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     

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?     

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.     

A Dominant-Negative Mutant of rab5 Inhibits Infection of Cells by Foot-and-Mouth Disease Virus: Implications for Virus Entry

Foot-and-mouth disease virus (FMDV) can use a number of different integrins (αvβ1, αvβ3, αvβ6, and αvβ8) as receptors to initiate infection. Infection mediated by αvβ6 is known to occur by clathrin-mediated endocytosis and is dependent on the acidic pH within endosomes. On internalization, virus is detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in late endosomal compartments. Due to the extreme sensitivity of FMDV to acidic pH, it is thought that EE can provide a pH low enough for infection to occur; however, definitive proof that infection takes place from within these compartments is still lacking. Here we have investigated the intracellular transport steps required for FMDV infection of IBRS-2 cells, which express αvβ8 as their FMDV receptor. These experiments confirmed that FMDV infection mediated by αvβ8 is also dependent on clathrin-mediate endocytosis and an acidic pH within endosomes. Also, the effect on FMDV infection of dominant-negative (DN) mutants of cellular rab proteins that regulate endosomal traffic was examined. Expression of DN rab5 reduced the number of FMDV-infected cells by 80%, while expression of DN rab4 or DN rab7 had virtually no effect on infection. Expression of DN rab11 inhibited infection by FMDV, albeit to a small extent (∼35%). These results demonstrate that FMDV infection takes place predominantly from within EE and does not require virus trafficking to the late endosomal compartments. However, our results suggest that infection may not be exclusive to EE and that a small amount of infection could occur from within PNRE.Foot-and-mouth disease virus (FMDV) is a member of the Aphthovirus genus of the family Picornaviridae and the etiological agent responsible for FMD, an economically important and severe vesicular condition of cloven-hoofed animals, including cattle, pigs, sheep, and goats (2). The mature virus particle consists of a positive-sense single-stranded RNA genome (vRNA) enclosed within a nonenveloped icosahedral capsid formed from 60 copies each of four virus-encoded proteins, VP1 to VP4 (1).The initial stage of FMDV infection is virus binding to cell surface integrins via a highly conserved RGD motif located on the GH loop of VP1. A number of different species of RGD-binding integrins (αvβ1, αvβ3, αvβ6, and αvβ8) have been reported to serve as receptors for FMDV (5, 23-26). Using pharmacological and dominant-negative (DN) inhibitors of specific endocytic pathways in combination with immunofluorescence confocal microscopy, the cell entry pathway used by FMDV has been determined for αvβ6-expressing cells (6, 36). These studies established that infection occurs by clathrin-mediated endocytosis and is dependent on the acidic pH within endosomes, which serves as the trigger for capsid disassembly and translocation of the vRNA across the endosomal membrane into the cytosol. Internalized virus was detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in late endosomes (LE) or lysosomes (Lys) (the late endosomal compartments). Due to the extreme sensitivity of FMDV to acidic pH (15), it is thought that EE can provide a pH low enough for virus disassembly to occur; however, definitive proof that infection takes place from within EE is still lacking. For example, the possibility cannot be excluded that a productive infection requires virus transport to late endosomal compartments, where, following capsid disassembly and viral genome transfer into the cytosol, the capsid proteins are rapidly degraded.rab proteins control multiple membrane trafficking events in the cell. They are members of the ras superfamily of small GTP-binding proteins and cycle between active GTP- and inactive GDP-bound states (22, 38, 39, 47, 50). Conversion between these states is regulated by guanine nucleotide exchange factors, which stimulate the binding of GTP, and GTPase-activating proteins that which accelerate GTP hydrolysis. Activated rab proteins are recruited onto membrane-bounded compartments where they regulate many steps of vesicle trafficking, including vesicle budding, movement, tethering, and fusion (35, 61). Each rab is recruited to a specific compartment and functions through interactions with specific effectors that mediate the downstream rab-associated functions (39). In mammalian cells, at least 12 rab proteins that regulate trafficking through the endosomal pathway have been identified (27). Of these, rab4, rab5, rab7, and rab11 play major roles in endocytic vesicle trafficking. rab5 is present on EE and regulates transport of incoming endocytic vesicles from the plasma membrane (PM) to EE and homotypic EE fusion events (3, 8, 10, 20, 30, 44, 52). Both rab4 and rab11 are regulators of receptor recycling from EE back to the PM (34); rab4 is localized primarily to EE and regulates rapid recycling directly back to the PM (16, 45, 48, 51, 56), and rab11 is localized primarily to the PNRE and regulates a slower recycling pathway through these compartments (21, 43, 54, 60). In addition rab11 also regulates membrane traffic from endocytic recycling compartments to the trans-Golgi network (55). rab7 is located primarily on LE and regulates traffic from EE to LE and between LE and Lys (7, 9, 18, 32, 40, 58, 59). The unique targeting of rab proteins to distinct cellular compartments and their specificity as regulators of vesicular trafficking has made them important tools for studying endocytosis. For example, expression of DN or constitutively active mutants of rab proteins that regulate endosomal traffic has been used to identify the intracellular transport steps that are required for infection by a number of different viruses (13, 14, 28, 31, 41, 42, 49, 53, 57, 59).Here we have investigated the intracellular transport steps required for FMDV infection using porcine IBRS-2 cells, which are derived from a natural host of FMDV. IBRS-2 cells use αvβ8, and not αvβ6, as the major FMDV receptor (11). Our initial experiments confirmed that FMDV infection mediated by αvβ8 is dependent on clathrin-mediated endocytosis and on an acidic pH within endosomes. The effect on FMDV infection within IBRS-2 cells of DN mutants of cellular rab proteins that regulate endosomal traffic was examined. These experiments show that rab5 is needed for FMDV infection, as expression of DN rab5 reduced the number of FMDV-infected cells by ∼80%. In contrast, expression of either DN rab4 or DN rab7 had virtually no effect on infection. Expression of DN rab11 inhibited infection by FMDV, albeit to a small extent (∼35%). These results demonstrate that FMDV infection takes place predominantly from within EE and does not require virus trafficking to the late endosomal compartments. However, our results suggest that infection may not be exclusive to EE and that a small amount of infection could occur from within PNRE.     

