Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.Key words: cell adhesion, membrane protein, integrin, platelet, transmembrane complex, transmembrane signalingThe communication of biological signals across the plasma membrane is fundamental to cellular function. The ubiquitous family of integrin adhesion receptors exhibits the unusual ability to convey signals bi-directionally (outside-in and inside-out signaling), thereby controlling cell adhesion, migration and differentiation.^1–5 Integrins are Type I heterodimeric receptors that consist of large extracellular domains (>700 residues), single-pass transmembrane (TM) domains, and mostly short cytosolic tails (<70 residues). The activation state of heterodimeric integrins is correlated to the association state of the TM domains of their α and β subunits.^6–10 TM dissociation initiated from the outside results in the transmittal of a signal into the cell, whereas dissociation originating on the inside results in activation of the integrin to bind ligands such as extracellular matrix proteins. The elucidation of the role of the TM domains in integrin-mediated adhesion and signaling has been the subject of extensive research efforts, perhaps commencing with the demonstration that the highly conserved GFFKR sequence motif of α subunits (Fig. 1), which closely follows the first charged residue on the intracellular face, αIIb(K989), constrains the receptor to a default low affinity state.¹¹ Despite these efforts, an understanding of this sequence motif had not been reached until such time as the structure of the αIIb TM segment was determined.¹² In combination with the structure of the β3 TM segment¹³ and available mutagenesis data,^6,9,10,14,15 this has allowed the first correct prediction of the overall association of an integrin αβ TM complex.¹² The predicted association was subsequently confirmed by the αIIbβ3 complex structure determined in phospholipid bicelles,¹⁶ as well as by the report of a similar structure based on molecular modeling using disulfide-based structural constraints.¹⁷ In addition to the structures of the dissociated and associated αβ TM domains, their membrane embedding was defined^{12,13,16,18,19} and it was experimentally recognized that, in the context of the native receptor, the TM complex is stabilized by the inactive, resting ectodomain.¹⁶ These advances in integrin membrane structural biology are complemented by the recent structures of a resting integrin ectodomain and an activating talin/β cytosolic tail complex that overlap with the αβ TM complex,^20,21 allowing detailed insight into integrin bi-directional TM signaling.Open in a separate windowFigure 1Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues^{12,13,16,18,19} are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

Matrix metalloproteinase (MMP) and TGFβ1-stimulated cell migration in skin and cornea wound healing

Cell migration during wound healing is a complex process that involves the expression of a number of growth factors and cytokines. One of these factors, transforming growth factor-beta (TGFβ) controls many aspects of normal and pathological cell behavior. It induces migration of keratinocytes in wounded skin and of epithelial cells in damaged cornea. Furthermore, this TGFβ-induced cell migration is correlated with the production of components of the extracellular matrix (ECM) proteins and expression of integrins and matrix metalloproteinases (MMPs). MMP digests ECMs and integrins during cell migration, but the mechanisms regulating their expression and the consequences of their induction remain unclear. It has been suggested that MMP-14 activates cellular signaling processes involved in the expression of MMPs and other molecules associated with cell migration. Because of the manifold effects of MMP-14, it is important to understand the roles of MMP-14 not only the cleavage of ECM but also in the activation of signaling pathways.Key words: wound healing, migration, matrix metalloproteinase, transforming growth factor, skin, corneaWound healing is a well-ordered but complex process involving many cellular activities including inflammation, growth factor or cytokine secretion, cell migration and proliferation. Migration of skin keratinocytes and corneal epithelial cells requires the coordinated expression of various growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), small GTPases, and macrophage stimulating protein (reviewed in refs. ¹ and ²). The epithelial cells in turn regulate the expression of matrix metalloproteinases (MMPs), extracellular matrix (ECM) proteins and integrins during cell migration.^1,3,4 TGF-β is a well-known cytokine involved in processes such as cell growth inhibition, embryogenesis, morphogenesis, tumorigenesis, differentiation, wound healing, senescence and apoptosis (reviewed in refs. ⁵ and ⁶). It is also one of the most important cytokines responsible for promoting the migration of skin keratinocytes and corneal epithelial cells.