The Role of PP2A A Subunits in Tumor Suppression

The protein phosphatase 2A (PP2A) family of heterotrimeric serine-threonine phosphatases participates in human cell transformation. Each functional PP2A complex contains one structural A subunit (Aα or Aβ), and mutations of both are found to occur at low frequency in human tumors. We have shown that Aα functions as haploinsufficient tumor suppressor gene by regulating in part phosphatidylinositol 3-kinase (PI3K) signaling. In contrast, loss of Aβ function due to biallelic alterations contributes to cancer progression through dysregulation of small GTPase RalA activity. These observations provide evidence that dysfunction of particular PP2A complexes regulate specific phosphorylation event necessary for cancer initiation.Key Words: protein phosphatase 2A, RalA, cancer, transformationReversible phosphorylation plays a key role in the regulation of signaling pathways relevant to cell transformation. Dysregulation of several kinase oncogenes have been shown to be required for cancer development, and several targeted therapies focused on inhibiting particular kinases have now been approved for clinical use. Although it is clear that phosphorylation is also regulated by phosphatases, initial biochemical studies suggested that unlike kinases, phosphatases act promiscuously and constitutively in vitro. However, recent work indicates that phosphatases play essential roles in malignant transformation by acting on specific substrates in vivo.Protein phosphatase 2A (PP2A) is a family of serine-threonine phosphatases implicated in the control of a diverse array of cellular processes. The PP2A core enzyme consists of a catalytic C subunit and a structural A subunit. In mammals, two distinct genes encode closely related versions of both the PP2A A and C subunits. The AC dimer recruits a third regulatory B subunit that has been predicted to dictate the substrate specificity and function of the PP2A heterotrimeric complex. Four unrelated families of B subunits have identified to date: B/B55/PR55/PPP2R2, B′/B56/PR61/PPP2R5, B″/PR72/PPP2R3 and Striatin¹ (Fig. 1). Recent genetic and proteomic studies implicate clear roles for PP2A subunits in regulating physiological functions and one emerging view is that specific PP2A complexes play critical roles in cell transformation by regulating particular substrates.Open in a separate windowFigure 1Disruption of PP2A complexes induces transformation. PP2A is a heterotrimeric protein complex, and several isoforms exist for each of the three subunits, creating a diverse family of related enzymes that regulate specific physiological functions. Alterations of PP2A structural subunits, Aα and Aβ, contribute to spontaneously arising human cancers by distinct mechanisms. Cancer-associated Aα haploinsufficiency may induce human cell transformation by activating PI3K/AKT pathway while PP2A Aβ loss-of-function permits the accumulation of activated RalA.Somatic alterations of the PP2A structural subunit Aβ (PPP2R1B) have been found to occur in colon, lung and breast cancers.^2–5 Notably, point mutations in one Aβ allele are commonly accompanied by loss of the second Aβ allele. We confirmed previous work⁶ that showed cancer-associated Aβ mutants form functionally null alleles.⁷ These studies indicate that Aβ is genetically inactivated in a subset of human cancers. In addition, we found that suppression of Aβ was found to cooperate with H-Ras, telomerase catalytic subunit hTERT and the SV40 Large T antigen to induce transformation of normal human cells while introduction of wild type Aβ into lung carcinoma cells lacking functional Aβ partially reverses this tumorigenic phenotype.⁷ Together, these data provide evidence that PP2A Aβ functions as a tumor suppressor gene.Previous work has shown cancer derived Aβ mutants exhibit markedly impaired ability to form complexes with the catalytic C subunit and the regulatory PR72 subunit.⁶ We have found that Aβ mutants also showed decreased ability to bind to regulatory Bα subunit and several members of B′ family. These data indicate that cancer-associated alterations of PP2A Aβ result in disruption of most if not all PP2A Aβ-containing complexes. Considering that distinct Aβ-B complexes are likely regulate the phosphorylation of particular substrates involved in transformation, further work is required to identify which B subunits participate in malignant transformation.Somatic mutations of the more abundant PP2A structural Aα subunit have also been reported in human cancers, although at low frequency.^2,8 We previously showed that cancer-associated PP2A Aα mutations contribute to cell transformation by creating a state of haploinsufficiency.⁹ Although these two distinct PP2A structural isoforms, Aα and Aβ, are 86% identical,¹⁰ it was unclear whether these two isoforms share overlapping functions.¹¹ We found that overexpression of Aα failed to revert the tumorigenic phenotype induced by Aβ suppression, suggesting that PP2A complexes containing Aα or Aβ are functionally distinct.To identify substrates specific for PP2A Aβ, we performed large scale immunopurification of PP2A Aα- and Aβ-containing complexes. We have found that PP2A Aβ complex, but not the PP2A Aα complex, binds to and inhibits activity of the small GTPase RalA through direct dephosphorylation at Ser183 and Ser 194. Cancer-associated Aβ mutants are unable to dephosphorylate RalA, suggesting that loss of Aβ function impairs the formation of complexes with RalA and deregulates its activity. Consistent with previous reports that implicated RalA in regulation of several signaling pathways relevant to cell transformation,^12–14 loss of function experiments revealed that RalA is crucial for transformation mediated by Aβ dysfunction. These findings strongly suggest that accumulation of phospho-RalA in PP2A Aβ deficient cells promotes tumorigenic phenotype (Fig. 1). However, we cannot exclude that other substrates of PP2A Aβ complexes also contribute to cell transformation.These observations also implicate phosphorylation of RalA as an alternative mechanism that may regulate RalA activity and cell transformation. Prior work has shown Aurora A kinase as one kinase that can induce RalA phosphorylation at Ser 194.¹⁵ However, further studies are required to identify the kinase(s) that are responsible for RalA phosphorylation at Ser 183 and Ser 194.While Aβ loss-of-function permits the accumulation of activated RalA, Aα haploinsufficiency seems to induce human cell transformation by activating AKT/PI3K signaling pathway⁹ (Fig. 1). However, it remains unclear whether PP2A A subunits determine the substrate specificity of heterotrimeric complexes by direct substrate binding, or by forming complex with particular set of B and C subunits. In consonance with the latter idea, Aα and Aβ have been reported to have different affinity to Cα, Bα, B''α1 and PR72 subunits.¹⁷ The systematic characterization of PP2A complex composition necessary for RalA dephosphorylation and Akt activation and further structural studies to resolve PP2A in complex with specific substrates will help elucidate the mechanistic details of how PP2A acts as a tumor suppressor.     

