition and how this recognition stimulates immune responses. Special emphasis is placed on Dectin-1, as we know the most about how this key β-glucan receptor translates recognition into intracellular signaling, stimulates cellular responses, and participates in orchestrating the adaptive immune response.
• Glycosyltransferase-programmed stereosubstitution (GPS) to create HCELL: engineering a roadmap for cell migration
  - Immunol Rev 230(1):51-74 (2009)
  Summary: During evolution of the vertebrate cardiovascular system, the vast endothelial surface area associated with branching vascular networks mandated the development of molecular processes to efficiently and specifically recruit circulating sentinel host defense cells and tissue repair cells at localized sites of inflammation/tissue injury. The forces engendered by high-velocity blood flow commensurately required the evolution of specialized cell surface molecules capable of mediating shear-resistant endothelial adhesive interactions, thus literally capturing relevant cells from the blood stream onto the target endothelial surface and permitting subsequent extravasation. The principal effectors of these shear-resistant binding interactions comprise a family of C-type lectins known as 'selectins' that bind discrete sialofucosylated glycans on their respective ligands. This review explains the 'intelligent design' of requisite reagents to convert native CD44 into the! sialofucosylated glycoform known as hematopoietic cell E-/L-selectin ligand (HCELL), the most potent E-selectin counter-receptor expressed on human cells, and will describe how ex vivo glycan engineering of HCELL expression may open the 'avenues' for the efficient vascular delivery of cells for a variety of cell therapies.
• PSGL-1 function in immunity and steady state homeostasis
  - Immunol Rev 230(1):75-96 (2009)
  Summary: The substantial importance of P-selectin glycoprotein ligand 1 (PSGL-1) in leukocyte trafficking has continued to emerge beyond its initial identification as a selectin ligand. PSGL-1 seemed to be a relatively simple molecule with an extracellular mucin domain extended as a flexible rod, teleologically consistent with its primary role in tethering leukocytes to endothelial selectins. The rolling interaction between leukocyte and endothelium mediated by this selectin-PSGL-1 interaction requires branched O-glycan extensions on specific PSGL-1 amino acid residues. In some cells, such as neutrophils, the glycosyltransferases involved in formation of the O-glycans are constitutively expressed, while in other cells, such as T cells, they are expressed only after appropriate activation. Thus, PSGL-1 supports leukocyte recruitment in both innate and adaptive arms of the immune response. A complex array of amino acids within the selectins engage multiple sugar residues! of the branched O-glycans on PSGL-1 and provide the molecular interactions responsible for the velcro-like catch bonds that support leukocyte rolling. Such binding of PSGL-1 can also induce signaling events that influence cell phenotype and function. Scrutiny of PSGL-1 has revealed a better understanding of how it performs as a selectin ligand and yielded unexpected insights that extend its scope from supporting leukocyte rolling in inflammatory settings to homeostasis including stem cell homing to the thymus and mature T-cell homing to secondary lymphoid organs. PSGL-1 has been found to bind homeostatic chemokines CCL19 and CCL21 and to support the chemotactic response to these chemokines. Surprisingly, the O-glycan modifications of PSGL-1 that support rolling mediated by selectins in inflammatory conditions interfere with PSGL-1 binding to homeostatic chemokines and thereby limit responsiveness to the chemotactic cues used in steady state T-cell traffic. The multi-level ! influence of PSGL-1 on cell traffic in both inflammatory and s! teady state settings is therefore substantially determined by the orchestrated addition of O-glycans. However, central as specific O-glycosylation is to PSGL-1 function, in vivo regulation of PSGL-1 glycosylation in T cells remains poorly understood. It is our purpose herein to review what is known, and not known, of PSGL-1 glycosylation and to update understanding of PSGL-1 functional scope.
• Glycosylation in immune cell trafficking
  - Immunol Rev 230(1):97-113 (2009)
  Summary: Leukocyte recruitment encompasses cell adhesion and activation steps that enable circulating leukocytes to roll, arrest, and firmly adhere on the endothelial surface before they extravasate into distinct tissue locations. This complex sequence of events relies on adhesive interactions between surface structures on leukocytes and endothelial cells and also on signals generated during the cell–cell contacts. Cell surface glycans play a crucial role in leukocyte recruitment. Several glycosyltransferases such as α1,3 fucosyltransferases, α2,3 sialyltransferases, core 2 N-acetylglucosaminlytransferases, β1,4 galactosyltransferases, and polypeptide N-acetylgalactosaminyltransferases have been implicated in the generation of functional selectin ligands that mediate leukocyte rolling via binding to selectins. Recent evidence also suggests a role of α2,3 sialylated carbohydrate determinants in triggering chemokine-mediated leukocyte arrest and influencing β1 int! egrin function. The recent discovery of galectin- and siglec-dependent processes further emphasizes the significant role of glycans for the successful recruitment of leukocytes into tissues. Advancing the knowledge on glycan function into appropriate pathology models is likely to suggest interesting new therapeutic strategies in the treatment of immune- and inflammation-mediated diseases.
• Galectin-3 regulates T-cell functions
  - Immunol Rev 230(1):114-127 (2009)
  Summary: Galectin-3 is absent in resting CD4+ and CD8+ T cells but is inducible by various stimuli. These include viral transactivating factors, T-cell receptor (TCR) ligation, and calcium ionophores. In addition, galectin-3 is constitutively expressed in human regulatory T cells and CD4+ memory T cells. Galectin-3 exerts extracellular functions because of its lectin activity and recognition of cell surface and extracellular matrix glycans. These include cell activation, adhesion, induction of apoptosis, and formation of lattices with cell surface glycoprotein receptors. Formation of lattices can result in restriction of receptor mobility and cause attenuation of receptor functions. Consistent with the presence of galectin-3 in intracellular locations, several functions have been described for this protein inside T cells. These include inhibition of apoptosis, promotion of cell growth, and regulation of TCR signal transduction. Studies of cell surface glycosylation hav! e led to convergence of glycobiology and galectin biology and provided new clues on how galectin-3 may participate in the regulation of cell surface receptor activities. The rapid expansion of the field of galectin research has positioned galectin-3 as a key regulator in T-cell functions.
• CD22 and Siglec-G: B-cell inhibitory receptors with distinct functions
  - Immunol Rev 230(1):128-143 (2009)
  Summary: Siglecs (sialic acid-binding immunoglobulin-like lectins) are sialic acid-binding proteins, which are expressed on many cell types of the immune system. B cells express two members of the Siglec family, CD22 (Siglec-2) and Siglec-G, both of which have been shown to inhibit B-cell signaling. CD22 recruits the tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) to immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and inhibits B-cell receptor (BCR)-induced Ca2+ signaling on normal B cells. CD22 interacts specifically with ligands carrying α2–6-linked sialic acids. Interaction with these ligands in cis regulates the association of CD22 with the BCR and thereby modulates the inhibitory function of CD22. Interaction of CD22 to ligands in trans can regulate both B-cell migration as well as the BCR signaling threshold. Siglec-G is a recently identified protein with an inhibitory function restricted to a B-cell subset, the B1 cells. Sig! lec-G inhibits Ca2+ signaling specifically in these cells. In addition, it controls the cellular expansion and antibody secretion of B1 cells. Thus, both Siglecs modulate BCR signaling on different B-cell populations in a mutually exclusive fashion.
• Conveying glycan information into T-cell homeostatic programs: a challenging role for galectin-1 in inflammatory and tumor microenvironments
  - Immunol Rev 230(1):144-159 (2009)
  Summary: The immune system has evolved sophisticated mechanisms composed of several checkpoints and fail-safe processes that enable it to orchestrate innate and adaptive immunity, while at the same time limiting aberrant or unfaithful T-cell function. These multiple regulatory pathways take place during the entire life-span of T cells including T-cell development, homing, activation, and differentiation. Galectin-1, an endogenous glycan-binding protein widely expressed at sites of inflammation and tumor growth, controls a diversity of immune cell processes, acting either extracellularly through specific binding to cell surface glycan structures or intracellularly through modulation of pathways that remain largely unexplored. In this review, we highlight the discoveries that have led to our current understanding of the role of galectin-1 in distinct immune cell process, particularly those associated with T-cell homeostasis. Also, we emphasize findings emerging from the ! study of experimental models of autoimmunity, chronic inflammation, fetomaternal tolerance, and tumor growth, which have provided fundamental insights into the critical role of galectin-1 and its specific saccharide ligands in immunoregulation. Challenges for the future will embrace the rational manipulation of galectin-1-glycan interactions both towards attenuating immune responses in autoimmune diseases, graft rejection, and recurrent fetal loss, while at the same overcoming immune tolerance in chronic infections and cancer.
• The regulation of inflammation by galectin-3
  - Immunol Rev 230(1):160-171 (2009)