^3,6,7TGFβ has two quite different effects on skin keratinocytes: it suppresses their multiplication and promotes their migration. The TGFβ-induced cell growth inhibition is usually mediated by Smad signaling, which upregulates expression of the cell cycle inhibitor p21^WAF1/Cip1 or p12^CDK2-AP1 in HaCaT skin keratinocyte cells and human primary foreskin keratinocytes.^8,9 Keratinocyte migration in wounded skin is associated with strong expression of TGFβ and MMPs,¹ and TGFβ stimulates the migration of manually scratched wounded HaCaT cells.¹⁰ TGFβ also induces cell migration and inhibits proliferation of injured corneal epithelial cells, whereas it stimulates proliferation of normal corneal epithelial cells via effects on the MAPK family and Smad signaling.^2,7 Indeed, skin keratinocytes and corneal epithelial cells display the same two physiological responses to TGFβ during wound healing; cell migration and growth inhibition. However as mentioned above, TGFβ has a different effect on normal cells. For example, it induces the epithelial to mesenchymal transition (EMT) of normal mammary cells and lens epithelial cells.^11,12 It also promotes the differentiation of corneal epithelial cells, and induces the fibrosis of various tissues.^2,6The MMPs are a family of structurally related zinc-dependent endopeptidases that are secreted into the extracellular environment.¹³ Members of the MMP family have been classified into gelatinases, stromelysins, collagenases and membrane type-MMPs (MT-MMPs) depending on their substrate specificity and structural properties. Like TGFβ, MMPs influence normal physiological processes including wound healing, tissue remodeling, angiogenesis and embryonic development, as well as pathological conditions such as rheumatoid arthritis, atherosclerosis and tumor invasion.^13,14The expression patterns of MMPs during skin and cornea wound healing are well studied. In rats, MMP-2, -3, -9, -11, -13 and -14 are expressed,¹⁵ and in mice, MMP-1, -2, -3, -9, -10 and -14 are expressed during skin wound healing.¹ MMP-1, -3, -7 and -12 are increased in corneal epithelial cells during Wnt 7a-induced rat cornea wound healing.¹⁶ Wound repair after excimer laser keratectomy is characterized by increased expression of MMP-1, -2, -3 and -9 in the rabbit cornea, and MMP-2, -9 in the rat cornea.^17,18 The expression of MMP-2 and -9 during skin keratinocyte and corneal epithelial cell migration has been the most thoroughly investigated, and it has been shown that their expression generally depends on the activity of MMP-14. MMP-14 (MT1-MMP) is constitutively anchored to the cell membrane; it activates other MMPs such as MMP-2, and also cleaves various types of ECM molecules including collagens, laminins, fibronectin as well as its ligands, the integrins.¹³ The latent forms of some cytokines are also cleaved and activated by MMP-14.¹⁹ Overexpression of MMP-14 protein was found to stimulate HT1080 human fibrosarcoma cell migration.²⁰ In contrast, the attenuation of MMP-14 expression using siRNA method decreased fibroblast invasiveness,²¹ angiogenesis of human microvascular endothelial cells,²² and human skin keratinocyte migration.¹⁰ The latter effect was shown to result from lowering MMP-9 expression. Other studies have shown that EGF has a critical role in MMP-9 expression during keratinocyte tumorigenesis and migration.^23,24 On the other hand, TGFβ modulates MMP-9 production through the Ras/MAPK pathway in transformed mouse keratinocytes and NFκB induces cell migration by binding to the MMP-9 promoter in human skin primary cultures.^25,26 Enhanced levels of pro-MMP-9 and active MMP-9 have also been noted in scratched corneal epithelia of diabetic rats.²⁷There is evidence that MMP-14 activates a number of intracellular signaling pathways including the MAPK family pathway, focal adhesion kinase (FAK), Src family, Rac and CD44, during cell migration and tumor invasion.^19,20,28 In COS-7 cells, ERK activation is stimulated by overexpression of MMP-14 and is essential for cell migration.²⁹ These observations all indicate that MMP-14 plays an important role in cell migration, not only by regulating the activity or expression of downstream MMPs but also by processing and activating migration-associated molecules such as integrins, ECMs and a variety of intracellular signaling pathays.³⁰Cell migration during wound healing is a remarkably complex phenomenon. TGFβ is just one small component of the overall process of wound healing and yet it triggers a multitude of reactions needed for cell migration. It is important to know what kinds of molecules are expressed when cell migration is initiated, but it is equally important to investigate the roles of these molecules and how their expression is regulated. Despite the availability of some information about how MMPs and signaling molecules can influence each other, much remains to be discovered in this area. It will be especially important to clarify how MMP-14 influences other signaling pathways since its role in cell migration is not restricted to digesting ECM molecules but also includes direct or indirect activation of cellular signaling pathways.     