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.     

The role of STEP in Alzheimer disease

Amyloid beta (Aβ), the putative causative agent in Alzheimer disease, is known to affect glutamate receptor trafficking. Previous studies have shown that Aβ downregulates the surface expression of N-methyl D-aspartate type glutamate receptors (NMDARs) by the activation of STriatal-Enriched protein tyrosine Phosphatase 61 (STEP₆₁). More recent findings confirm that STEP₆₁ plays an important role in Aβ-induced NMDAR endocytosis. STEP levels are elevated in human AD prefrontal cortex and in the cortex of several AD mouse models. The increase in STEP₆₁ levels and activity contribute to the removal of GluN1/GluN2B receptor complexes from the neuronal surface membranes. The elevation of STEP₆₁ is due to disruption in the normal degradation of STEP₆₁ by the ubiquitin proteasome system. Here, we briefly discuss additional studies in support of our hypothesis that STEP₆₁ contributes to aspects of the pathophysiology in Alzheimer''s disease. Exogenous application of Aβ-enriched conditioned medium (7PA2-CM) to wild-type cortical cultures results in a loss of GluN1/GluN2B subunits from neuronal membranes. Abeta-mediated NMDAR internalization does not occur in STEP knock-out cultures, but is rescued by the addition of active TAT-STEP to the cultures prior to Aβ treatment.Key words: Alzheimer disease, amyloid beta, NMDA receptor, protein tyrosine phosphatases, STEP, synaptic plasticityIn Alzheimer disease (AD), the abnormal accumulation of soluble Aβ peptides has a profound impact on cognitive function.¹ Aβ peptides disrupt synaptic plasticity, a molecular mechanism involved in learning and memory.^2,3 N-methyl D-aspartate type glutamate receptors (NMDAR) play an important role in the development of synaptic strengthening. Aβ downregulates the surface expression of NMDARs by activation of STriatal-Enriched protein tyrosine Phosphatase 61 (STEP₆₁).⁴ STEP₆₁ is a brain-specific phosphatase that opposes the development of synaptic strengthening.⁵ STEP₆₁ is present in postsynaptic terminals, immunoprecipitates with the NMDAR complex and decreases NMDA channel function.^6,7 The reduced channel function is mediated, at least in part, by an increased internalization of the NMDAR complex, as STEP dephosphorylates the GluN2B subunit at a regulatory tyrosine (tyr¹⁴⁷²) leading to NMDAR endocytosis. Knocking down STEP with interfering RNA increases NMDAR trafficking to synaptic membranes.^4,8 A previous study suggested that Aβ leads to the activation of STEP through a calcineurin-mediated pathway, which subsequently increased internalization of surface NMDAR.⁴ A recent study has demonstrated that STEP is also regulated by the ubiquitin proteasome system, and an Aβ-mediated disruption of the proteasome leads to increased STEP₆₁ levels in human Alzheimer''s disease (AD) brains and AD mouse models.⁹ Taken together, these studies suggest that an increase in the activity of STEP₆₁ contributes to the cognitive deficits in AD by increasing the internalization of NMDAR from synaptic membrane surfaces.     