In Vivo Retargeting of Adenovirus Type 5 to αvβ6 Integrin Results in Reduced Hepatotoxicity and Improved Tumor Uptake following Systemic Delivery

A key impediment to successful cancer therapy with adenoviral vectors is the inefficient transduction of malignant tissue in vivo. Compounding this problem is the lack of cancer-specific targets, coupled with a shortage of corresponding high-efficiency ligands, permitting selective retargeting. The epithelial cell-specific integrin αvβ6 represents an attractive target for directed therapy since it is generally not expressed on normal epithelium but is upregulated in numerous carcinomas, where it plays a role in tumor progression. We previously have characterized a high-affinity, αvβ6-selective peptide (A20FMDV2) derived from VP1 of foot-and-mouth disease virus. We generated recombinant adenovirus type 5 (Ad5) fiber knob, incorporating A20FMDV2 in the HI loop, for which we validated the selectivity of binding and functional inhibition of αvβ6. The corresponding αvβ6-retargeted virus Ad5-EGFP_A20 exhibited up to 50-fold increases in coxsackievirus- and-adenovirus-receptor-independent transduction and up to 480-fold-increased cytotoxicity on a panel of αvβ6-positive human carcinoma lines compared with Ad5-EGFP_WT. Using an αvβ6-positive (DX3-β6) xenograft model, we observed a ∼2-fold enhancement in tumor uptake over Ad5-EGFP_WT following systemic delivery. Furthermore, ∼5-fold-fewer Ad5-EGFP_A20 genomes were detected in the liver (P = 0.0002), correlating with reduced serum transaminase levels and E1A expression. Warfarin pretreatment, to deplete coagulation factors, did not improve tumor uptake significantly with either virus but did significantly reduce liver sequestration and hepatic toxicity. The ability of Ad5-EGFP_A20 to improve delivery to αvβ6, combined with its reduced hepatic tropism and toxicity, highlights its potential as a prototype virus for future clinical investigation.The aim of cancer gene therapy is to achieve targeted delivery of therapeutic transgenes to malignant tissue, with negligible effects on surrounding healthy tissue. Efforts in the development of adenoviruses as therapeutic agents have been persistent. However, several challenges still remain. Inefficient transduction of diseased tissue and the innate hepatotropism and toxicity of adenovirus type 5 (Ad5) in vivo following intravenous delivery represent major issues to be addressed. Additionally, the use of adenoviral vectors for cancer therapy is thought to be incompatible with the broad distribution of the primary adenovirus receptor, the coxsackievirus and adenovirus receptor (CAR), in normal tissues (6). Furthermore, it recently has emerged that human, but not murine, erythrocytes express CAR on their surface, which promotes the sequestration of Ad5 in the circulation and may represent another restriction to efficient tumor delivery in vivo (8).The predominant adenoviral serotype currently used in gene therapy applications is human Ad5. Ad5 binds to cells through a docking process in which the distal knob domain of the fiber structural protein binds to CAR (6, 23). This is followed by the exposure of an arginine-glycine-aspartate (RGD) motif in the penton base which promotes viral internalization, mediated primarily by αvβ3 and αvβ5 integrins (49). Binding to CAR represents the initial event in cell attachment in vitro, and therefore CAR expression levels long have been thought to be critical in determining the transduction efficiency of Ad5 in vivo. Several studies have reported low expression of CAR in primary carcinoma lines and tumor explants (3, 21, 30, 32, 37), highlighting the necessity for CAR-independent targeting strategies. However, the nonspecific sequestration of Ad5 in the liver remains the major obstacle to achieving high-efficiency tumor targeting following systemic delivery.A preeminent role for coagulation factors (i.e., FVII, FIX, FX, protein C, and C4BP) in directing liver uptake following systemic delivery has been demonstrated in recent years (36, 41, 48), and hepatocyte transduction now has been shown to be mediated predominantly by a direct Ad5 hexon-FX interaction (22, 48). This discovery has prompted the experimental use of anticoagulants, such as warfarin, in an attempt to avoid liver sequestration, with the aim of increasing the bioavailability of the virus for the tumor. However, it recently has emerged that coagulation factors may also be required for efficient tumor delivery in vivo and that the depletion of blood factors may in fact preclude successful tumor uptake (16). Accordingly, Ad vector constructs which combine liver detargeting with high-efficiency, CAR-independent gene delivery to cancer-specific receptors are highly desirable.The epithelial cell-specific integrin αvβ6 generally is undetectable in normal adult tissue but is upregulated significantly in numerous carcinomas, where high expression often correlates with poor prognosis (1, 4, 13). We have shown previously that over 90% of oral squamous cell carcinomas express αvβ6 strongly (35, 45) and that high αvβ6 expression promotes tumor progression (35, 43, 46). Binding to αvβ6 is via the RGD motif in its ligands, which include the latency-associated peptides (LAP) of transforming growth factor β1 (TGF-β1) and TGF-β3 (33), in addition to the VP1 structural protein of foot-and-mouth disease virus (FMDV), for which αvβ6 is a native receptor (20). We have shown that specificity for αvβ6 is dependent on the inclusion of a DLXXL motif in an extended carboxy α-helical loop, with the RGD motif situated at the apex of a hairpin loop domain (10). Functional analysis of known αvβ6 ligands identified a candidate peptide, A20FMDV2, which had high affinity and selective binding to αvβ6 (10). Recent studies have further supported this finding, demonstrating that this peptide forms a highly stable, EDTA-resistant complex with αvβ6 integrin, in a manner analogous to the highly infectious FMDV (11).Over the past decade, significant attempts to increase the delivery of Ad5 to target tissues have been made by modifying structural tropism determinants. Retargeting strategies have included the insertion of RGD motifs (12), polylysine (pK7) motifs (50) and TAT peptide from human immunodeficiency virus type 1 (26) in attempts to improve delivery to malignant tissue or to the endothelial networks that supply the tumors. The resolution of the crystal structure of the Ad5 knob domain by X-ray crystallography identified the HI loop region as being suitable for peptide incorporation (51), and to date this site has been shown to tolerate the insertion of ligands of up to 83 amino acids with negligible effects on structural integrity (5). In order to redirect the native tropism of Ad5 to αvβ6, we genetically incorporated A20FMDV2 into the HI loop region of the fiber knob domain. We hypothesized that Ad5-EGFP_A20 would permit significant improvements in selective delivery to αvβ6, both in vitro and in vivo.Here, we describe the successful subversion of Ad5 infection to αvβ6 expressed on human carcinoma cell lines in vitro and demonstrate that the enhanced transduction observed with Ad5-EGFP_A20 is CAR independent. We have confirmed that the improved infectivity is due to the insertion of the A20FMDV2 peptide and that cell entry is mediated predominantly through αvβ6 integrin. Additionally, using an αvβ6-positive (DX3-β6) xenograft model, we observed ∼2-fold enhancement in tumor uptake over Ad5-EGFP_WT in vivo following systemic delivery. Furthermore, ∼5-fold-fewer Ad5-EGFP_A20 genomes were detected in the liver (P = 0.0002), correlating with reduced serum transaminase levels and E1A expression. These data show that redirecting Ad5 to αvβ6 can increase tumor delivery while simultaneously limiting hepatotoxicity; this may therefore represent a means of overcoming some of the limitations of current Ad5 therapy.     