The TRPA1 channel and oral hypoglycemic agents

Diabetes mellitus type 2 (DM2) results from the combination of insulin unresponsiveness in target tissues and the failure of pancreatic β cells to secrete enough insulin.¹ It is a highly prevalent chronic disease that is aggravated with time, leading to major complications, such as cardiovascular disease and peripheral and ocular neuropathies.² Interestingly, therapies to improve glucose homeostasis in diabetic patients usually involve the use of glibenclamide, an oral hypoglycemic drug that blocks ATP-sensitive K⁺ channels (K_ATP),^3,4 forcing β cells to release more insulin to overcome peripheral insulin resistance. However, sulfonylureas are ineffective for long-term treatments and ultimately result in the administration of insulin to control glucose levels.⁵ The mechanisms underlying β-cell failure to respond effectively with glibenclamide after long-term treatments still needs clarification. A recent study demonstrating that this drug activates TRPA1,⁶ a member of the Transient Receptor Potential (TRP) family of ion channels and a functional protein in insulin secreting cells,^7,8 has highlighted a possible role for TRPA1 as a potential mediator of sulfonylurea-induced toxicity.     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

Protein Misfolding and the Serpinopathies

The serpins are the largest superfamily of protease inhibitors. They are found in almost all branches of life including viruses, prokaryotes and eukaryotes. They inhibit their target protease by a unique mechanism that involves a large conformational transition and the translocation of the enzyme from the upper to the lower pole of the protein. This complex mechanism, and the involvement of serpins in important biological regulatory processes, makes them prone to mutation-related diseases. For example the polymerization of mutant α₁-antitrypsin leads to the accumulation of ordered polymers within the endoplasmic reticulum of hepatocytes in association with cirrhosis. An identical process in the neuron specific serpin, neuroserpin, results in the accumulation of polymers in neurons and the dementia FENIB. In both cases there is a clear correlation between the molecular instability, the rate of polymer formation and the severity of disease. A similar process underlies the hepatic retention and plasma deficiency of antithrombin, C1 inhibitor, α₁-antichymotrypsin and heparin co-factor II. The common mechanism of polymerization has allowed us to group these conditions together as a novel class of disease, the serpinopathies.Key Words: serpins, α1-antitrypsin, neuroserpin, polymerization, dementia, conformational disease, serpinopathiesSerpins (or serine protease inhibitors) are the largest family of protease inhibitors. They have been found in all major branches of life including viruses, prokaryotes and eukaryotes.^1–3 Despite their name there is increasing evidence that serpins can also inhibit other classes of proteases as demonstrated by the viral serpin CrmA and recently by a plant serpin, serpin1.^4,5 They can even play a non-inhibitory role in events as diverse as blood pressure regulation (angiotensinogen), chromatin condensation (MENT), tumor progression (maspin), protein folding (hsp47) and hormone transport (cortisol and thyroxine binding globulin).⁶One of the most important roles of serpins is the regulation of enzymes involved in proteolytic cascades. Among these serpins are α₁-antitrypsin, α₁-antichymotrypsin, C1 inhibitor, antithrombin and plasminogen activator inhibitor-1, which play an important role in the control of proteases involved in the inflammatory, complement, coagulation and fibrinolytic pathways, respectively.^1,3 The serpin superfamily is characterised by more than 30% homology with the archetypal serpin α₁-antitrypsin and conservation of tertiary structure.^7,8 Serpins adopt a metastable conformation composed in most cases of 9 α-helices, three β-sheet (A to C) and an exposed mobile reactive centre loop (RCL). This flexible RCL typically contains 20 residues that act as a pseudo substrate for the target protease (Fig. 1A).