Plant defensins: Defense,development and application

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

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

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

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?     

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.     

Molecular functions of the PP2A regulatory subunit Tap46 in plants

Tap42/α4 is a regulatory subunit of the protein phosphatase 2A (PP2A) family of phosphatases and plays a role in the target of rapamycin (TOR) pathway that regulates cell growth, ribosome biogenesis, translation and cell cycle progression in both yeast and mammals. We determined the cellular functions of Tap46, the plant homolog of Tap42/α4, in both Arabidopsis thaliana and Nicotiana benthamiana. Tap46 associated with the catalytic subunits of PP2A and the PP2A-like phosphatases PP4 and PP6 in vivo. Tap46 was phosphorylated by TOR in vitro, indicating that Tap46 is a direct substrate of TOR kinase. Tap46 deficiency caused cellular phenotypes that are similar to TOR-depletion phenotypes, including repression of global translation and activation of both autophagy and nitrogen recycling. Furthermore, Tap46 depletion regulated total PP2A activity in a time-dependent manner similar to TOR deficiency. These results suggest that Tap46 acts as a positive effector of the TOR signaling pathway in controlling diverse metabolic processes in plants. However, Tap46 silencing caused acute cell death, while TOR silencing only hastened senescence. Furthermore, mitotic cells with reduced Tap46 levels exhibited chromatin bridges at anaphase, while TOR depletion did not cause a similar defect. These findings suggest that Tap46 may have TOR-independent functions as well as functions related to TOR signaling in plants.Key words: acute cell death, autophagy, chromatin bridge, nitrogen mobilization, protein phosphatases, target of rapamycin (TOR)Yeast type 2A phosphatase-associated protein 42 kDa (Tap42) is a regulatory subunit that directly associates with catalytic subunits of the protein phosphatase 2A (PP2A) family of protein phosphatases to make a heterodimer and regulates the activity and substrate specificity of the intact enzyme complex.¹ Functions of Tap42 as a component of the target of rapamycin (TOR) signaling pathway have been well characterized in yeast.^1–3 Tap42-regulated phosphatase activities play a major role in signal transduction mediated by TOR. Accumulating evidence suggest that TOR regulates phosphorylation of target proteins by restraining PP2A activity through Tap42 phosphorylation.^1–3 Rapamycin inhibits TOR activity and also influences Tap42-mediated phosphatase regulation in yeast.^3–5α4, the mammalian homolog of Tap42, also associates with the catalytic subunits of PP2A, PP4 and PP6 to make a heterodimer.⁶ Rapamycin inhibits mammalian TOR (mTOR) activity, but it is not clear whether rapamycin prevents the formation of the α4/PP2Ac complex or whether α4 stimulates or represses PP2Ac activity.^7–9 Interestingly, loss of Tap42 function in Drosophila does not affect TOR-regulated activities, including cell growth, metabolism and S6 kinase activity, but results in mitotic arrest caused by spindle anomalies and subsequent activation of c-Jun N-terminal kinase signaling and apoptosis.¹⁰ Similarly, α4 deletion in mice leads to the rapid onset of apoptosis in both proliferating and differentiated cells, while rapamycin itself does not severely affect adult cells.¹¹ Furthermore, while TOR depletion causes developmental arrest and organ degeneration at the L3 stage in Caenorhabditis elegans, loss of α4 does not reproduce TOR deficiency phenotypes, but mainly leads to a fertility defect.