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     

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).     

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.     

NTPase and 5′ to 3′ RNA Duplex-Unwinding Activities of the Hepatitis E Virus Helicase Domain

Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae. The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5′ overhangs showing 5′-to-3′ polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg²⁺ binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5′ overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.Viruses with single-strand positive-sense RNA genomes represent the largest class of viruses, which includes numerous pathogens of humans, plants, and animals. In these viruses, RNA replication occurs through negative-strand RNA intermediate, which may also act as the template for synthesis of subgenomic RNAs in some viruses. During replication, various nonstructural proteins remain associated with the viral polymerase in a small compartmentalized replisome. Most of the other accessory proteins are obtained from the cellular machinery.Helicase seems to be essential for RNA replication by many positive-sense RNA viruses (19). Many positive-strand RNA viruses encode their own RNA helicases and besides RNA-dependent RNA polymerase, helicase is the most conserved viral sequence in these viruses. It has been shown by direct mutagenesis studies in poliovirus (26, 39), alphaviruses (31), brome mosaic virus (2, 41), nidoviruses (40), and flaviviruses (15) that helicase functions are essential for viral replication. In addition, it may be involved in RNA translocation, genome packaging, protection of RNA at the replication center, modulating RNA-protein interactions, etc.Helicases are classified into six superfamilies, SF-1 to SF-6 (11, 35), and can be classified further into subfamilies, A (3′→5′) or B (5′→3′) depending on their unwinding directionality. Classic helicases (exhibiting both NTPase and unwinding activities) are referred to as subtype α, while translocases (with no unwinding activity) are referred to as subtype β (35). SF-1 and SF-2 constitute largest of these superfamilies with seven signature motifs (I, Ia, II, III, IV, V, and VI), which form core of the enzyme. Although these motifs are not comparable between SF-1 and SF-2, universal features of core domains include (i) conserved residues involved in binding and hydrolysis of the NTP and (ii) an arginine finger that plays a key role in energy coupling.Hepatitis E virus (HEV) is a nonenveloped virus in the genus Hepevirus of the family Hepeviridae. Hepatitis E is an important public health disease in many developing countries and is also endemic in some industrialized countries (8). Infection by HEV has a known association with increased mortality during pregnancy (22, 23). HEV has a positive-sense RNA genome of ∼7.2 kb, consisting of a 5′ noncoding region (5′NCR) of 27 to 35 nucleotides (nt), followed by three open reading frames (ORFs)—ORF1, ORF2, and ORF3—and a 3′NCR of 65 to 74 nt, ending with a poly(A) tail of variable length (37). The 5′ end has m⁷G cap (18). ORF1 is known to encode for the viral nonstructural polyprotein with a proposed molecular mass of ∼186 kDa (3). Based on protein sequence homology, the ORF1 polyprotein is proposed to contain four putative domains indicative of methyltransferase, papain-like cysteine protease, RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) (24). ORF2 encodes the major structural protein (capsid protein), which has N-terminal signal peptide and three glycosylation sites and is translocated across the endoplasmic reticulum (ER). ORF2 protein associates with the 5′ end of the viral RNA, suggesting its regulatory role in the virus replication (36, 37, 44, 45). ORF3 encodes a protein which gets phosphorylated by the cellular mitogen activated protein kinase and is associated with cellular membranes and cytoskeleton fractions (43).HEV belongs to an “alpha-like” supergroup of positive-sense single-stranded RNA (+ssRNA) viruses with conserved motifs of replication-related proteins in the ORF1, with typical signature sequences homologous with the other members of the family (11, 12, 13). ORF1 of HEV encodes additional domains such as the Y domain, papainlike protease, “proline-rich hinge,” and the X domain. Methyltransferase (25), RdRp (1), and X domain (binding to poly-ADP-ribose) (9) in ORF1 have been characterized, whereas the functions of the other domains are yet to be identified. Intracellularly expressed RdRp localizes itself in the ER membranes (30), suggesting that HEV replicates probably in ER in the cytosolic compartment of the cells. It is still unknown whether ORF1 polyprotein undergoes cleavages to form separate functional units of the replication machinery or functions as a single protein with multiple functional domains.The putative RNA helicase of HEV contains all of the seven conserved segments typical of the SF-1 helicase (12, 13). Putative SF-1 helicases are extremely widespread among +ssRNA viruses. Based on sequence comparisons, such helicases have been identified in a variety of plant virus families, as well as in animal viruses such as alphavirus, rubivirus, hepatitis E virus, and coronavirus (11). When compared to other +ssRNA viral helicases belonging to SF-1, HEV helicase showed the highest overall similarity with the helicase of beet necrotic yellow vein virus, a plant furovirus. HEV helicase was speculated to have N-terminal NTPase and C-terminal RNA-binding domains (24). A major obstacle in studying HEV replication has been lack of cell culture system. We report here experimental verification of the helicase activity of the recombinant helicase domain protein of HEV.     