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.     

Tracking protein aggregate interactions

Amyloid fibrils share a structural motif consisting of highly ordered β-sheets aligned perpendicular to the fibril axis.^{1, 2} At each fibril end, β-sheets provide a template for recruiting and converting monomers.³ Different amyloid fibrils often co-occur in the same individual, yet whether a protein aggregate aids or inhibits the assembly of a heterologous protein is unclear. In prion disease, diverse prion aggregate structures, known as strains, are thought to be the basis of disparate disease phenotypes in the same species expressing identical prion protein sequences.^4–7 Here we explore the interactions reported to occur when two distinct prion strains occur together in the central nervous system.Key words: prion, prions, strain, TSE, interaction, amyloid, LCP, neurodegeneration, aggregation     

Mouse model for probing tumor suppressor activity of protein phosphatase 2A in diverse signaling pathways

Evidence that protein phosphatase 2A (PP2A) is a tumor suppressor in humans came from the discovery of mutations in the genes encoding the Aα and Aβ subunits of the PP2A trimeric holoenzymes, Aα-B-C and Aβ-B-C. One point mutation, Aα-E64D, was found in a human lung carcinoma. It renders Aα specifically defective in binding regulatory B′ subunits. Recently, we reported a knock-in mouse expressing Aα-E64D and an Aα knockout mouse. The mutant mice showed a 50–60% increase in the incidence of lung cancer induced by benzopyrene. Importantly, PP2A''s tumor suppressor activity depended on p53. These data provide the first direct evidence that PP2A is a tumor suppressor in mice. In addition, they suggest that PP2A is a tumor suppressor in humans. Here, we report that PP2A functions as a tumor suppressor in mice that develop lung cancer triggered by oncogenic K-ras. We discuss whether PP2A may function as a tumor suppressor in diverse tissues, with emphasis on endometrial and ovarian carcinomas, in which Aα mutations were detected at a high frequency. We propose suitable mouse models for examining whether PP2A functions as tumor suppressor in major growth-stimulatory signaling pathways, and we discuss the prospect of using the PP2A activator FTY720 as a drug against malignancies that are driven by these pathways.Key words: lung cancer, oncogenic K-ras, p53, Aα mutations in endometrial cancerUnderstanding how protein phosphatase 2A (PP2A) functions as a tumor suppressor requires knowledge of its complex structure and the roles its numerous regulatory subunits play. The trimeric holoenzyme is composed of a catalytic C subunit, a scaffolding A subunit and one of many regulatory B subunits. The catalytic C subunit exists as two isoforms, Cα and Cβ, that are 96% identical. The scaffolding A subunit also exists as two isoforms, Aα and Aβ, and they are 87% identical. The B subunits fall into four families designated B, B′, B″ and B‴. The B or PR55 family has four members; the B'' family (also designated B56 or PR61) consists of five isoforms and additional splice variants, and the B” or PR72 family has four members including splice variants. B, B′ and B″ are largely unrelated by sequence. The combination of all subunits could give rise to over 70 distinct holoenzymes. In addition, the ability of PP2A to associate with approximately 150 other proteins further increases its regulatory potential.^1–5 Figure 1B shows a schematic diagram of the holoenzyme whose subunit interactions and structure have been revealed initially by biochemical studies^17,18 and subsequently in great detail by crystal structure analyses.^19–23 Through this work and numerous other investigations, it has become increasingly clear over the past 25 years that PP2A is not just a nonspecific phosphatase, as it was thought to be initially, but a highly sophisticated enzyme involved in most, if not all, fundamental cellular processes. One of the most challenging properties of PP2A is its role as a tumor suppressor, which has been covered by excellent reviews in references ^24–28. The present report highlights recently developed mouse models for investigating PP2A''s tumor suppressor activity.Open in a separate windowFigure 1Model of PP2A holoenzyme; location of human cancer-associated Aα mutations; high frequency of Aα mutations in endometrial cancer. (B) Trimeric PP2A holoenzyme consists of one catalytic subunit (Cα or Cβ), one scaffolding subunit (Aα or Aβ) and one of several regulatory subunits (B, B'' or B”). Aα and Aβ consist of 15 repeats connected by inter-repeat loops. Each repeat consists of two antiparallel α-helices connected by intra-repeat loops. (A) Aα mutations in endometrial (endo) or ovarian (ovary) cancer are clustered at or near intra-repeat loop 5 of repeat 5 (from P179 to R183) and at or near intra-repeat loop 7 of repeat 7 (from R249 to R258). Numbers in parentheses represent number of tumors with a mutation at a particular site.