  Summary: Galectin-3 is a β-galactoside-binding animal lectin of appro- ximately 30 kDa and is evolutionarily highly conserved. Galectin-3 is promiscuous, its localization within the tissue micro-environment may be extracellular, cytoplasmic, or nuclear, and it has a concentration-dependent ability to be monomeric or form oligomers. These properties impart great flexibility on galectin-3 as a specific regulator of many biological systems including inflammation. For example, in acute tissue damage galectin-3 is a key component in the host defense against microbes such as Streptococcus pneumoniae. However, if tissue injury becomes repetitive galectin-3 also appears to be intimately involved in the transition to chronic inflammation, facilitating the walling off of tissue injury with fibrogenesis and organ scarring. Therefore galectin-3 can be viewed as a regulatory molecule acting at various stages along the continuum from acute inflammation to chronic inflammation and t! issue fibrogenesis. In this review, we examine the role of galectin-3 in inflammation, and discuss the manipulation of galectin-3 expression as a potentially novel therapeutic strategy in the treatment of a broad range of inflammatory diseases.
• Galectins in innate immunity: dual functions of host soluble -galactoside-binding lectins as damage-associated molecular patterns (DAMPs) and as receptors for pathogen-associated molecular patterns (PAMPs)
  - Immunol Rev 230(1):172-187 (2009)
  Summary: The glycocalyx is a glycan layer found on the surfaces of host cells as well as microorganisms and enveloped virus. Its thickness may easily exceed 50 nm. The glycocalyx does not only serve as a physical protective barrier but also contains various structurally different glycans, which provide cell- or microorganism-specific 'glycoinformation'. This information is decoded by host glycan-binding proteins, lectins. The roles of lectins in innate immunity are well established, as exemplified by collectins, dectin-1, and dendritic cell (DC)-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). These mammalian lectins are synthesized in the secretory pathway and presented on the cell surface to bind to specific glycan 'epitopes'. As they recognize non-self glycans presented by microorganisms, they can be considered as receptors for pathogen-associated molecular patterns (PAMPs), i.e. pattern recognition receptors (PRRs). One notable exception ! is the galectin family. Galectins are synthesized and stored in the cytoplasm, but upon infection-initiated tissue damage and/or following prolonged infection, cytosolic galectins are either passively released by dying cells or actively secreted by inflammatory activated cells through a non-classical pathway, the 'leaderless' secretory pathway. Once exported, galectins act as PRR, as well as immunomodulators (or cytokine-like modulators) in the innate response to some infectious diseases. As galectins are dominantly found in the lesions where pathogen-initiated tissue damage signals appear, this lectin family is also considered as potential damage-associated molecular pattern (DAMP) candidates that orchestrate innate immune responses alongside the PAMP system.
• Carbohydrate specificity of the recognition of diverse glycolipids by natural killer T cells
  - Immunol Rev 230(1):188-200 (2009)
  Summary: Most T lymphocytes recognize peptide antigens bound to or presented by molecules encoded in the major histocompatibility complex (MHC). The CD1 family of antigen-presenting molecules is related to the MHC-encoded molecules, but CD1 proteins present lipid antigens, mostly glycolipids. Here we review T-lymphocyte recognition of glycolipids, with particular emphasis on the subpopulation known as natural killer T (NKT) cells. NKT cells influence many immune responses, they have a T-cell antigen receptor (TCR) that is restricted in diversity, and they share properties with cells of the innate immune system. NKT cells recognize antigens presented by CD1d with hexose sugars in α-linkage to lipids, although other, related antigens are known. The hydrophobic alkyl chains are buried in the CD1d groove, with the carbohydrate exposed for TCR recognition, together with the surface of the CD1d molecule. Therefore, understanding the biochemical basis for antigen recognition! by NKT cells requires an understanding of how the trimolecular complex of CD1d, glycolipid, and the TCR is formed, which is in part a problem of carbohydrate recognition by the TCR. Recent investigations from our laboratories as well as studies from other groups have provided important information on the structural basis for NKT-cell specificity.
• Regulation of Notch signaling during T- and B-cell development by O-fucose glycans
  - Immunol Rev 230(1):201-215 (2009)
  Summary: Notch signaling is required for the development of all T cells and marginal zone (MZ) B cells. Specific roles in T- and B-cell differentiation have been identified for different Notch receptors, the canonical Delta-like (Dll) and Jagged (Jag) Notch ligands, and downstream effectors of Notch signaling. Notch receptors and ligands are post-translationally modified by the addition of glycans to extracellular domain epidermal growth factor-like (EGF) repeats. The O-fucose glycans of Notch cell-autonomously modulate Notch–ligand interactions and the strength of Notch signaling. These glycans are initiated by protein O-fucosyltransferase 1 (Pofut1), and elongated by the transfer of N-acetylglucosamine (GlcNAc) to the fucose by β1,3GlcNAc-transferases termed lunatic, manic, or radical fringe. This review discusses T- and B-cell development from progenitors deficient in O-fucose glycans. The combined data show that Lfng and Mfng regulate T-cell development by enhan! cing the interactions of Notch1 in T-cell progenitors with Dll4 on thymic epithelial cells. In the spleen, Lfng and Mfng cooperate to modify Notch2 in MZ B progenitors, enhancing their interaction with Dll1 on endothelial cells and regulating MZ B-cell production. Removal of O-fucose affects Notch signaling in myelopoiesis and lymphopoiesis, and the O-fucose glycan in the Notch1 ligand-binding domain is required for optimal T-cell development.
• Immunological functions of hyaluronan and its receptors in the lymphatics
  - Immunol Rev 230(1):216-231 (2009)
  Summary: The lymphatic system is best known for draining interstitial fluid from the tissues and returning it to the blood circulation. However, the lymphatic system also provides the means for immune surveillance in the immune system, acting as conduits that convey soluble antigens and antigen-presenting cells from the tissues to the lymph nodes, where primary lymphocyte responses are generated. One macromolecule that potentially unites these two functions is the large extracellular matrix glycosaminoglycan hyaluronan (HA), a chemically simple copolymer of GlcNAc and GlcUA that fulfills a diversity of functions from danger signal to adhesive substratum, depending upon chain length and particular interaction with its many different binding proteins and a small but important group of receptors. The two most abundant of these receptors are CD44, which is expressed on leukocytes that traffic through the lymphatics, and LYVE-1, which is expressed almost exclusively on lymp! hatic endothelium. Curiously, much of the HA within the tissues is turned over and degraded in lymph nodes, by a poorly understood process that occurs in the medullary sinuses. Indeed there are several mysterious aspects to HA in the lymphatics. Here we cover some of these by reviewing recent findings in the biology of lymphatic endothelial cells and their possible roles in HA homeostasis together with fresh insights into the complex and enigmatic nature of LYVE-1, its regulation of HA binding by sialylation and self-association, and its potential function in leukocyte trafficking.
• T-cell growth, cell surface organization, and the galectin–glycoprotein lattice
  - Immunol Rev 230(1):232-246 (2009)
  Summary: Basal, activation, and arrest signaling in T cells determines survival, coordinates responses to pathogens, and, when dysregulated, leads to loss of self-tolerance and autoimmunity. At the T-cell surface, transmembrane glycoproteins interact with galectins via their N-glycans, forming a molecular lattice that regulates membrane localization, clustering, and endocytosis of surface receptors. Galectin–T-cell receptor (TCR) binding prevents ligand-independent TCR signaling via Lck by blocking spontaneous clustering and CD4-Lck recruitment to TCR, and in turn F-actin transfer of TCR/CD4-Lck complexes to membrane microdomains. Peptide–major histocompatibility complexes overcome galectin–TCR binding to promote TCR clustering and signaling by Lck at the immune synapse. Galectin also localizes the tyrosine phosphatase CD45 to microdomains and the immune synapse, suppressing basal and activation signaling by Lck. Following activation, membrane turnover increases ! and galectin binding to cytotoxic T-lymphocyte antigen-4 (CTLA-4) enhances surface expression by inhibiting endocytosis, thereby promoting growth arrest. Galectins bind surface glycoproteins in proportion to the branching and number of N-glycans per protein, the latter an encoded feature of protein sequence. N-glycan branching is conditional to the activity of Golgi N-acetylglucosaminyl transferases I, II, IV and V (Mgat1, 2, 4, and 5) and metabolic supply of their donor substrate UDP-GlcNAc. Genetic and metabolic control of N-glycan branching co-regulate homeostatic set-points for basal, activation, and arrest signaling in T cells and, when disturbed, result in T-cell hyperactivity and autoimmunity.
• Modulation of host immune responses by helminth glycans
  - Immunol Rev 230(1):247-257 (2009)
  Summary: Parasitic infections regulate/alter host immune responses. Among parasitic infections, helminth infection often leads to systemic immune suppression or anergy. Helminth infection or helminth extracts drive CD4+ T-helper (Th) cell responses towards Th2 type and activate antigen-presenting cells (APCs) such that these cells express an anti-inflammatory phenotype. Among the myriad molecules present on or secreted by helminth parasites, glycans have been shown to be key in inducing Th2-type and anti-inflammatory immune responses. The majority of studies on immune modulatory helminth glycans have focused on Lacto-N-fucopentaose III and LewisX. When presented as glycol-conjugates, with multiple copies of the sugars conjugated to a carrier molecule, these compounds activate APCs, inducing an alternative activation pattern, whose phenotypic profile is substantially different than that seen using pro-inflammatory activators such as lipopolysaccharide. Though the mechan! ism of APC activation by LNFPIII/LewisX glycoconjugates has not been fully elucidated, it involves C-type lectin ligation on the surface of APCs, with subsequent antagonism of Toll-like receptor signaling. In this article, we discuss the APC surface receptors known to play roles in LNFPIII/LewisX induced alternative activation of APCs. We also discuss what is currently known regarding downstream signaling pathways, closing with a discussion of future research directions for this field of investigation including the potential use of immune modulatory glycans as vaccine adjuvants and anti-inflammatory therapeutics.