^9–15 After formation of a Michaelis complex^16,17 the enzyme cleaves the P1-P1′ bond of the serpin, releasing the P1'' residue and forming an ester bond between the protease and the serpin.^18,19 This is then followed by a dramatic conformational transition from a stressed to relaxed conformation with the enzyme being pulled from the upper to the lower pole of the serpin and the insertion of the reactive loop as an extra strand in β-sheet A.^20–25 As a consequence of this conformational change the thermal stability of the serpin is greatly enhanced. Whereas a typical serpin in its native state exhibits a midpoint of thermal denaturation of around 50–60°C, a cleaved serpin with its RCL fully incorporated into β-sheet A denatures at temperatures >120°C.^9,26,27 Another consequence is the inactivation of the enzyme, stabilised at the acyl-intermediate and unable to proceed further to deacylation of the complex.^24,28 This serpin-protease complex then binds to members of the lipoprotein receptor family and is cleared from the circulation.^29–31Open in a separate windowFigure 1Inhibition of neutrophil elastase by α₁-antitrypsin and the structural basis of polymerization. (A) After docking (left) the neutrophil elastase (grey) is inactivated by movement from the upper to the lower pole of the protein (right). This is associated with the insertion of the RCL (red) as an extra strand into β-sheet A (green). (B) The structure of α₁-antitrypsin is centred on β-sheet A (green) and the mobile reactive centre loop (red). Polymer formation results from the Z variant of α₁-antitrypsin (Glu342Lys at P17; indicated by arrow) or mutations in the shutter domain (blue circle) that open β-sheet A to favour partial loop insertion and the formation of an unstable intermediate (M*). The patent β-sheet A then accepts the loop of another molecule to form a dimer (D), which then extends into polymers (P). The individual molecules of α₁-antitrypsin within the polymer, although identical, are coloured red, yellow and blue for clarity. Figure reproduced with permission from Lomas et al.⁹⁷Despite the evolutionary advantage conferred upon serpins by the remarkable mobility of the native state, their complexity is also their weak point.^19,32 Mutations affecting the serpins can lead to a variety of diseases, resulting from either a gain or loss of function.^6,19 For example mutations can cause aberrant conformational transitions that result in the retention of the serpin within the cell of synthesis. This will lead to either protein overload and death of the cell in which the serpin is synthesised, or disease as a consequence of the resulting plasma deficiency. Such a mechanism underlies diseases as diverse as cirrhosis, thrombosis, angio-oedema, emphysema and dementia. We review here the common mechanism underlying these diseases that we have grouped together as the serpinopathies.^33–35 The aggregation and accumulation of conformationally destabilized proteins is an important feature of many neurodegenerative diseases, including Alzheimer''s and Parkinson''s disease and the spongiform encephalopathies. Indeed we have used the serpinopathies as a paradigm for these other ‘conformational diseases’.³⁶     

RALFs: Peptide regulators of plant growth

Peptide signaling regulates a variety of developmental processes and environmental responses in plants.^1–6 For example, the peptide systemin induces the systemic defense response in tomato⁷ and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants.^8,9 The CLAVATA3 peptide regulates meristem size¹⁰ and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae.^11,12 LURE peptides produced by synergid cells attract pollen tubes to the embryo sac.⁹ RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.Key words: peptide, growth factor, alkalinization     

Insights from Fc receptor biology: A route to improved antibody reagents