^6–9 E64D, E64G and R418W were found in lung, breast and skin cancer, respectively.¹⁰ Shown in (C and D) are C-terminal truncations, Δ171–589 from breast cancer missing repeats 6 to 15¹⁰ and Δ375–589 from kidney cancer missing repeats 11 to 15.¹¹ (E) Frequency of Aα mutations in endometrial (18%, 31/171) and ovarian (6%, 27/470) cancers in comparison to K-ras, Arf, p53 and PI3K.^6–9 (F) Loss of Bα, B''γ3 (formerly known as B''α1),¹² and B”/PR72 binding to mutant Aα. Note: All Aα mutants are defective in B''γ3 binding.^13,14 For E393Q, see reference ¹⁵; for R183W in pancreatic (pa) cancer, see reference ¹⁶; *indicates synthetic mutant.     

Activation of PKC-ε counteracts maturation and apoptosis of HL-60 myeloid leukemic cells in response to TNF family members

Protein kinase C (PKC)-ε, a component of the serine/threo-nine PKC family, has been shown to influence the survival and differentiation pathways of normal hematopoietic cells. Here, we have modulated the activity of PKC-ε with specific small molecule activator or inhibitor peptides. PKC-ε inhibitor and activator peptides showed modest effects on HL-60 maturation when added alone, but PKC-ε activator peptide significantly counteracted the pro-maturative activity of tumor necrosis factor (TNF)-α towards the monocytic/macrophagic lineage, as evaluated in terms of CD14 surface expression and morphological analyses. Moreover, while PKC-ε inhibitor peptide showed a reproducible increase of TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis, PKC-ε activator peptide potently counteracted the pro-apoptotic activity of TRAIL. Taken together, the anti-maturative and anti-apoptotic activities of PKC-ε envision a potentially important proleukemic role of this PKC family member.Key words: acute myeloid leukemia, surface antigens, HL-60 cells, apoptosis, maturation.Activation of all protein kinase C (PKC) family of serine and threonine isoenzymes is associated with binding to the negatively charged phospholipids, phosphatidylserine, while different PKC isozymes have varying sensitivities to Ca²⁺ and lipid-derived second messengers such as diacylglycerol (Gonelli et al., 2009). Upon activation, PKC isozymes translocate from the soluble to the particulate cell fraction, including cell membrane, nucleus and mitochondria (Gonelli et al., 2009). PKC primary sequence can be broadly separated into two domains: the N-terminal regulatory domain and the conserved C-terminal catalytic domain.The regulatory domain of PKC is composed of the C1 and C2 domains that mediate PKC interactions with second messengers, phospholipids, as well as inter and intramolecular protein-protein interactions. Differences in the order and number of copies of signaling domains, as well as sequence differences that affect binding affinities, result in the distinct activity of each PKC isozyme (Gonelli et al., 2009).In recent years, a series of peptides derived from PKC have been shown to modulate its activity by interfering with critical protein-protein interactions within PKC and between PKC and PKC-binding proteins (Brandman et al., 2007, Souroujon and Mochly-Rosen, 1998). Focusing on PKC-ε isozyme and using a rational approach, one C2-derived peptide that acts as an isozyme-selective activator (Dorn et al., 1999) and another that acts as a selective inhibitor (Johnson et al., 1996) of PKC-ε, have been identified.These findings are particularly interesting since besides being involved in the physiology of normal cardiac (Braun and Mochly-Rosen, 2003, Johnson et al., 1996, Li et al., 2006), hematopoietic (Gobbi et al., 2009, Mirandola et al., 2006, Racke et al., 2001), and neuronal (Borgatti et al., 1996) cell models, mounting experimental evidences have linked altered PKC-ε functions to solid tumor development (Okhrimenko et al., 2005, Gillespie et al., 2005, Lu et al., 2006). Therefore, taking advantage of the recent availability of small molecule peptides able to activate or inhibit specifically PKC-ε by disrupting protein/protein interactions (Dorn et al., 1999, Johnson et al., 1996), which open important therapeutic perspectives, we have investigated the effects of both PKC-ε activator and PKC-ε inhibitor peptides on the maturation and survival of leukemic cells, using as a model system the HL-60 myeloblastic leukemia cell line, which can be induced to undergo terminal differentiation or apoptotic cell death by a variety of chemical and biological agents (Breitman et al., 1980, Zauli et al., 1996).     