Hot off the presses! Apr 21 Immunol Rev

The Apr 21 issue of the Immunol Rev is now up on Pubget (About Immunol Rev): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Mechanisms of costimulation
  Sharpe AH - Immunol Rev 229(1):5-11 (2009)
  
• CD28 and CTLA-4 coreceptor expression and signal transduction
  Rudd CE Taylor A Schneider H - Immunol Rev 229(1):12-26 (2009)
  Summary: T-cell activation is mediated by antigen-specific signals from the TCRζ/CD3 and CD4–CD8–p56lck complexes in combination with additional co-signals provided by coreceptors such as CD28, inducible costimulator (ICOS), cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death (PD-1), and others. CD28 and ICOS provide positive signals that promote and sustain T-cell responses, while CTLA-4 and PD-1 limit responses. The balance between stimulatory and inhibitory co-signals determines the ultimate nature of T-cell responses where response to foreign pathogen is achieved without excess inflammation and autoimmunity. In this review, we outline the current knowledge of the CD28 and CTLA-4 signaling mechanisms [involving phosphatidylinositol 3 kinase (PI3K), growth factor receptor-bound protein 2 (Grb2), Filamin A, protein kinase C θ (PKCθ), and phosphatases] that control T-cell immunity. We also present recent findings on T-cell receptor-interacting molecule (! TRIM) regulation of CTLA-4 surface expression, and a signaling pathway involving CTLA-4 activation of PI3K and protein kinase B (PKB)/AKT by which cell survival is ensured under conditions of anergy induction.
• Dynamic regulation of T-cell costimulation through TCR–CD28 microclusters
  Yokosuka T Saito T - Immunol Rev 229(1):27-40 (2009)
  Summary: T-cell activation requires contact between T cells and antigen-presenting cells (APCs) to bring T-cell receptors (TCRs) and major histocompatibility complex peptide (MHCp) together to the same complex. These complexes rearrange to form a concentric circular structure, the immunological synapse (IS). After the discovery of the IS, dynamic imaging technologies have revealed the details of the IS and provided important insights for T-cell activation. We have redefined a minimal unit of T-cell activation, the 'TCR microcluster', which recognizes MHCp, triggers an assembly of assorted molecules downstream of the TCR, and induces effective signaling from TCRs. The relationship between TCR signaling and costimulatory signaling was analyzed in terms of the TCR microcluster. CD28, the most valuable costimulatory receptor, forms TCR–CD28 microclusters in cooperation with TCRs, associates with protein kinase C θ, and effectively induces initial T-cell activation. Afte! r mature IS formation, CD28 microclusters accumulate at a particular subregion of the IS, where they continuously assemble with the kinases and not TCRs, and generate sustained T-cell signaling. We propose here a 'TCR–CD28 microcluster' model in which TCR and costimulatory microclusters are spatiotemporally formed at the IS and exhibit fine-tuning of T-cell responses by assembling with specific players downstream of the TCR and CD28.
• Regulating the regulators: costimulatory signals control the homeostasis and function of regulatory T cells
  Bour-Jordan H Bluestone JA - Immunol Rev 229(1):41-66 (2009)
  Summary: Costimulation is a concept that goes back to the early 1980s when Lafferty and others hypothesized that cell surface and soluble molecules must exist that are essential for initiating immune responses subsequent to antigen exposure. The explosion in this field of research ensued as over a dozen molecules have been identified to function as second signals following T-cell receptor engagement. By 1994, it seemed clear that the most prominent costimulatory pathway CD28 and functionally related costimulatory molecules, such as CD154, were the major drivers of a positive immune response. Then the immunology world turned upside down. CD28 knockout mice, which were, in most cases, immunodeficient, led to increased autoimmunity when bred into the non-obese diabetic background. Another CD28 family member, cytotoxic T-lymphocyte-associated protein 4, which was presumed to be a costimulatory molecule on activated T cells, turned out to be critical in downregulating immun! ity. These results, coupled with the vast suppressor cell literature which had been largely rebuked, suggested that the immune system was not poised for response but controlled in such a way that regulation was dominant. Over the last decade, we have learned that these costimulatory molecules play a key role in the now classical CD4+CD25+Foxp3+ regulatory T cells (Tregs) that provide critical control of unwanted autoimmune responses. In this review, we discuss the connections between costimulation and Tregs that have changed the costimulation paradigm.
• Negative regulators of T-cell activation: potential targets for therapeutic intervention in cancer, autoimmune disease, and persistent infections
  Pentcheva-Hoang T Corse E Allison JP - Immunol Rev 229(1):67-87 (2009)
  Summary: The generation of productive adaptive immune responses depends on the antigen-specific activation of T and B cells. The outcome of T-cell receptor engagement is influenced by signals from both positive and negative regulatory molecules that can either activate or inhibit T-cell function. CD28 and cytotoxic T-lymphocyte antigen-4 are the prototypical members of an immunoglobulin domain-containing protein family that play important roles in the control of T-cell responses against infection, cancer, and in autoimmune disease. Although the precise molecular details of their functions are still under active investigation, tumors and chronic pathogens seem to have exploited these pathways to achieve immune evasion. Furthermore, malfunction of the inhibitory arm of the immune response appears responsible for the development of multiple autoimmune pathologies. As a result, the negative regulators of T-cell activation have become attractive targets for therapeutic inte! rvention in cancer, chronic infection, and autoimmune disease. The application of findings from basic research has provided insight into the manipulation of these pathways in the clinic and offers promising strategies for the treatment of disease.
• Yin–Yang of costimulation: crucial controls of immune tolerance and function
  Nurieva RI Liu X Dong C - Immunol Rev 229(1):88-100 (2009)
  Summary: In addition to signals from the T-cell receptor complex, it has been recognized for many years that a 'second' signal, most notably from CD28, is also important in T-cell activation. In the recent years, many new members of CD28 family as well as the molecules that share structural homology to CD28 ligands CD80 and CD86 have been discovered. Interestingly, some of these proteins function to dampen T-cell activation and regulate the induction of T-cell tolerance. Therefore, positive and negative costimulation are the two sides of the coin to fine tune T-cell receptor signaling to determine the outcome of T-cell receptor engagement-tolerance versus function.
• The role of costimulation in antibody deficiencies: ICOS and common variable immunodeficiency
  Yong PF Salzer U Grimbacher B - Immunol Rev 229(1):101-113 (2009)
  Summary: The identification of mutations in the inducible costimulator (ICOS) gene in nine patients with common variable immunodeficiency (CVID) was a major breakthrough. CVID is a complex, highly heterogeneous primary immunodeficiency disease, and the discovery of these mutations revealed a molecular basis. ICOS belongs to the CD28 family of costimulatory molecules and is expressed exclusively on activated T cells. It has at least three critical functions: germinal center formation, isotype class switching, and the development of memory B cells. The discovery of human ICOS deficiency showed that a monogenic disorder could account for the full spectrum of manifestations seen in childhood and adulthood-onset CVID, including autoimmune, inflammatory, and malignant disease complications, as well as recurrent infections. Moreover, this discovery showed that a disorder which had previously been perceived as a B-cell disease might in fact have its genetic origin in human T c! ells. In this article, we review the role of ICOS in the mammalian immune system and human disease, as well as the discovery and characteristics of patients with ICOS deficiency. Finally, we also discuss how these 'human knockouts' have contributed to our understanding of ICOS functions and have suggested potential avenues for using therapeutic ICOS manipulation to treat other diseases.
• PD-1 signaling in primary T cells
  Riley JL - Immunol Rev 229(1):114-125 (2009)
  Summary: Programmed death-1 (PD-1) is a cell surface molecule that regulates the adaptive immune response. Engagement of PD-1 by its ligands PD-L1 or PD-L2 transduces a signal that inhibits T-cell proliferation, cytokine production, and cytolytic function. While a great deal is known concerning the biologic roles PD-1 plays in regulating the primary immune response and in T-cell exhaustion, comparatively little is known regarding how PD-1 ligation alters signaling pathways. PD-1 ligation is known to inhibit membrane-proximal T-cell signaling events, while ligation of the related inhibitory molecule cytotoxic T-lymphocyte antigen-4 appears to target more downstream signaling pathways. A major obstacle to an in-depth understanding of PD-1 signaling is the lack of physiologic models in which to study signal transduction. This review focuses on: (i) signaling pathways altered by PD-1 ligation, (ii) factors recruited upon PD-1 phosphorylation, and (iii) exploring the hypoth! esis that PD-1 ligation induces distinct signals during various stages of immune-cell differentiation. Lastly, we describe models to dissect the function of the PD-1 cytoplasmic tail using primary cells in the absence of agonist antibodies.
• Costimulatory and coinhibitory receptors in anti-tumor immunity
  Driessens G Kline J Gajewski TF - Immunol Rev 229(1):126-144 (2009)