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?     

Inhibition of Herpes Simplex Virus Replication by a 2-Amino Thiazole via Interactions with the Helicase Component of the UL5-UL8-UL52 Complex

With the use of a high-throughput biochemical DNA helicase assay as a screen, T157602, a 2-amino thiazole compound, was identified as a specific inhibitor of herpes simplex virus (HSV) DNA replication. T157602 inhibited reversibly the helicase activity of the HSV UL5-UL8-UL52 (UL5/8/52) helicase-primase complex with an IC₅₀ (concentration of compound that yields 50% inhibition) of 5 μM. T157602 inhibited specifically the UL5/8/52 helicase and not several other helicases. The primase activity of the UL5/8/52 complex was also inhibited by T157602 (IC₅₀ = 20 μM). T157602 inhibited HSV growth in a one-step viral growth assay (IC₉₀ = 3 μM), and plaque formation was completely prevented at concentrations of 25 to 50 μM T157602. Vero, human foreskin fibroblast (HFF), and Jurkat cells could be propagated in the presence of T157602 at concentrations exceeding 100 μM with no obvious cytotoxic effects, indicating that the window between antiviral activity and cellular toxicity is at least 33-fold. Seven independently derived T157602-resistant mutant viruses (four HSV type 2 and three HSV type 1) carried single base pair mutations in the UL5 that resulted in single amino acid changes in the UL5 protein. Marker rescue experiments demonstrated that the UL5 gene from T157602-resistant viruses conferred resistance to T157602-sensitive wild-type viruses. Recombinant UL5/8/52 helicase-primase complex purified from baculoviruses expressing mutant UL5 protein showed complete resistance to T157602 in the in vitro helicase assay. T157602 and its analogs represent a novel class of specific and reversible anti-HSV agents eliciting their inhibitory effects on HSV replication by interacting with the UL5 helicase.Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) each comprise at least 77 genes whose expression is tightly regulated (42). These genes are assigned to four kinetic classes, designated as α, β, γ1, and γ2 on the basis of the timing of and requirements for their expression (46). The five α genes, α0, α4, α22, α27, and α47, are expressed first in the absence of viral protein synthesis and are responsible for the regulated expression of the other viral genes. The β genes require functional α gene products for their expression and encode proteins and enzymes that are directly involved in DNA synthesis and nucleotide metabolism. The γ genes form the last set of viral genes to be expressed, with the γ2 class having viral DNA replication as a strict requirement for their expression.The HSV genome contains three origins of replication (44, 45, 47, 48, 50, 54) and encodes seven viral proteins that are essential for DNA replication (34, 59). These include an origin binding protein (OBP) encoded by open reading frame (ORF) UL9 (14, 15, 17, 35), a DNA binding protein encoded by UL29 (40, 53, 54), a DNA polymerase encoded by ORF UL30 and its accessory factor encoded by UL42 (1, 4, 8, 18, 19, 21, 24, 37), and a heterotrimeric complex consisting of proteins encoded by ORFs UL5, UL8, and UL52, which include both 5′-to-3′ helicase activity and primase activity (10–12). Although extensively studied, the roles of the individual subunits of the helicase-primase complex and their specific interactions with each other have not been completely defined. However, several lines of evidence suggest that the UL5 gene encodes the helicase activity of the complex. Examination of the amino acid sequence of the UL5 protein revealed that it contains six conserved motifs that are found in many DNA and RNA helicases, two of these motifs defining an ATP binding site (20, 25, 32, 52, 61). Site-specific mutagenesis of amino acids within each of the six motifs revealed that all six are critical for the function of the UL5 protein as a helicase in transient replication assays (60, 61).The observation that recombinant UL5, UL52, and UL8 proteins could be purified from baculovirus-infected insect cells as a complex that displays DNA-dependent ATPase, helicase, and primase activities that are identical to those produced during