Immunotherapy for Alzheimer disease

Immunotherapy approaches for Alzheimer disease currently are among the leading therapeutic directions for the disease. Active and passive immunotherapy against the β-amyloid peptides that aggregate and accumulate in the brain of those afflicted by the disease have been shown by numerous groups to reduce plaque pathology and improve behavior in transgenic mouse models of the disease. Several ongoing immunotherapy clinical trials for Alzheimer disease are in progress. The background and ongoing challenges for these immunological approaches for the treatment of Alzheimer disease are discussed.Key words: Alzheimer disease, amyloid, tau, immunotherapy, vaccineThe publication in Nature on a vaccine approach for Alzheimer disease (AD) by Schenk and colleagues in 1999 initiated a push for treatment for this major disease of aging. AD neuropathology is characterized by the progressive loss of synapses and neurons, and the aberrant accumulation in the brain of β-amyloid peptides in plaques and the microtubule associated protein tau in neurofibrillary tangles. Mutations in familial forms of AD have been associated with elevated β-amyloid levels, whereas mutations in tau have been linked to familial forms of frontotemporal dementia. Remarkably, injection of β-amyloid peptides with Freund''s adjuvant into transgenic mice harboring a human AD mutation that develop AD-like neuropathology and progressive cognitive decline led to reduced β-amyloid plaque pathology.¹ This study was subsequently confirmed and extended by multiple groups to show also behavioral improvement in AD transgenic mice with active β-amyloid immunization.^2,3 Passive immunotherapy with antibodies directed at β-amyloid were similarly effective in reducing plaques and improving behavior in AD transgenic mice.⁴ A temporary setback occurred when the first clinical trial with β-amyloid vaccination was halted after 6% of patients developed an inflammatory reaction in the brain (chemical meningoencephalitis). A subsequent study supported clinical benefits among patients in this active vaccination trial.⁵ A more recent postmortem study on a subset of patients who had participated in the aborted trial supported active removal of β-amyloid plaques by inflammatory cells, but also indicated that 7 of the 8 patients who were studied at autopsy continued to have progressive cognitive decline despite the removal of amyloid plaques.⁶The critical mechanisms whereby active or passive vaccination against β-amyloid can affect the disease process remain uncertain. Recruitment and activation of microglia, the macrophage of the central nervous system, by β-amyloid antibodies is thought to lead to β-amyloid plaque removal. At the same time, fibrillar β-amyloid containing plaques, formerly viewed as the major toxic entities in AD, are increasingly viewed as potentially only pathological remnants of the disease. Smaller assemblies, particularly of two to twelve β-amyloid peptides (oligomers), are considered pathogenic, although the site of pathogenesis remains controversial. Secreted, extracellular β-amyloid oligomers have been shown to damage synapses.⁷ Some groups stress the aberrant accumulation of β-amyloid within neurons and synapses leading to subsequent extracellular localization following destruction of neurites and synapses.⁸ Evidence has been presented that antibodies targeting β-amyloid peptides up to 42–43 amino acids can block the toxic effects of extracellular β-amyloid oligomers on synapses.⁷ Interestingly, β-amyloid immunotherapy was also shown to clear intraneuronal β-amyloid in an AD transgenic mouse; the intraneuronal variety is a pool of β-amyloid that correlates with the onset of cognitive decline prior to plaques and tangles in these mice.⁹ Intriguingly, antibodies directed at the β-amyloid domain exposed to the extracellular space within the amyloid precursor protein (APP) were shown to be internalized by neurons, where they reduced the intraneuronal pool of β-amyloid and protected against synaptic damage in neurons cultured from AD transgenic mice.^10,11 It is possible that inefficient clearance of the intracellular pool of β-amyloid played a role in the continued cognitive decline in the seven of eight patients in the aborted active vaccination clinical trial studied at autopsy who showed clearance of β-amyloid plaques.Work on β-amyloid immunotherapy in AD contributed to a reevaluation of the role of the immune system in the brain. Previously, it was considered that the brain was immune privileged, and that antibodies entered the brain only with the breakdown of the blood brain barrier. Rare neuroimmunological disorders had suggested more complex interactions. Pathological antibodies directed at neuronal proteins could be found localizing to specific types of neurons in paraneoplastic diseases linked to diverse systemic cancers^12,13 or collagen-vascular diseases such as lupus.¹⁴ Such pathological antibodies can be directed at synaptic or even intracellular proteins in selective neurons in the brain, leading to localized neurological symptoms. For paraneoplastic diseases it is hypothesized that antibodies directed at the cancer cells cross-react with neuronal antigens. Since titers of antibodies can be higher in brain than in the blood, intrathecal synthesis of antibodies from sequestration of B cells has been proposed to occur in the brain.¹⁵ The interaction between the immune system and the brain is therefore viewed as increasingly complex, with antibodies not only gaining access to the brain but also nerve cells, where they can even alter intracellular biology.¹⁰ These findings open up new possibilities for antibody-directed therapies for diseases of the nervous system.Currently, leading concerns for β-amyloid immunotherapy are the potential development of chemical meningoencephalitis and micro-hemorrhages in the brain. Involvement of T cells in damage to the brain vasculature is considered to contribute to these potential side effects. In addition, the β-amyloid released upon antibody-induced removal of plaques may damage blood vessels as β-amyloid is cleared from the brain via the vasculature.¹⁶ Recently, a phase 2 Elan/Wyeth study using passive β-amyloid immunotherapy with a humanized monoclonal antibody described (at the 2008 International Conference on Alzheimer''s disease) significant benefits in patients not harboring the apolipoprotein E4 (apoE4) allele genetic risk factor for late onset AD. In contrast, no clear therapeutic benefit and more cases with brain inflammation occurred in those with the apoE4 allele linked with an increased risk for AD. Why apoE4 carriers did not benefit in this β-amyloid immunotherapy trial is unknown, but has prompted separation of patients into E4 negative and positive groups in subsequent clinical trials. The less robust than hoped for effects even in the apoE4 negative patients has further dampened expectations. The reason for why the human studies are not showing the protection seen in the transgenic mouse studies could relate to β-amyloid playing less of a role in the more typical late onset AD than it does in the rare autosomal dominant familial forms used to generate the AD transgenic mice. It is also not clear which β-amyloid epitope(s) should be targeted by antibodies to maximize potential benefits while minimizing side effects in AD patients. Optimizing antibody specificity for immunotherapy is further complicating by the varied conformations of different β-amyloid aggregation states. In addition, β-amyloid immunotherapy may be more challenging in patients with AD because it is not effective in reducing tau tangle pathology.⁶ Most immunotherapy studies were done on transgenic AD mouse models that deposit β-amyloid plaques, but not tau tangles. In the more recently generated triple transgenic AD mouse that develops both plaques and tangles, β-amyloid antibodies reversed β-amyloid pathology and early pre-tangle tau pathology, but not hyperphosphorylated tau aggregates.⁸ Recent evidence supports that β-amyloid neurotoxicity acts synergistic with tau,¹⁷ and that both pathologies begin at synapses.¹⁸ Interestingly, tau immunotherapy was reported to protect against tau pathology in transgenic mice harboring mutant tau.¹⁹ Thus, dual immunotherapy targeting of both β-amyloid and tau can be considered. Finally, immunotherapy at earlier stages of the disease process may be more effective.In summary, the β-amyloid vaccine heralded a new era of therapeutic research for AD and despite some setbacks is actively being pursued in several ongoing clinical trials. It continues to be among the leading hopes in the AD research community. Another major effort to specifically block the generation of β-amyloid is also progressing, although not without setbacks along the way. For example, the protease involved in the final cleavage to liberate β-amyloid was found to be involved in multiple other important activities, such as cleavage of Notch. Antibody approaches are also being applied in efforts to block secretase cleavage to generate β-amyloid.²⁰ Finally, there remains some worry that β-amyloid peptides have an as yet unknown normal biological function, although cumulative immunotherapy and other therapeutic studies in animal models have provided sufficient support for the continued pursuit of β-amyloid lowering as a treatment for AD.     