  Summary: Despite the expression of antigens by tumor cells, spontaneous immune-mediated rejection of cancer seems to be a rare event. T-cell receptor engagement by peptide/major histocompatibility complexes constitutes the main signal for the activation of naive T cells but is not sufficient to initiate a productive generation and maintenance of effector cells. Full activation of T cells requires additional signals driven by costimulatory molecules present on activated antigen-presenting cells but rarely on tumors. Following the discovery of B7-1 (CD80), several other costimulatory molecules have been shown to contribute to T-cell activation and have relevance for improving anti-tumor immunity. Moreover, increasing the understanding of coinhibitory receptors has highlighted key additional pathways that can dominantly inhibit anti-tumor T-cell function. Improving positive costimulation, and interfering with negative regulation, continues to represent an attractive immun! otherapeutic approach for the treatment of cancer. This review focuses upon those pathways with the highest potential for clinical application in human cancer patients.
• Fine tuning the immune response through B7-H3 and B7-H4
  Yi KH Chen L - Immunol Rev 229(1):145-151 (2009)
  Summary: B7-H3 and B7-H4 belong to a new class of immune regulatory molecules, which primarily execute their functions in peripheral tissues to fine tune immune responses in target organs. In normal circumstances, while the mRNA for both molecules is broadly distributed, tight control at the post-transcriptional level is imposed. Under a pathogenic environment, such as inflammation and cancer, the control is often aberrant. Upon engaging their receptors, these molecules regulate the immune response in positive or negative ways depending on the expression and type of cells bearing the receptors. Thus, manipulation of the expression of these molecules and/or their receptors may represent a realistic opportunity to fine tune immune responses and to design new immunotherapeutic approaches.
• Molecular mechanism and function of CD40/CD40L engagement in the immune system
  Elgueta R Benson MJ de Vries VC Wasiuk A Guo Y Noelle RJ - Immunol Rev 229(1):152-172 (2009)
  Summary: During the generation of a successful adaptive immune response, multiple molecular signals are required. A primary signal is the binding of cognate antigen to an antigen receptor expressed by T and B lymphocytes. Multiple secondary signals involve the engagement of costimulatory molecules expressed by T and B lymphocytes with their respective ligands. Because of its essential role in immunity, one of the best characterized of the costimulatory molecules is the receptor CD40. This receptor, a member of the tumor necrosis factor receptor family, is expressed by B cells, professional antigen-presenting cells, as well as non-immune cells and tumors. CD40 binds its ligand CD40L, which is transiently expressed on T cells and other non-immune cells under inflammatory conditions. A wide spectrum of molecular and cellular processes is regulated by CD40 engagement including the initiation and progression of cellular and humoral adaptive immunity. In this review, we desc! ribe the downstream signaling pathways initiated by CD40 and overview how CD40 engagement or antagonism modulates humoral and cellular immunity. Lastly, we discuss the role of CD40 as a target in harnessing anti-tumor immunity. This review underscores the essential role CD40 plays in adaptive immunity.
• The significance of OX40 and OX40L to T-cell biology and immune disease
  Croft M So T Duan W Soroosh P - Immunol Rev 229(1):173-191 (2009)
  Summary: OX40 (CD134) and its binding partner, OX40L (CD252), are members of the tumor necrosis factor receptor/tumor necrosis factor superfamily and are expressed on activated CD4+ and CD8+ T cells as well as on a number of other lymphoid and non-lymphoid cells. Costimulatory signals from OX40 to a conventional T cell promote division and survival, augmenting the clonal expansion of effector and memory populations as they are being generated to antigen. OX40 additionally suppresses the differentiation and activity of T-regulatory cells, further amplifying this process. OX40 and OX40L also regulate cytokine production from T cells, antigen-presenting cells, natural killer cells, and natural killer T cells, and modulate cytokine receptor signaling. In line with these important modulatory functions, OX40–OX40L interactions have been found to play a central role in the development of multiple inflammatory and autoimmune diseases, making them attractive candidates for in! tervention in the clinic. Conversely, stimulating OX40 has shown it to be a candidate for therapeutic immunization strategies for cancer and infectious disease. This review provides a broad overview of the biology of OX40 including the intracellular signals from OX40 that impact many aspects of immune function and have promoted OX40 as one of the most prominent costimulatory molecules known to control T cells.
• Immune regulation by 4-1BB and 4-1BBL: complexities and challenges
  Wang C Lin GH McPherson AJ Watts TH - Immunol Rev 229(1):192-215 (2009)
  Summary: The tumor necrosis factor receptor family member 4-1BB plays a key role in the survival of activated and memory CD8+ T cells. Depending on the disease model, 4-1BB can participate at different stages and influence different aspects of the immune response, likely due to the differential expression of receptor and ligand relative to other costimulatory molecules. Studies comparing mild versus severe influenza infection of mice suggest that the immune system uses inducible receptors such as 4-1BB to prolong the immune response when pathogens take longer to clear. The expression of 4-1BB on diverse cell types, evidence for bidirectional as well as receptor-independent signaling by 4-1BBL, the unexpected hyperproliferation of 4-1BB-deficient T cells, and complex effects of agonistic anti-4-1BB therapy have revealed additional roles for the 4-1BB/4-1BBL receptor/ligand pair in the immune system. In this review, we discuss these diverse roles of 4-1BB and its ligand ! in the immune response, exploring possible mechanisms for the observed complexities and implications for therapeutic applications of 4-1BB/4-1BBL.
• Timing and tuning of CD27–CD70 interactions: the impact of signal strength in setting the balance between adaptive responses and immunopathology
  Nolte MA van Olffen RW van Gisbergen KP van Lier RA - Immunol Rev 229(1):216-231 (2009)
  Summary: After binding its natural ligand cluster of differentiation 70 (CD70), CD27, a tumor necrosis factor receptor (TNFR)-associated factor-binding member of the TNFR family, regulates cellular activity in subsets of T, B, and natural killer cells as well as hematopoietic progenitor cells. In normal immune responses, CD27 signaling appears to be limited predominantly by the restricted expression of CD70, which is only transiently expressed by cells of the immune system upon activation. Studies performed in CD27-deficient and CD70-transgenic mice have defined a non-redundant role of this receptor–ligand pair in shaping adaptive T-cell responses. Moreover, adjuvant properties of CD70 have been exploited for the design of anti-cancer vaccines. However, continuous CD27–CD70 interactions may cause immune dysregulation and immunopathology in conditions of chronic immune activation such as during persistent virus infection and autoimmune disease. We conclude that opti! mal tuning of CD27–CD70 interaction is crucial for the regulation of the cellular immune response. We provide a detailed comparison of costimulation through CD27 with its closely related family members 4-1BB (CD137), CD30, herpes virus entry mediator, OX40 (CD134), and glucocorticoid-induced TNFR family-related gene, and we argue that these receptors do not have a unique function per se but that rather the timing, context, and intensity of these costimulatory signals determine the functional consequence of their activity.
• Immunoregulation by tumor necrosis factor superfamily member LIGHT
  Wang Y Zhu M Miller M Fu YX - Immunol Rev 229(1):232-243 (2009)
  Summary: LIGHT (homologous to lymphotoxins, inducible expression, competes with herpesvirus glycoprotein D for herpesvirus entry mediator, a receptor expressed on T lymphocytes) is a member of the tumor necrosis factor superfamily that contributes to the regulation of immune responses. LIGHT can influence T-cell activation both directly and indirectly by engagement of various receptors that are expressed on T cells and on other types of cells. LIGHT, LIGHT receptors, and their related binding partners constitute a complicated molecular network in the regulation of various processes. The molecular cross-talk among LIGHT and its related molecules presents challenges and opportunities for us to study and to understand the full extent of the LIGHT function. Previous research from genetic and functional studies has demonstrated that dysregulation of LIGHT expression can result in the disturbance of T-cell homeostasis and activation, changing the ability of self-tolerance an! d of the control of infection. Meanwhile, blockade of LIGHT activity can ameliorate the severity of various T-cell-mediated diseases. These observations indicate the importance of LIGHT and its involvement in many physiological and pathological conditions. Understanding LIGHT interactions offers promising new therapeutic strategies that target LIGHT-engaged pathways to fight against cancer and various infectious diseases.
• The CD160, BTLA, LIGHT/HVEM pathway: a bidirectional switch regulating T-cell activation
  Cai G Freeman GJ - Immunol Rev 229(1):244-258 (2009)
  Summary: CD160 is a newly identified ligand for HVEM (herpes virus entry mediator). Previously identified HVEM ligands include BTLA (B- and T-lymphocyte attenuator), LIGHT (lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes) and LTα (lymphotoxin-α). The binding of LIGHT or LTα to HVEM delivers a costimulatory signal, whereas the binding of BTLA or CD160 to HVEM delivers a coinhibitory signal. Thus, HVEM is a bidirectional switch regulating T-cell activation in a costimulatory or coinhibitory fashion whose outcome depends on the ligand engaged. The cysteine-rich domain 1 (CRD1) of HVEM is essential for the binding of coinhibitory ligands CD160 and BTLA but not costimulatory ligand LIGHT. Deletion or blockade of HVEM CRD1 abolishes the binding of CD160 and BTLA, but not LIGHT, and converts HVEM to a dominant costimulatory molecule, possibly through the loss of negative ! signaling by CD160/BTLA. Therapies targeting the CRD1 of HVEM to block BTLA and CD160 binding are being developed to enhance immune responses and vaccination.
• The costimulatory role of TIM molecules
  Rodriguez-Manzanet R Dekruyff R Kuchroo VK Umetsu DT - Immunol Rev 229(1):259-270 (2009)