Fc receptors and their interaction with antibodies will be a major theme at the forthcoming FASEB Science Research Conference on Immunoreceptors to be held in Snowmass this July (details available at www.faseb.org/src/home.aspx, follow the tabs for Immunoreceptors). Since its inception in the mid 1980s, this meeting series has maintained a focus on Fc receptors, and this year’s meeting will be no exception.From a therapeutic viewpoint, there is much to be gained from a detailed understanding of the biology of effector molecules such as Fc receptors and complement. Indeed, knowledge of the interaction of IgG with such molecules has been central to the development of improved mAbs with altered functions and transformed half-lives, tailored for particular therapeutic applications. Examples include mAbs designed to maximise complement recruitment¹ or to enhance Fc receptor engagement and triggering of ADCC,^2-5 or conversely, variants engineered to be unable to engage complement⁶ or Fc receptors.⁷ Glycoengineering of IgG Fc offers an alternative means to modify effector function capabilities,⁸ while development of IgG mutants that display extended or altered serum half-lives has been driven through exhaustive analysis of the interaction with FcRn.^9,10Despite the appreciable advances that have been made in unravelling the various facets of Fc receptor biology, new information pertinent to mAb engineering continues to emerge. A flavour of some of these new advances will be given below. They span novel receptors and receptor roles, structure-function relationships, the molecular architecture of signaling complexes, the influence of the membrane lipid environment and scaffolding interactions, isotype considerations, through to technical innovations likely to inform the field.Remarkably, new receptors that have previously eluded characterization are now being described. These include the IgM receptor, which evidence indicates is a molecule also known as TOPO/Fas apoptotic inhibitory molecule 3 whose gene lies close to other known immunoglobulin receptors on chromosome 1,¹¹ and a receptor for IgD recently documented on basophils.¹² Moreover, we are seeing an appreciation of new roles for existing Fc receptors. An example is the demonstration in a transgenic study that human FcγRIIa can trigger active and passive anaphylaxis and airway inflammation. Moreover, human mast cells, monocytes and neutrophils were shown to produce anaphylactogenic mediators when FcγRIIA was engaged.¹³ Hence IgG may contribute to allergic and anaphylactic reactions in humans by engaging FcγRIIa.Exciting new structural information on Fc receptors and their ligands is emerging. An important example is the solving of the X-ray crystal structure for human FcγRI.¹⁴ While the structural information supports a ligand binding mode similar to those of FcγRII or FcγRIII, the FG-loop in domain 2 of FcγRI with its conserved one-residue deletion appears critical for high affinity IgG binding. A second example concerns the high responder/low responder (HR/LR) polymorphisms of FcγRIIa, which are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. New insights into these differences have been provided by the recent solving of the structure for the complex of the HR allele with IgG Fc.¹⁵ Third, understanding of the human IgE-FcεRI interaction has moved forward significantly through the solving of the X-ray crystal structure of the complex of FcεRI and the entire Fc region of IgE (comprising domains Cε2, Cε3 and Cε4).¹⁶ In a final example, the structural basis for the improved efficacy of nonfucosylated mAbs has been investigated.¹⁷ The X-ray crystal structure of the complex between nonfucosylated IgG Fc and a soluble form of FcγRIIIa carrying two N-linked glycans showed that one of two receptor glycans interacts with nonfucosylated Fc to stabilize the complex. It is proposed that when the Fc glycan is fucosylated this interaction is inhibited due to steric hindrance and, together with the negative effects of Fc fucosylation on the dynamics of the receptor binding site, this provides a rationale for the improved ADCC displayed by nonfucosylated IgG.A question of interest is precisely how Fc receptors bound to antibody ligands organize themselves within signaling complexes in the cell membrane. Some intriguing clues to this conundrum of molecular architecture are now surfacing. In mast cells, FcεRI molecules loaded with IgE form a synapse when presented with antigen that is mobile within a lipid bilayer, via coalescence into large cholesterol-rich clusters.