Integrin linked kinase (ILK) expression and function in vascular smooth muscle cells

Vascular smooth muscle cell (SMC) migration and proliferation contribute to arterial wound repair and thickening of the intimal layer in atherosclerosis, restenosis and transplant vascular disease. These processes are influenced by cell adhesion to molecules present in the extracellular matrix, and regulated by the integrin family of cell-surface matrix receptors. An important signaling molecule acting downstream of integrin receptors is integrin-linked kinase (ILK), a serine/threonine kinase and scaffolding protein. ILK has been implicated in cancer cell growth and survival through modulation of downstream targets, notably Akt and glycogen synthase kinase-3β (GSK3β). Evidence also exists to establish ILK as a molecular adaptor protein linking integrins to the actin cytoskeleton and regulating actin polymerization, and this function may not necessarily depend upon the kinase activity of ILK. ILK has been implicated in anchorage-independent growth, cell cycle progression, epithelial-mesenchymal transition (EMT), invasion and migration. In addition, ILK has been shown to be involved in vascular development, tumor angiogenesis and cardiac hypertrophy. Despite the documented involvement of integrin signaling in vascular pathologies, the function of ILK has not been well characterized in the SMC response to vascular injury. This brief review summarizes and puts into context the current literature on ILK expression and function in the vascular smooth muscle cell.Key words: smooth muscle cell, migration, extracellular matrix, atherosclerosis, cytoskeletonA large body of research is dedicated to elucidating the mechanisms by which smooth muscle cells (SMCs) contribute to thickening of the arterial wall in pathologies such as atherosclerosis and restenosis. After arterial injury and during neointimal hyperplasia, SMCs undergo a phenotypic switch characterized by the transition from a quiescent to an active/synthetic phenotype, and they begin to synthesize an abundant extracellular matrix.¹ In turn, interactions between cells and the matrix govern the process of neointimal thickening.² Cell surface integrin receptors play important roles in signaling proliferative and migratory cellular responses during arterial wound repair. Integrin-linked kinase (ILK) is an important downstream mediator of integrin signaling, yet little is known of its function in the arterial response to injury.Integrin-linked kinase (ILK) was originally identified as a serine-threonine kinase binding to the cytoplasmic domain of β₁- and β₃-integrin subunits.³ ILK functions to activate Akt and inhibit glycogen synthase kinase-3β (GSK3β),^4–6 and has been implicated in cancer cell growth and survival through modulation of these downstream targets. Given its role in anchorage-independent growth, survival and cell cycle progression,⁷ epithelial-mesenchymal transition (EMT), and invasion and migration,^8,9 it is often suggested that ILK be targeted for cancer treatment.¹⁰ ILK is also involved in vascular development^11,12 and tumor angiogenesis.^13,14Concurrent studies in model organisms and cell cultures point to a role for ILK as a molecular scaffold linking integrins to the actin cytoskeleton and regulating actin polymerization.^15–17 Furthermore, this scaffolding function may be independent of the kinase activity of ILK. In C. elegans, genetic ablation of pat-4/ilk (ILK homologue) leads to severe adhesion defects, muscle detachment and embryonic lethality.¹⁵ However PAT-4/ILK does not phosphorylate GSK3β in C. elegans.¹⁵ Similarly, in Drosophila melanogaster, loss of function mutants for ILK resulted in severe embryonic muscle-attachment defects and detachment of F-actin from the cell membrane, and the muscle attachment defect was rescued by expressing a kinase-deficient ILK.^15,17 Finally, tissue-specific conditional knockout of ILK in mouse chondrocytes results in defects in the skeleton,^18,19 and inhibition of cell adhesion, spreading and cytoskeletal assembly in chondrocytes in culture.¹⁸ These deficiencies were not attributable to impaired Akt or GSK3β signaling. In fact, the importance of ILK kinase function appears to be cell type-dependent. Inhibition of ILK activity in transformed cells resulted in a decrease in Akt phosphorylation and apoptosis, but had no effect in non-transformed cell types including vascular SMCs, thus calling into question the importance of ILK as a kinase in non-cancerous cell types.²⁰We have studied the function of ILK in vascular smooth muscle cell wound repair and found that ILK acted as a scaffolding protein at focal adhesion sites.²¹ In our experiments, immunostaining of cultured SMCs revealed co-localization of ILK and paxillin at focal adhesions, a finding which is consistent with a previous study.²² Several proteins such as PINCH1, parvins and paxillin interact directly with ILK to facilitate its localization to focal adhesions and coordinate actin organization and cell spreading.^23–25 Overexpression of an ILK-binding-deficient PINCH protein in tracheal SMCs led to decreased recruitment of ILK and PINCH to focal adhesions, and decreased association between ILK, paxillin and vinculin.²⁶We hypothesized that ILK acting as a scaffolding protein might regulate the SMC response to vascular injury. To study this, we examined ILK using in vitro models mimicking vascular injury. Silencing ILK expression with siRNA decreased cell adhesion to fibronectin, and accelerated cell proliferation and wound closure.