  Summary: The T-cell immunoglobulin domain and mucin domain (TIM) family, including TIM-1, TIM-2, TIM-3, and TIM-4, is a relatively newly described group of molecules with a conserved structure and important immunological functions, including T-cell activation, induction of T-cell apoptosis and T-cell tolerance, and the clearance of apoptotic cells. TIM-1 costimulates T-cell activation and enhances cytokine production. In humans, TIM-1 also serves as a susceptibility gene for allergy and asthma. TIM-3, expressed on T cells and dendritic cells, regulates T-cell apoptosis and immune tolerance. By contrast, TIM-4, which is expressed primarily on antigen-presenting cells and which is a receptor for phosphatidylserine, regulates T-cell activation and tolerance, in part by mediating the uptake and engulfment of apoptotic cells. The TIM molecules thus have surprisingly broad activities affecting multiple aspects of immunology.
• Costimulatory pathways in transplantation: challenges and new developments
  Li XC Rothstein DM Sayegh MH - Immunol Rev 229(1):271-293 (2009)
  Summary: T cells are central to graft rejection, and therefore preventing T cells from recognizing and destroying allografts remains an important area of transplant research. However, T cells are also required for transplant tolerance; a subset can enforce a state of tolerance by functioning as regulatory cells. As both rejection and regulation directed against alloantigens require T-cell activation, costimulatory molecules undoubtedly play an important role in regulating both processes and ultimately the fate of the allograft. However, costimulation involves an incredibly complex array of interactions that may act contemporaneously or at different times; these interactions can have additive or opposing effects on T-cell activation or differentiation. While some costimulatory molecules mediate activation of naive T cells or generation of memory T cells, others inhibit T-cell activation and promote anergy or apoptosis. Moreover, a given pathway can have diametrically di! fferent effects on T-effector cells and regulatory T cells (Tregs). Such a complexity presents both challenges and opportunities in targeting T-cell costimulatory pathways to promote transplant tolerance. In this review article, we provide a summary of recent advances in our understanding of T-cell costimulatory pathways in regulating different phases of the T-cell response in transplant models. We focus specifically on costimulatory molecules in the immunoglobulin (Ig) superfamily, tumor necrosis factor (TNF)/TNF receptor superfamily, and in the emerging T-cell Ig domain and mucin domain family (TIM), highlighting their unique and redundant roles in regulating the T-effector and Treg responses after transplantation. Finally, we summarize emerging approaches toward inducing tolerance by tipping the balance between cytopathic T-effector cells and protective Tregs by selectively targeting specific T-cell costimulatory pathways that are critically involved.
• Translating costimulation blockade to the clinic: lessons learned from three pathways
  Ford ML Larsen CP - Immunol Rev 229(1):294-306 (2009)
  Summary: As the recognition that costimulatory signals are critical for optimal T-cell activation, proliferation, and differentiation, there has been an explosion in the study of costimulatory molecules and their roles in enhancing anti-donor T-cell responses following transplantation. Here, we focus on the bench-to-beside translation of blocking agents designed to target three critical costimulatory pathways: the CD28/CD80/CD86 pathway, the CD154/CD40 pathway, and the lymphocyte function associated antigen-1/intercellular adhesion molecule pathway. While blockade of each of these pathways proved promising in inhibiting donor-reactive T-cell responses and promoting long-term graft survival in murine models of transplantation, the progression of development of therapeutic agents to block these pathways has each taken a slightly different course. Both logistical and biological pitfalls have accompanied the translation of blockers of all three pathways into clinically app! licable therapies, and the development of costimulatory blockade as a substitute for current standard-of-care calcineurin inhibitors has by no means reached completion. Collaboration between both the basic and clinical arenas will further propel the development of costimulation blockers currently in the pipeline, as well as of novel methods to target these critical pathways during transplantation.
• The clinical utility of inhibiting CD28-mediated costimulation
  Linsley PS Nadler SG - Immunol Rev 229(1):307-321 (2009)
  Summary: This volume covers many topics in the field of T-cell costimulation. The need for such a volume is testament to the growth of the field. From its beginning as a concept in the 1980s, we have now progressed to the point where many molecules now have functionally defined roles in T-cell costimulation. In addition, the field has progressed 'from bench to bedside'. Abatacept [cytotoxic T-lymphocyte antigen-4 (CTLA-4)-immunoglobulin (Ig) (CTLA-4-Ig)], an inhibitor of CD28-mediated T-cell costimulation, was approved for the treatment of moderate-to-severe rheumatoid arthritis in 2006 by the Food and Drug Administration and in 2007 by the European Medicines Agency. This chapter first presents a personal historical perspective on the early basic studies on the elucidation of the CD28/B7 T-cell costimulatory pathway and the discovery of CTLA-4-Ig. We next present an overview of studies of CTLA-4-Ig in preclinical animal studies. The material discussed in these first tw! o sections is selective rather than exhaustive; their purpose is to provide context for the final section, a summary of human clinical studies performed with abatacept.
• Autoimmunity risk alleles in costimulation pathways
  Maier LM Hafler DA - Immunol Rev 229(1):322-336 (2009)
  Summary: The basis for susceptibility to common autoimmune diseases is a complex interplay between multiple genetic and environmental risk factors. We have now entered a new generation of genetic study designs which has not only furthered our understanding of the individual mechanisms involved in the common human autoimmune diseases but also has pointed towards common pathways. In this review we focus on costimulatory mechanisms with the most convincing association results in large collections of patients and control subjects. These include the genes encoding cytotoxic T-lymphocyte antigen-4, CD58, CD40, inducible T-cell costimulator ligand, CD244, CD226, tumor necrosis factor (TNF) (ligand) superfamily member 4, TNF superfamily member 15, and programmed cell death 1. The unbiased genome-wide association scans suggest that indeed immune related genes underlie the pathogenesis of human autoimmune disease with common involvement of costimulatory pathways. The identificat! ion of allelic variants associated with disease risk followed by understanding their functional outcomes and affected pathways provides a rationale approach for drug design.
• Molecular mechanisms of T-cell receptor and costimulatory molecule ligation/blockade in autoimmune disease therapy
  Podojil JR Miller SD - Immunol Rev 229(1):337-355 (2009)
  Summary: Pro-inflammatory CD4+ T-cell-mediated autoimmune diseases, such as multiple sclerosis and type 1 diabetes, are hypothesized to be initiated and maintained by activated antigen-presenting cells presenting self antigen to self-reactive interferon-γ and interleukin-17-producing CD4+ T-helper (Th) type 1/Th17 cells. To date, the majority of Food and Drug Administration-approved therapies for autoimmune disease primarily focus on the global inhibition of immune inflammatory activity. The goal of ongoing research in this field is to develop both therapies that inhibit/eliminate activated autoreactive cells as well as antigen-specific treatments, which allow for the directed blockade of the deleterious effects of self-reactive immune cell function. According to the two-signal hypothesis, activation of a naive antigen-specific CD4+ T cell requires both stimulation of the T-cell receptor (TCR) (signal 1) and stimulation of costimulatory molecules (signal 2). There als! o exists a balance between pro-inflammatory and anti-inflammatory immune cell activity, which is regulated by the type and strength of the activating signal as well as the local cytokine milieu in which the naive CD4+ T cell is activated. To this end, the majority of ongoing research is focused on the delivery of suboptimal TCR stimulation in the absence of costimulatory molecule stimulation, or potential blockade of stimulatory accessory molecules. Therefore, the signaling pathways involved in the induction of CD4+ T-cell anergy, as apposed to activation, are topics of intense interest.
• Sequence, structure, function, immunity: structural genomics of costimulation
  Chattopadhyay K Lazar-Molnar E Yan Q Rubinstein R Zhan C Vigdorovich V Ramagopal UA Bonanno J Nathenson SG Almo SC - Immunol Rev 229(1):356-386 (2009)
  Summary: Costimulatory receptors and ligands trigger the signaling pathways that are responsible for modulating the strength, course, and duration of an immune response. High-resolution structures have provided invaluable mechanistic insights by defining the chemical and physical features underlying costimulatory receptor:ligand specificity, affinity, oligomeric state, and valency. Furthermore, these structures revealed general architectural features that are important for the integration of these interactions and their associated signaling pathways into overall cellular physiology. Recent technological advances in structural biology promise unprecedented opportunities for furthering our understanding of the structural features and mechanisms that govern costimulation. In this review, we highlight unique insights that have been revealed by structures of costimulatory molecules from the immunoglobulin and tumor necrosis factor superfamilies and describe a vision for fut! ure structural and mechanistic analysis of costimulation. This vision includes simple strategies for the selection of candidate molecules for structure determination and highlights the critical role of structure in the design of mutant costimulatory molecules for the generation of in vivo structure–function correlations in a mammalian model system. This integrated 'atoms-to-animals' paradigm provides a comprehensive approach for defining atomic and molecular mechanisms.
• Corrigendum
  - Immunol Rev 229(1):387-387 (2009)
  