¹⁸ Of particular relevance to the therapeutic setting, clustering of receptors into immune synapses is also seen with FcγR. For instance, during in vivo ADCC mediated by tumor-specific mAb, clustering of FcγR, actin and phosphotyrosines has been noted at contact zones between tumor cells and macrophages or neutrophils.¹⁹ The theme of the influence of the membrane lipid domain environment on Fc receptor function is taken up elsewhere. It has been shown, for example, that serine phosphorylation of FcγRI influences membrane mobility and function. The cytoplasmic tail of FcγRI interacts with protein 4.1G,²⁰ and it is proposed that this is mediated via a phosphoserine-dependent mechanism critical for localization of the receptor to lipid rafts.²¹ With regard to FcγRIIa, a major role for lipid rafts in the regulation of IgG binding to FcγRIIa has been revealed.²² Notably, exclusion of FcγRIIa from lipid raft membrane microdomains is able to suppress IgG binding in myeloid cells.Increased knowledge of the capabilities of Fc receptors specific for other antibody classes is opening up new options for therapy. For example, IgA antibodies may offer a highly useful and efficacious alternative approach of particular relevance to treatment at mucosal sites. Human IgA mAbs have been demonstrated to mediate efficient tumor cell killing^23,24 and to have the capability to control certain infectious diseases.^25,26 The detailed understanding of functional sites in IgA that has resulted from numerous mutagenesis studies,²⁷ coupled with improved ways to produce and isolate recombinant IgA mAbs²⁸ should facilitate developments toward therapeutics based on this immunoglobulin class. Similarly, recent studies indicate that IgE may serve as an alternative to the classic IgG backbone for therapeutic antibodies.²⁹Finally, technical innovations seem poised to further inform the field and advances are arriving or may be anticipated from techniques such as solution nuclear magnetic resonance (NMR) spectroscopy,³⁰ cryo-electron tomography,³¹ single particle tracking,³² and ultrasensitive force techniques such as adhesion frequency assays.^33,34Interest in Fc receptors continues unabated, and the contribution that the field can make to mAb development and optimisation is unquestionable. The FASEB SRC on Immunoreceptors will serve as a forum for discourse on the above issues and much more, providing invaluable information and networking opportunities for all those interested in ways to maximise the efficacy of mAbs and mAb-based reagents. Registration is open until 24 June 2012.     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

Prions: En route from structural models to structures

The prion hypothesis^1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.Key words: prion, NMR, solid-state NMR, MAS, structure, Ure2p, HET-sDespite the large interest in the basic mechanisms of fibril formation and prion propagation, little is known about the molecular structure of prions at atomic resolution and the mechanism of propagation. Prions with related properties to the ones responsible for mammalian diseases were also discovered in yeast and funghi^8,9 which provide convenient model system for their studies. Prion proteins described include the mammalian prion protein PrP, Ure2p,¹⁰ Rnq1p,¹¹ Sup35,¹² Swi1,¹³ and Cyc8,¹⁴ from bakers yeast (S. cervisiae) and HET-s from the filamentous fungus P. anserina. The soluble non-prion form of the proteins characterized in vitro is a globular protein with an unfolded, dynamically disordered N- or C-terminal tail.