²¹ However, silencing ILK in wounded SMCs did not attenuate the increase in Akt and GSK3β phosphorylation observed after wounding.²¹ Nonetheless, we observed rearrangement of focal adhesions and stress fibers in ILK-silenced SMCs, which may have contributed to the reduced adhesion to fibronectin and enhanced cell migration and proliferation. Thus it seems that the scaffolding role of ILK may be more important for focal adhesion dynamics and remodeling in SMCs than the kinase function of ILK. These results were also surprising because they imply that ILK functions to inhibit cell growth and motility, unlike several studies which have suggested that ILK signals to increase these processes.^7,8,10To address in vivo arterial wound repair, we studied ILK expression after balloon catheter injury of the rat carotid artery. Following balloon injury, SMCs undergo a process of dedifferentiation which includes enhanced proliferation and migration from the media to the intima. We found that ILK protein expression was dramatically decreased in the media during the SMC proliferative and migratory responses.21 The rapid decrease in ILK protein expression is consistent with the effects of silencing ILK in cultured SMCs. We propose that the decrease in ILK following injury facilitates the rearrangement of focal adhesions, altering cell adhesion to facilitate SMC migration and proliferation. The decrease in ILK expression in SMCs following injury may be related to the transition of these cells to a de-differentiated state. A recent study has shown that increased ILK expression correlates with cell differentiation in the luminal layers of the epithelium in the esophagus, colon and intestines when compared to the basal layers.²⁷ ILK was also prominent in more differentiated areas of malignant tumors. In our studies, we noted an increase in ILK expression in the layers of the intima closest to the vascular lumen. This was consistent with findings in another recent study reporting increased ILK protein expression in the intima of balloon-injured rat carotid arteries in vivo and in the developing intima of human saphenous veins cultured ex vivo.²⁸ We suggest that ILK is upregulated here in coincidence with the re-establishment of SMC quiescence.In addition to maintaining stable cell adhesion to matrix, in the quiescent differentiated SMC, ILK may function to mediate contraction and aid the cell in exerting force on surrounding extracellular matrix fibers. In SMCs, ILK is localized to myofilaments, and promotes cell contraction by directly phosphorylating myosin light chain (MLC) or myosin light chain phosphatase (MLCP).^9,29,30 Alternatively, ILK may activate smooth-muscle contraction indirectly via phosphorylation and activation of MLCP inhibitors including CPI-17 and PHI-1.²⁹ Consistent with a role for ILK in mediating contraction, stimulation of tracheal SMCs with acetycholine recruits ILK and PINCH to the cell membrane, and overexpression of an ILK-binding-deficient mutant PINCH attenuated the localization of ILK at adhesion sites, and attenuated actin polymerization, the activation of the actin nucleation initiator N-WASP, and the development of tension.²⁶ ILK has also been identified as a key regulator of cardiac myocyte contractility.³¹ Likewise, ILK is required in the skeletal muscle of zebrafish for integrin-matrix adhesion to maintain the stability of muscle fibres.³² Mice with a skeletal muscle-specific deletion of ILK develop muscular dystrophy and detachment of muscle cells from basement membranes.³³ ILK mutants also showed displacement of several focal adhesion proteins and reorganization of the actin cytoskeleton.³⁴Our results after silencing ILK expression differ somewhat from a previous study of ILK in vascular SMCs. Overexpression of wild- type ILK in SMCs increased cell migration in response to stromal derived factor-1 or angiotensin II, while overexpression of a kinase-dead mutant of ILK (E359K) suppressed SMC migration in Boyden chamber assays.³⁵ In contrast to this study, we have shown the effects of inhibiting endogenous ILK by siRNA. ILK-induced quiescence of SMC may require tight regulation of intracellular ILK levels such that both its suppression and its upregulation promote cell motility.Taken together, these studies reveal that the functions of ILK are broader and more complex than originally thought. This molecule has the potential to function as an adapter protein regulating cytoskeletal assembly and signal transduction from focal adhesion sites, as a protein kinase activating several signaling axes, and as a regulator of the mitotic spindle.^36,37 The breadth of ILK function in regulating cell-matrix interactions, cytoskeletal organization and cell signaling is of great importance to normal development and disease progression. Functional studies using both kinase-deficient ILK variants and ILK siRNA will allow researchers to specifically attribute cellular behaviors to the proposed functions of ILK, and to determine their relative importance in different cells and pathologies. Based on our studies using injury models mimicking cellular events in occlusive vascular disease, we propose that ILK functions to maintain SMCs in a stationary, contractile phenotype in the normal artery. Following arterial injury, decreased ILK expression facilitates the reorganization of focal adhesions and the actin cytoskeleton, allowing for more efficient SMC migration and proliferation to establish a thickened neointima.     