• Erratum
  - Immunol Rev 229(1):388-388 (2009)
  

Hot off the presses! Jun 30 PLoS Biol

The Jun 30 issue of the PLoS Biol is now up on Pubget (About PLoS Biol): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Bad Taste Protects Fruit Flies from Eating a Toxic Amino Acid in Plants
  - PLoS Biol 7(6):e1000128 (2009)
  
• Engineering Sexless Seeds as a Path to High-Yield Crops
  - PLoS Biol 7(6):e1000118 (2009)
  
• Change for the Better: Phosphoregulation of Proteins Drives Evolution
  - PLoS Biol 7(6):e1000127 (2009)
  
• Zap1 Sticks It to Candida Biofilms
  - PLoS Biol 7(6):e1000117 (2009)
  
• The Hedgehog Signaling Pathway: Where Did It Come From?
  - PLoS Biol 7(6):e1000146 (2009)
  
• Mind the Gap!
  - PLoS Biol 7(6):e122 (2009)
  
• Parks and Tourism
  - PLoS Biol 7(6):e1000143 (2009)
  
• Light, Sleep, and Circadian Rhythms: Together Again
  Dijk DJ Archer SN - PLoS Biol 7(6):e1000145 (2009)
  
• The Finer Points of Filopodia
  - PLoS Biol 7(6):e1000142 (2009)
  