a herpesvirus infection allowed functional and biochemical analyses of the individual components of the complex (10, 13, 38). Although the UL5 protein alone contained the defining helicase amino acid sequence motifs, the UL5 protein does not display helicase activity in vitro in the absence of the UL52 protein. Purified UL5 protein has less than 1% of the ATPase activity of the complex UL5-UL8-UL52 (UL5/8/52) complex (2, 43). In addition, studies with recombinant herpesviruses carrying mutations in the UL5 gene that abolish helicase activity revealed that the UL5 protein could still form specific interactions with UL8 and UL52 proteins (60). These results indicate that the functional domains of UL5 protein required for helicase activity are separate from those involved in protein-protein interactions and that UL5 and UL52 must interact to yield efficient helicase activity. Further mutagenesis studies with the UL52 protein identified mutations that abolish the primase activity of the complex, while the helicase and ATPase activities are unaffected, suggesting that the UL52 protein is responsible for the primase activity of the complex (27). The third component of the helicase-primase complex, the UL8 protein, interacts with other viral replication proteins, including the OBP, the single-stranded DNA binding protein, and the viral DNA polymerase (30, 33). It has been postulated that the interaction of the UL8 protein with the OBP (encoded by the UL9 gene) may function to recruit helicase-primase complexes to initiation complexes at viral origins (30). The UL8 protein is also required for stimulation of primer synthesis by the UL52 protein and for stimulation of the helicase activity of the helicase-primase complex which is crucial to allow efficient unwinding of long stretches of duplex DNA (16, 43, 49). Additionally, UL8 appears to be required for efficient nuclear entry of the helicase-primase complex (1, 3, 31).As the UL5, UL8, and UL52 gene products are essential for HSV replication and have not been exploited previously for antiviral drug discovery, they represent attractive targets for the development of novel anti-HSV agents. Current anti-HSV drugs include vidarabine (adenine arabinoside; Ara-A), foscarnet (phosphonoformic acid; PFA), and a wide variety of nucleoside analogs, the most clinically successful being acyclovir (ACV) and its analogs valacyclovir and famciclovir. ACV is phosphorylated by viral thymidine kinase (TK) to its monophosphate form, an event that occurs to a much lesser extent in uninfected cells. Subsequent phosphorylation events by cellular enzymes convert the ACV monophosphate to its triphosphate form. The ACV triphosphate derivative directly inhibits the DNA polymerase by competing as a substrate with dGTP. Because the ACV triphosphate lacks the 3′ hydroxyl group required to elongate the DNA chain, DNA replication is terminated. The triphosphorylated form of ACV is a much better substrate for the viral DNA polymerase than it is for the cellular DNA polymerase; thus, very little ACV triphosphate is incorporated into cellular DNA. Although ACV has proven to be safe and successful at reducing the duration, severity, and in some cases recurrence of HSV infections, eradication of the infection symptoms is far from complete and latent virus can reactivate frequently (55–58). In addition, primarily as a result of poor patient compliance with inconvenient ACV dosage regimens, virulent HSV strains resistant to ACV that contain mutations in either the viral TK or DNA polymerase gene have arisen (6, 7, 9, 26, 39). More potent and efficacious drugs that target other essential components of the virus replicative cycle would be invaluable as therapeutic agents to treat HSV and ACV-resistant HSV infections.To identify novel inhibitors of the HSV helicase-primase enzyme, we developed a high-throughput in vitro helicase assay and screened >190,000 samples. Using this biochemical approach, we identified T157602, a 2-amino thiazole, as a specific inhibitor of HSV replication. By generating and analyzing T157602-resistant viruses, we further demonstrate genetically that the molecular target of T157602 is the UL5 component of the HSV helicase-primase complex.     