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins.^{1, 2, 3} Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices.^{4, 5} They are activated by BH3-only proteins binding to the α2–α5 surface groove,^{6, 7, 8, 9, 10, 11, 12} or for Bax, to the α1/α6 ‘rear pocket''.¹³ Binding triggers dissociation of the latch domain (α6–α8) from the core domain (α2–α5), together with exposure of N-terminal epitopes and the BH3 domain.^{6, 7, 14, 15, 16} The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers.^{6, 7, 14, 17, 18} In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer.¹⁹ In Bax, the α5 and α6 helices may insert into the MOM,²⁰ although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface.^{7, 21, 22, 23} The dimers can be linked via cysteine residues placed in α6,^{18, 24, 25} and more recently via cysteine residues in either α3 or α5,^{6, 21} allowing detection of the higher-order oligomers associated with pore formation.^{26, 27} However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the MOM in healthy cells,²⁸ whereas the Bax C terminus is either exposed²⁹ or sequestered within the hydrophobic groove until apoptotic signals trigger Bax translocation.^{5, 30, 31} The hydrophobic stretch is important, as substituting polar or charged residues decreased targeting of Bak and Bax.^{10, 32} Mitochondrial targeting is also controlled by basic residues at the far C termini,^{32, 33, 34} and by interaction with VDAC2^{35, 36} via the Bak and Bax C termini.^{37, 38} Retrotranslocation of Bak and Bax was also altered by swapping the C termini.³⁹The membrane topology of the Bak and Bax C termini before and after apoptosis has not been examined directly, due in part to difficulty in reconstituting oligomers of full-length Bak in artificial membranes. Nor is it known whether the C termini contribute to pore formation by promoting oligomerization or disturbing the membrane. To address these questions synthetic peptides based on the Bak and Bax C termini have been studied in model membranes. The peptides adopt a predominantly α-helical secondary structure,^{40, 41, 42, 43} with orientation affected by lipid composition.^{42, 44, 45} The peptides could also permeabilize lipid vesicles,^{41, 43, 46, 47} suggesting that the C termini in full-length Bak and Bax may contribute to pore formation.Here we examined the membrane topology of the C termini within full-length Bak and Bax in the MOM, both before and after apoptotic pore formation. After pore formation the α9 helices of Bak (and of Bax) became juxtaposed but did not line the surface of a pore. The α9:α9 interaction occurred after Bak activation and conformation change, but was promoted by formation of BH3:groove dimers. Combining linkage at more than one interface indicated that the Bak α9:α9 interface can link BH3:groove dimers to high-order oligomers, and moreover, that the α6–α9 region is flexible in oligomerized Bak.     

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.