• A Global Perspective on Trends in Nature-Based Tourism
  - PLoS Biol 7(6):e1000144 (2009)
  Reports of rapid growth in nature-based tourism and recreation add significant weight to the economic case for biodiversity conservation but seem to contradict widely voiced concerns that people are becoming increasingly isolated from nature. This apparent paradox has been highlighted by a recent study showing that on a per capita basis, visits to natural areas in the United States and Japan have declined over the last two decades. These results have been cited as evidence of "a fundamental and pervasive shift away from nature-based recreation"—but how widespread is this phenomenon? We address this question by looking at temporal trends in visitor numbers at 280 protected areas (PAs) from 20 countries. This more geographically representative dataset shows that while PA visitation (whether measured as total or per capita visit numbers) is indeed declining in the United States and Japan, it is generally increasing elsewhere. Total visit numbers are growing in 15 of! the 20 countries for which we could get data, with the median national rate of change unrelated to the national rate of population growth but negatively associated with wealth. Reasons for this reversal of growth in the richest countries are difficult to pin down with existing data, but the pattern is mirrored by trends in international tourist arrivals as a whole and so may not necessarily be caused by disaffection with nature. Irrespective of the explanation, it is clear that despite important downturns in some countries, nature-related tourism is far from declining everywhere, and may still have considerable potential both to generate funds for conservation and to shape people's attitudes to the environment.
• Management Effectiveness of the World's Marine Fisheries
  Mora C Myers RA Coll M Libralato S Pitcher TJ Sumaila RU Zeller D Watson R Gaston KJ Worm B - PLoS Biol 7(6):e1000131 (2009)
  Ongoing declines in production of the world's fisheries may have serious ecological and socioeconomic consequences. As a result, a number of international efforts have sought to improve management and prevent overexploitation, while helping to maintain biodiversity and a sustainable food supply. Although these initiatives have received broad acceptance, the extent to which corrective measures have been implemented and are effective remains largely unknown. We used a survey approach, validated with empirical data, and enquiries to over 13,000 fisheries experts (of which 1,188 responded) to assess the current effectiveness of fisheries management regimes worldwide; for each of those regimes, we also calculated the probable sustainability of reported catches to determine how management affects fisheries sustainability. Our survey shows that 7% of all coastal states undergo rigorous scientific assessment for the generation of management policies, 1.4% also have a participa! tory and transparent processes to convert scientific recommendations into policy, and 0.95% also provide for robust mechanisms to ensure the compliance with regulations; none is also free of the effects of excess fishing capacity, subsidies, or access to foreign fishing. A comparison of fisheries management attributes with the sustainability of reported fisheries catches indicated that the conversion of scientific advice into policy, through a participatory and transparent process, is at the core of achieving fisheries sustainability, regardless of other attributes of the fisheries. Our results illustrate the great vulnerability of the world's fisheries and the urgent need to meet well-identified guidelines for sustainable management; they also provide a baseline against which future changes can be quantified.
• Allometry of the Duration of Flight Feather Molt in Birds
  Rohwer S Ricklefs RE Rohwer VG Copple MM - PLoS Biol 7(6):e1000132 (2009)
  We used allometric scaling to explain why the regular replacement of the primary flight feathers requires disproportionately more time for large birds. Primary growth rate scales to mass (M) as M0.171, whereas the summed length of the primaries scales almost twice as fast (M0.316). The ratio of length (mm) to rate (mm/day), which would be the time needed to replace all the primaries one by one, increases as the 0.14 power of mass (M0.316/M0.171 = M0.145), illustrating why the time required to replace the primaries is so important to life history evolution in large birds. Smaller birds generally replace all their flight feathers annually, but larger birds that fly while renewing their primaries often extend the primary molt over two or more years. Most flying birds exhibit one of three fundamentally different modes of primary replacement, and the size distributions of birds associated with these replacement modes suggest that birds that replace their primaries in a ! single wave of molt cannot approach the size of the largest flying birds without first transitioning to a more complex mode of primary replacement. Finally, we propose two models that could account for the 1/6 power allometry between feather growth rate and body mass, both based on a length-to-surface relationship that transforms the linear, cylindrical growing region responsible for producing feather tissue into an essentially two-dimensional structure. These allometric relationships offer a general explanation for flight feather replacement requiring disproportionately more time for large birds.
• Melanopsin as a Sleep Modulator: Circadian Gating of the Direct Effects of Light on Sleep and Altered Sleep Homeostasis in Opn4−/− Mice
  Tsai JW Hannibal J Hagiwara G Colas D Ruppert E Ruby NF Heller HC Franken P Bourgin P - PLoS Biol 7(6):e1000125 (2009)
  Light influences sleep and alertness either indirectly through a well-characterized circadian pathway or directly through yet poorly understood mechanisms. Melanopsin (Opn4) is a retinal photopigment crucial for conveying nonvisual light information to the brain. Through extensive characterization of sleep and the electrocorticogram (ECoG) in melanopsin-deficient (Opn4−/−) mice under various light–dark (LD) schedules, we assessed the role of melanopsin in mediating the effects of light on sleep and ECoG activity. In control mice, a light pulse given during the habitual dark period readily induced sleep, whereas a dark pulse given during the habitual light period induced waking with pronounced theta (7–10 Hz) and gamma (40–70 Hz) activity, the ECoG correlates of alertness. In contrast, light failed to induce sleep in Opn4−/− mice, and the dark-pulse-induced increase in theta and gamma activity was delayed. A 24-h recording under a LD 1-h∶1-h schedule rev! ealed that the failure to respond to light in Opn4−/− mice was restricted to the subjective dark period. Light induced c-Fos immunoreactivity in the suprachiasmatic nuclei (SCN) and in sleep-active ventrolateral preoptic (VLPO) neurons was importantly reduced in Opn4−/− mice, implicating both sleep-regulatory structures in the melanopsin-mediated effects of light. In addition to these acute light effects, Opn4−/− mice slept 1 h less during the 12-h light period of a LD 12∶12 schedule owing to a lengthening of waking bouts. Despite this reduction in sleep time, ECoG delta power, a marker of sleep need, was decreased in Opn4−/− mice for most of the (subjective) dark period. Delta power reached after a 6-h sleep deprivation was similarly reduced in Opn4−/− mice. In mice, melanopsin's contribution to the direct effects of light on sleep is limited to the dark or active period, suggesting that at this circadian phase, melanopsin compensates for circadian va! riations in the photo sensitivity of other light-encoding path! ways such as rod and cones. Our study, furthermore, demonstrates that lack of melanopsin alters sleep homeostasis. These findings call for a reevaluation of the role of light on mammalian physiology and behavior.
• Interaction between Attention and Bottom-Up Saliency Mediates the Representation of Foreground and Background in an Auditory Scene
  Elhilali M Xiang J Shamma SA Simon JZ - PLoS Biol 7(6):e1000129 (2009)
  The mechanism by which a complex auditory scene is parsed into coherent objects depends on poorly understood interactions between task-driven and stimulus-driven attentional processes. We illuminate these interactions in a simultaneous behavioral–neurophysiological study in which we manipulate participants' attention to different features of an auditory scene (with a regular target embedded in an irregular background). Our experimental results reveal that attention to the target, rather than to the background, correlates with a sustained (steady-state) increase in the measured neural target representation over the entire stimulus sequence, beyond auditory attention's well-known transient effects on onset responses. This enhancement, in both power and phase coherence, occurs exclusively at the frequency of the target rhythm, and is only revealed when contrasting two attentional states that direct participants' focus to different features of the acoustic stimulus. The ! enhancement originates in auditory cortex and covaries with both behavioral task and the bottom-up saliency of the target. Furthermore, the target's perceptual detectability improves over time, correlating strongly, within participants, with the target representation's neural buildup. These results have substantial implications for models of foreground/background organization, supporting a role of neuronal temporal synchrony in mediating auditory object formation.
• Attention Enhances the Retrieval and Stability of Visuospatial and Olfactory Representations in the Dorsal Hippocampus
  - PLoS Biol 7(6):e1000140 (2009)
  A key question in the analysis of hippocampal memory relates to how attention modulates the encoding and long-term retrieval of spatial and nonspatial representations in this region. To address this question, we recorded from single cells over a period of 5 days in the CA1 region of the dorsal hippocampus while mice acquired one of two goal-oriented tasks. These tasks required the animals to find a hidden food reward by attending to either the visuospatial environment or a particular odor presented in shifting spatial locations. Attention to the visuospatial environment increased the stability of visuospatial representations and phase locking to gamma oscillations—a form of neuronal synchronization thought to underlie the attentional mechanism necessary for processing task-relevant information. Attention to a spatially shifting olfactory cue compromised the stability of place fields and increased the stability of reward-associated odor representations, which were mos! t consistently retrieved during periods of sniffing and digging when animals were restricted to the cup locations. Together, these results suggest that attention selectively modulates the encoding and retrieval of hippocampal representations by enhancing physiological responses to task-relevant information.
• Long-Term Relationships between Synaptic Tenacity, Synaptic Remodeling, and Network Activity
  Minerbi A Kahana R Goldfeld L Kaufman M Marom S Ziv NE - PLoS Biol 7(6):e1000136 (2009)
  Synaptic plasticity is widely believed to constitute a key mechanism for modifying functional properties of neuronal networks. This belief implicitly implies, however, that synapses, when not driven to change their characteristics by physiologically relevant stimuli, will maintain these characteristics over time. How tenacious are synapses over behaviorally relevant time scales? To begin to address this question, we developed a system for continuously imaging the structural dynamics of individual synapses over many days, while recording network activity in the same preparations. We found that in spontaneously active networks, distributions of synaptic sizes were generally stable over days. Following individual synapses revealed, however, that the apparently static distributions were actually steady states of synapses exhibiting continual and extensive remodeling. In active networks, large synapses tended to grow smaller, whereas small synapses tended to grow larger, ma! inly during periods of particularly synchronous activity. Suppression of network activity only mildly affected the magnitude of synaptic remodeling, but dependence on synaptic size was lost, leading to the broadening of synaptic size distributions and increases in mean synaptic size. From the perspective of individual neurons, activity drove changes in the relative sizes of their excitatory inputs, but such changes continued, albeit at lower rates, even when network activity was blocked. Our findings show that activity strongly drives synaptic remodeling, but they also show that significant remodeling occurs spontaneously. Whereas such spontaneous remodeling provides an explanation for "synaptic homeostasis" like processes, it also raises significant questions concerning the reliability of individual synapses as sites for persistently modifying network function.
• Positional Cues in the Drosophila Nerve Cord: Semaphorins Pattern the Dorso-Ventral Axis
  Zlatic M Li F Strigini M Grueber W Bate M - PLoS Biol 7(6):e1000135 (2009)
  During the development of neural circuitry, neurons of different kinds establish specific synaptic connections by selecting appropriate targets from large numbers of alternatives. The range of alternative targets is reduced by well organised patterns of growth, termination, and branching that deliver the terminals of appropriate pre- and postsynaptic partners to restricted volumes of the developing nervous system. We use the axons of embryonic Drosophila sensory neurons as a model system in which to study the way in which growing neurons are guided to terminate in specific volumes of the developing nervous system. The mediolateral positions of sensory arbors are controlled by the response of Robo receptors to a Slit gradient. Here we make a genetic analysis of factors regulating position in the dorso-ventral axis. We find that dorso-ventral layers of neuropile contain different levels and combinations of Semaphorins. We demonstrate the existence of a central to dorsal ! and central to ventral gradient of Sema 2a, perpendicular to the Slit gradient. We show that a combination of Plexin A (Plex A) and Plexin B (Plex B) receptors specifies the ventral projection of sensory neurons by responding to high concentrations of Semaphorin 1a (Sema 1a) and Semaphorin 2a (Sema 2a). Together our findings support the idea that axons are delivered to particular regions of the neuropile by their responses to systems of positional cues in each dimension.