^15–18 In the prion form, the proteins form fibrillar aggregates, in which the tail adopts a different conformation and is thought to be the dominant structural element for fibril formation.Fibrills are difficult to structurally characterize at atomic resolution, as X-ray diffraction and liquid-state NMR cannot be applied because of the non-crystallinity and the mass of the fibrils. Solid-state NMR, in contrast, is nowadays well suited for this purpose. The size of the monomer, between 230 and 685 amino-acid residues for the prions of Figure 1, and therefore the number of resonances in the spectrum—that used to be large for structure determination—is now becoming tractable by this method.Open in a separate windowFigure 1Prions identified today and characterized as consisting of a prion domain (blue) and a globular domain (red).Prion proteins characterized so far were found to be usually constituted of two domains, namely the prion domain and the globular domain (see Fig. 1). This architecture suggests a divide-and-conquer approach to structure determination, in which the globular and prion domain are investigated separately. In isolation, the latter, or fragments thereof, were found to form β-sheet rich structures (e.g., Ure2p(1-89),^6,19 Rnq1p(153-405)²⁰ and HET-s(218-289)²¹). The same conclusion was reached by investigating Sup35(1-254).²² All these fragements have been characterized as amyloids, which we define in the sense that a significant part of the protein is involved in a cross-beta motif.²³ An atomic resolution structure however is available presently only for the HET-s prion domain, and was obtained from solid-state NMR²⁴ (vide infra). It contains mainly β-sheets, which form a triangular hydrophobic core. While this cross-beta structure can be classified as an amyloid, its triangular shape does deviate significantly from amyloid-like structures of smaller peptides.²³Regarding the globular domains, structures have been determined by x-ray crystallography (Ure2p^25,26 and HET-s²⁷), as well as NMR (mammal prions^15,28–30). All reveal a protein fold rich in α-helices, and dimeric structures for the Ure2 and HET-s proteins. The Ure2p fold resembles that of the β-class glutathione S-transferases (GST), but lacks GST activity.²⁵It is a central question for the structural biology of prions if the divide-and-conquer approach imposed by limitations in current structural approaches is valid. Or in other words: can the assembly of full-length prions simply be derived from the sum of the two folds observed for the isolated domains?     

A role for PCNA2 in translesion synthesis by Arabidopsis thaliana DNA polymerase η

Eukaryotic DNA polymerase η (Polη) confers ultraviolet (UV) resistance by catalyzing translesion synthesis (TLS) past UV photoproducts. Polη has been studied extensively in budding yeast and mammalian cells, where its interaction with monoubiquitylated proliferating cell nuclear antigen (PCNA) is necessary for its biological activity. Recently, in collaboration with other investigators, our laboratory demonstrated that Arabidopsis thaliana Polη is required for UV resistance in plants. Furthermore, the purified enzyme can perform TLS opposite a cyclobutane pyrimidine dimer and interacts with PCNA. Intriguingly, the biological activity of Polη in a heterologous yeast assay depends on co-expression with Arabidopsis PCNA2 and Polη sequences implicated in binding PCNA or ubiquitin. We suggest that interaction of Arabidopsis Polη with ubiquitylated PCNA2 is required for TLS past UV photoproducts by Polη.Key words: polymerase η, proliferating cell nuclear antigen, translesion synthesis, ubiquitin, Arabidopsis thaliana, ultraviolet radiationUltraviolet (UV)-induced pyrimidine dimers can block the progression of DNA replication forks potentially disrupting the replication machinery and resulting in cell death. For this reason, cells have evolved non-essential, low fidelity DNA polymerases (Pols) capable of copying damaged templates,^1,2 a process termed translesion DNA synthesis (TLS). In budding yeast, TLS past UV photoproducts is catalyzed by Polη and Polζ (composed of the Rev3 catalytic and Rev7 accessory subunits), but also involves the Rev1 protein in an as yet undetermined role linked to Polζ.^1,3,4 Yeast and human Polη replicates cyclobutane pyrimidine dimers (CPDs), in particular thymine-thymine (TT) CPDs, in a relatively error-free manner whereas Polζ is essential for UV mutagenesis implicating it in error-prone TLS.