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.     

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.     

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.     

α1-AMP-Activated Protein Kinase Regulates Hypoxia-Induced Na,K-ATPase Endocytosis via Direct Phosphorylation of Protein Kinase Cζ

Hypoxia promotes Na,K-ATPase endocytosis via protein kinase Cζ (PKCζ)-mediated phosphorylation of the Na,K-ATPase α subunit. Here, we report that hypoxia leads to the phosphorylation of 5′-AMP-activated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK α subunit (AMPK-DN) construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. The overexpression of the reactive oxygen species (ROS) scavenger catalase prevented hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondrion-deficient ρ⁰-A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKCζ translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK α phosphorylates PKCζ on residue Thr410 within the PKCζ activation loop. Importantly, the activation of AMPK α was necessary for hypoxia-induced AMPK-PKCζ binding in alveolar epithelial cells. The overexpression of T410A mutant PKCζ prevented hypoxia-induced Na,K-ATPase endocytosis, confirming that PKCζ Thr410 phosphorylation is essential for this process. PKCζ activation by AMPK is isoform specific, as small interfering RNA targeting the α1 but not the α2 catalytic subunit prevented PKCζ activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK α1 isoform, which binds and directly phosphorylates PKCζ at Thr410, thereby promoting Na,K-ATPase endocytosis.When exposed to low oxygen levels (hypoxia), cells develop adaptative strategies to maintain adequate levels of ATP (21). These strategies include increasing the efficiency of energy-producing pathways, mostly through anaerobic glycolysis, while decreasing energy-consuming processes such as Na,K-ATPase activity (30). Alveolar hypoxia occurs in many respiratory disorders, and it has been shown to decrease epithelial active Na⁺ transport, leading to impaired fluid reabsorption (37, 41, 42). Active Na⁺ transport and, thus, alveolar fluid reabsortion are effected mostly via apical sodium channels and the basolateral Na,K-ATPase (32, 38, 42). We have reported previously that hypoxia inhibits Na,K-ATPase activity by promoting its endocytosis from the plasma membrane by a mechanism that requires the generation of mitochondrial reactive oxygen species (ROS) and the phosphorylation of the Na,K-ATPase α subunit at Ser18 by protein kinase Cζ (PKCζ) (8, 9).The 5′-AMP-activated protein kinase (AMPK) is a heterotrimeric Ser/Thr kinase composed of a catalytic α subunit and regulatory β and γ subunits. Both isoforms of the AMPK catalytic subunit (α1 and α2) form complexes with noncatalytic subunits. The α1 subunit is ubiquitously expressed, whereas the α2 subunit isoform is expressed predominantly in tissues like the liver, heart, and skeletal muscle (36). The α1 and α2 subunit isoforms have ∼90% homology in their N-terminal catalytic domains and ∼60% homology in their C-terminal domains (36), suggesting that they may have distinct downstream targets (31). AMPK activation requires phosphorylation at Thr172 in the activation loop of the α subunit by upstream kinases (12, 19). Findings from recent studies suggest that AMPK is an important signaling intermediary in coupling ion transport and metabolism (15). Indeed, it has been reported that the pharmacological activation of AMPK inhibits amiloride- and ouabain-sensitive epithelial Na⁺ transport (15). Moreover, the activities of the epithelial Na⁺ channel (ENaC) (2, 17), the Na,K-ATPase (40), and the cystic fibrosis transmembrane conductance regulator (17) have been shown to be inhibited by AMPK. Here, we provide evidence that hypoxia, via mitochondrial ROS, leads to AMPK activation and that AMPK binds to and directly phosphorylates PKCζ in an isoform-specific manner, thus promoting Na,K-ATPase endocytosis in alveolar epithelial cells (AEC).     

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