• Eps8 Regulates Axonal Filopodia in Hippocampal Neurons in Response to Brain-Derived Neurotrophic Factor (BDNF)
  - PLoS Biol 7(6):e1000138 (2009)
  The regulation of filopodia plays a crucial role during neuronal development and synaptogenesis. Axonal filopodia, which are known to originate presynaptic specializations, are regulated in response to neurotrophic factors. The structural components of filopodia are actin filaments, whose dynamics and organization are controlled by ensembles of actin-binding proteins. How neurotrophic factors regulate these latter proteins remains, however, poorly defined. Here, using a combination of mouse genetic, biochemical, and cell biological assays, we show that genetic removal of Eps8, an actin-binding and regulatory protein enriched in the growth cones and developing processes of neurons, significantly augments the number and density of vasodilator-stimulated phosphoprotein (VASP)-dependent axonal filopodia. The reintroduction of Eps8 wild type (WT), but not an Eps8 capping-defective mutant, into primary hippocampal neurons restored axonal filopodia to WT levels. We further sh! ow that the actin barbed-end capping activity of Eps8 is inhibited by brain-derived neurotrophic factor (BDNF) treatment through MAPK-dependent phosphorylation of Eps8 residues S624 and T628. Additionally, an Eps8 mutant, impaired in the MAPK target sites (S624A/T628A), displays increased association to actin-rich structures, is resistant to BDNF-mediated release from microfilaments, and inhibits BDNF-induced filopodia. The opposite is observed for a phosphomimetic Eps8 (S624E/T628E) mutant. Thus, collectively, our data identify Eps8 as a critical capping protein in the regulation of axonal filopodia and delineate a molecular pathway by which BDNF, through MAPK-dependent phosphorylation of Eps8, stimulates axonal filopodia formation, a process with crucial impacts on neuronal development and synapse formation.
• Regulation of Mammary Stem/Progenitor Cells by PTEN/Akt/β-Catenin Signaling
  Korkaya H Paulson A Charafe-Jauffret E Ginestier C Brown M Dutcher J Clouthier SG Wicha MS - PLoS Biol 7(6):e1000121 (2009)
  Recent evidence suggests that many malignancies, including breast cancer, are driven by a cellular subcomponent that displays stem cell-like properties. The protein phosphatase and tensin homolog (PTEN) is inactivated in a wide range of human cancers, an alteration that is associated with a poor prognosis. Because PTEN has been reported to play a role in the maintenance of embryonic and tissue-specific stem cells, we investigated the role of the PTEN/Akt pathway in the regulation of normal and malignant mammary stem/progenitor cell populations. We demonstrate that activation of this pathway, via PTEN knockdown, enriches for normal and malignant human mammary stem/progenitor cells in vitro and in vivo. Knockdown of PTEN in normal human mammary epithelial cells enriches for the stem/progenitor cell compartment, generating atypical hyperplastic lesions in humanized NOD/SCID mice. Akt-driven stem/progenitor cell enrichment is mediated by activation of the Wnt/β-catenin pa! thway through the phosphorylation of GSK3-β. In contrast to chemotherapy, the Akt inhibitor perifosine is able to target the tumorigenic cell population in breast tumor xenografts. These studies demonstrate an important role for the PTEN/PI3-K/Akt/β-catenin pathway in the regulation of normal and malignant stem/progenitor cell populations and suggest that agents that inhibit this pathway are able to effectively target tumorigenic breast cancer cells.
• Direct Binding of pRb/E2F-2 to GATA-1 Regulates Maturation and Terminal Cell Division during Erythropoiesis
  Kadri Z Shimizu R Ohneda O Maouche-Chretien L Gisselbrecht S Yamamoto M Romeo PH Leboulch P Chretien S - PLoS Biol 7(6):e1000123 (2009)
  How cell proliferation subsides as cells terminally differentiate remains largely enigmatic, although this phenomenon is central to the existence of multicellular organisms. Here, we show that GATA-1, the master transcription factor of erythropoiesis, forms a tricomplex with the retinoblastoma protein (pRb) and E2F-2. This interaction requires a LXCXE motif that is evolutionary conserved among GATA-1 orthologs yet absent from the other GATA family members. GATA-1/pRb/E2F-2 complex formation stalls cell proliferation and steers erythroid precursors towards terminal differentiation. This process can be disrupted in vitro by FOG-1, which displaces pRb/E2F-2 from GATA-1. A GATA-1 mutant unable to bind pRb fails to inhibit cell proliferation and results in mouse embryonic lethality by anemia. These findings clarify the previously suspected cell-autonomous role of pRb during erythropoiesis and may provide a unifying molecular mechanism for several mouse phenotypes and human ! diseases associated with GATA-1 mutations.
• Plant Insecticide L-Canavanine Repels Drosophila via the Insect Orphan GPCR DmX
  - PLoS Biol 7(6):e1000147 (2009)
  For all animals, the taste sense is crucial to detect and avoid ingesting toxic molecules. Many toxins are synthesized by plants as a defense mechanism against insect predation. One example of such a natural toxic molecule is l-canavanine, a nonprotein amino acid found in the seeds of many legumes. Whether and how insects are informed that some plants contain l-canavanine remains to be elucidated. In insects, the taste sense relies on gustatory receptors forming the gustatory receptor (Gr) family. Gr proteins display highly divergent sequences, suggesting that they could cover the entire range of tastants. However, one cannot exclude the possibility of evolutionarily independent taste receptors. Here, we show that l-canavanine is not only toxic, but is also a repellent for Drosophila. Using a pharmacogenetic approach, we find that flies sense food containing this poison by the DmX receptor. DmXR is an insect orphan G-protein–coupled receptor that has partially diverg! ed in its ligand binding pocket from the metabotropic glutamate receptor family. Blockade of DmXR function with an antagonist lowers the repulsive effect of l-canavanine. In addition, disruption of the DmXR encoding gene, called mangetout (mtt), suppresses the l-canavanine repellent effect. To avoid the ingestion of l-canavanine, DmXR expression is required in bitter-sensitive gustatory receptor neurons, where it triggers the premature retraction of the proboscis, thus leading to the end of food searching. These findings show that the DmX receptor, which does not belong to the Gr family, fulfills a gustatory function necessary to avoid eating a natural toxin.
• RIN4 Functions with Plasma Membrane H+-ATPases to Regulate Stomatal Apertures during Pathogen Attack
  - PLoS Biol 7(6):e1000139 (2009)
  Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4-mediated immune signal transduction, we purified components of the RIN4 protein complex. We identified six novel proteins that had not previously been implicated in RIN4 signaling, including the plasma membrane (PM) H+-ATPases AHA1 and/or AHA2. RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo. RIN4 overexpression and knockout lines exhibit differential PM H+-ATPase activity. PM H+-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H+-ATPase activity and, importantly, its stomata could not be re-opened by virulent Pseudomonas syringae. We also demonstrate t! hat RIN4 is expressed in guard cells, highlighting the importance of this cell type in innate immunity. These results indicate that the Arabidopsis protein RIN4 functions with the PM H+-ATPase to regulate stomatal apertures, inhibiting the entry of bacterial pathogens into the plant leaf during infection.
• Turning Meiosis into Mitosis
  d'Erfurth I Jolivet S Froger N Catrice O Novatchkova M Mercier R - PLoS Biol 7(6):e1000124 (2009)
  Apomixis, or asexual clonal reproduction through seeds, is of immense interest due to its potential application in agriculture. One key element of apomixis is apomeiosis, a deregulation of meiosis that results in a mitotic-like division. We isolated and characterised a novel gene that is directly involved in controlling entry into the second meiotic division. By combining a mutation in this gene with two others that affect key meiotic processes, we created a genotype called MiMe in which meiosis is totally replaced by mitosis. The obtained plants produce functional diploid gametes that are genetically identical to their mother. The creation of the MiMe genotype and apomeiosis phenotype is an important step towards understanding and engineering apomixis.
• Evolution of Phosphoregulation: Comparison of Phosphorylation Patterns across Yeast Species
  Beltrao P Trinidad JC Fiedler D Roguev A Lim WA Shokat KM Burlingame AL Krogan NJ - PLoS Biol 7(6):e1000134 (2009)
  The extent by which different cellular components generate phenotypic diversity is an ongoing debate in evolutionary biology that is yet to be addressed by quantitative comparative studies. We conducted an in vivo mass-spectrometry study of the phosphoproteomes of three yeast species (Saccharomyces cerevisiae, Candida albicans, and Schizosaccharomyces pombe) in order to quantify the evolutionary rate of change of phosphorylation. We estimate that kinase–substrate interactions change, at most, two orders of magnitude more slowly than transcription factor (TF)–promoter interactions. Our computational analysis linking kinases to putative substrates recapitulates known phosphoregulation events and provides putative evolutionary histories for the kinase regulation of protein complexes across 11 yeast species. To validate these trends, we used the E-MAP approach to analyze over 2,000 quantitative genetic interactions in S. cerevisiae and Sc. pombe, which demonstrated tha! t protein kinases, and to a greater extent TFs, show lower than average conservation of genetic interactions. We propose therefore that protein kinases are an important source of phenotypic diversity.
• Biofilm Matrix Regulation by Candida albicans Zap1
  Nobile CJ Nett JE Hernday AD Homann OR Deneault JS Nantel A Andes DR Johnson AD Mitchell AP - PLoS Biol 7(6):e1000133 (2009)
  A biofilm is a surface-associated population of microorganisms embedded in a matrix of extracellular polymeric substances. Biofilms are a major natural growth form of microorganisms and the cause of pervasive device-associated infection. This report focuses on the biofilm matrix of Candida albicans, the major fungal pathogen of humans. We report here that the C. albicans zinc-response transcription factor Zap1 is a negative regulator of a major matrix component, soluble β-1,3 glucan, in both in vitro and in vivo biofilm models. To understand the mechanistic relationship between Zap1 and matrix, we identified Zap1 target genes through expression profiling and full genome chromatin immunoprecipitation. On the basis of these results, we designed additional experiments showing that two glucoamylases, Gca1 and Gca2, have positive roles in matrix production and may function through hydrolysis of insoluble β-1,3 glucan chains. We also show that a group of alcohol dehydrogen! ases Adh5, Csh1, and Ifd6 have roles in matrix production: Adh5 acts positively, and Csh1 and Ifd6, negatively. We propose that these alcohol dehydrogenases generate quorum-sensing aryl and acyl alcohols that in turn govern multiple events in biofilm maturation. Our findings define a novel regulatory circuit and its mechanism of control of a process central to infection.
• Self-Organization of the Escherichia coli Chemotaxis Network Imaged with Super-Resolution Light Microscopy
  Greenfield D McEvoy AL Shroff H Crooks GE Wingreen NS Betzig E Liphardt J - PLoS Biol 7(6):e1000137 (2009)
  The Escherichia coli chemotaxis network is a model system for biological signal processing. In E. coli, transmembrane receptors responsible for signal transduction assemble into large clusters containing several thousand proteins. These sensory clusters have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here, we use photoactivated localization microscopy (PALM) to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW) with a precision of 15 nm. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters. Analysis of the relative cellular locations of 1! .1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.
• ATP and MO25α Regulate the Conformational State of the STRADα Pseudokinase and Activation of the LKB1 Tumour Suppressor
  Zeqiraj E Filippi BM Goldie S Navratilova I Boudeau J Deak M Alessi DR van Aalten DM - PLoS Biol 7(6):e1000126 (2009)
  Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADα in complex with MO25α. The structure reveals an intricate web of interactions between STRADα and MO25α involving the αC-helix of STRADα, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADα binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADα for MO25α, and conversely, binding of MO25α promotes interaction of STRADα with ATP. Mutagenesis studies reveal that association of STRADα with eit! her ATP or MO25α is essential for LKB1 activation. We conclude that ATP and MO25α cooperate to maintain STRADα in an "active" closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADα that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE) syndrome. We demonstrate this mutation destabilizes STRADα and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADα to activate LKB1 is dependent on a closed "active" conformation, aided by ATP and MO25α binding. Thus, the function of STRADα is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations.
• Crystal Structure of a Yeast Aquaporin at 1.15 Å Reveals a Novel Gating Mechanism
  Fischer G Kosinska-Eriksson U Aponte-Santamaría C Palmgren M Geijer C Hedfalk K Hohmann S de Groot BL Neutze R Lindkvist-Petersson K - PLoS Biol 7(6):e1000130 (2009)
  Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.