^1,4,5Both UV resistance due to TLS and the polymerases responsible have been well-studied in yeast and mammalian cells over the past decade. Only more recently has evidence emerged that TLS may also contribute to UV resistance in plants. Arabidopsis thaliana POLH, REV1, REV3 and REV7 encode homologs of Polη, Rev1, Rev3 and Rev7, respectively.^6–10 T-DNA insertions in POLH, REV1 or REV3 sensitise root growth to acute UV doses,^6–8,10 and these mutations, as well as inactivation of REV7, increase the sensitivity of whole plants to longer term UV treatment.^6,8 Interestingly, polh rev3 double mutants show an additive increase in UV sensitivity over that observed for polh and rev3 single mutants,^6,10 potentially pointing to differences in the UV photoproducts bypassed by the two polymerases. That the enhanced UV sensitivity of the mutants may reflect a TLS deficiency is suggested by the finding that purified Arabidopsis Polη catalyzes primer extension and TLS past a TT CPD in vitro.⁶For TLS to occur, Polη must gain access to the replication machinery arrested at a UV photoproduct. It does so in yeast and mammalian cells by interacting with proliferating cell nuclear antigen (PCNA), the eukaryotic sliding clamp required for processive DNA replication.^1,3,11, DNA damage or stalling of the replicative polymerase triggers monoubiquitylation of PCNA at lysine 164 by a complex of the E2 ubiquitin conjugase Rad6 and the E3 ubiquitin ligase Rad18.^1,3,11,12 This modification increases the affinity of Polη for PCNA, with which it interacts via a single PCNA interacting peptide (PIP) box and a single ubiquitin-binding zinc finger (UBZ) domain.^1,3In contrast to its yeast and mammalian counterparts, Polη from Arabidopsis and Oryza sativa (rice) has two PIP boxes and lacks a UBZ.^6,9,10 Instead the two polymerases each possess two ubiquitin-binding motifs (UBMs) similar to those present in the Arabidopsis Rev1 protein and a vertebrate TLS polymerase, Pol., for which there is no homolog in Arabidopsis.^6,13 Considerable differences in the sequences flanking the UBMs in Polη and Rev1 argue that Polη did not acquire its UBMs from Rev1, and so, although perhaps unique to plant Polη, their origin remains a mystery.The presence of PCNA- and ubiquitin-binding sequences in plant Polη hint that it may operate in TLS in a manner similar to that for Polη from yeast or mammalian cells. Indeed, three lines of evidence⁶ lead us to suggest that the Polη PIP boxes and UBMs likely function in binding ubiquitylated PCNA and this interaction is probably required for TLS past UV photoproducts by Arabidopsis Polη. First, Arabidopsis Polη interacts physically and in yeast two-hybrid assays with Arabidopsis PCNA1 and PCNA2. Second, expression in yeast of Arabidopsis cDNAs encoding Polη and PCNA2, but not PCNA1, fully complements the UV sensitivity conferred by elimination of yeast Polη. In vitro mutagenesis suggests the inability of Polη plus PCNA1 to restore UV resistance is due to a lysine at position 201 in PCNA1 but not PCNA2. In the three-dimensional structure of PCNA, amino acid 201 lies adjacent to lysine-164, the residue that is ubiquitylated in yeast and human PCNA. Thus, one possibility is that lysine-201 in PCNA1 prevents complementation of UV sensitivity by inhibiting ubiquitylation of lysine-164. Third, altering presumed critical residues in either of the two PIP boxes or UBM2 in Arabidopsis Polη also prevents restoration of UV resistance in Polη-deficient yeast cells.Several important parts of the puzzle remain to be solved. In particular, the ubiquitylation of plant PCNA has yet to be demonstrated, and the identity of the proteins that might monoubiquitylate plant PCNA is uncertain. Although Arabidopsis Rad6 homologs can ubiquitylate target proteins in vitro, there is no evidence that Arabidopsis PCNA1 or PCNA2 is a substrate, and Arabidopsis lacks a Rad18 homolog.^14,15 Finally, if PCNA is ubiquitylated in planta, does this occur at lysine-164 in response to DNA damage or replication fork stalling, is the interaction of Polη with PCNA stimulated by this modification, and is an enhanced interaction mediated by the Polη UBMs?