¹² Taken together, these results suggest that the yeast Tap42/TOR paradigm is not completely conserved in higher eukaryotes and that Tap42/α4 functions may not be exclusively dependent on the Tor signaling pathway.In this study, we investigated the in vivo functions and phosphatase regulation of Tap46, the plant Tap42/α4 homolog, in relation to TOR in Nicotiana benthamiana, Arabidopsis and tobacco BY2 cells. Tap46 was shown to interact with the catalytic subunits of PP2A, PP4 and PP6 in vivo. Recombinant Tap46 protein was phosphorylated by immunoprecipitated TOR kinase and its deletion forms in vitro. Dexamethasone-induced RNAi of Tap46 caused dramatic repression of global translation and activation of both autophagy and nitrogen mobilization in the early stages of gene silencing. These phenotypes mimic those of TOR inactivation or TOR deficiency in Arabidopsis, yeast and mammals, indicating that Tap46 is a critical mediator of the Tor pathway in the regulation of these metabolic processes in plants. However, these early phenotypes of Tap46-deficient plants were soon followed by an acute and rapid programmed cell-death (PCD), while TOR silencing only led to growth retardation and premature senescence in Arabidopsis and N. benthamiana, confirming results from a previous study.¹³ The PCD caused by Tap46 deficiency is consistent with the apoptosis induced by loss of Tap42/α4 function in both Drosophila and mice.^10,11 Thus Tap42/α4/Tap46 appears to have a strong anti-apoptotic activity in higher eukaryotes. The underlying mechanisms of PCD activation caused by Tap46 depletion remain to be revealed, but it is possible that the inappropriate modulation of phosphatase activity and aberrant protein phosphorylation led to stress signaling and PCD activation.Another interesting phenotype of Tap46 deficiency is the formation of chromatin bridges in anaphase during mitosis, suggesting a role for Tap46 in plant cell mitotic progression. However, there have been no reports of anaphase bridge formation in tor mutants of any organisms. In Drosophila, loss of Tap42 function causes spindle disorganization and pre-anaphase arrest prior to the onset of apoptosis.¹⁰ In addition, Drosophila mutants with a defective regulatory subunit of PP2A exhibit an increased number of lagging chromosomes and chromatin bridges in anaphase.^14,15 Tap46 likely regulates the functions of PP2A family phosphatases during mitosis by direct association with their catalytic subunits, thereby modulating both the activity and specificity of the enzyme. Accumulating evidence reveals dynamic functions of PP2A during mitosis in both yeast and mammals: PP2A regulates kinetochore function, sister chromatid cohesion, spindle bipolarity and progression to anaphase.^15–17 Counteracting the activity of protein kinases, PP4 has also been implicated in both centrosome maturation and function during mitosis.¹⁸ Based on immunolabeling results, Tap46 was visualized as distinct spots around chromatin and mitotic spindles during mitosis in tobacco BY2 cells (Lee HS and Pai HS, unpublished results). Further studies will address the interacting partners and dynamic relocation of Tap46 during the cell cycle.Our results in this study demonstrated that Tap46 plays an important regulatory role in plant growth and metabolism; a major part of its function appears related to TOR signaling. However, we consistently observed certain phenotypic differences between Tap46-silenced and TOR-silenced Arabidopsis and N. benthamiana plants: an acute and rapid PCD occurred upon Tap46 silencing but not upon TOR silencing, despite a similar degree of gene silencing. Furthermore, we did not observe anaphase bridge formation in mitotic root-tip cells of ethanol-induced TOR RNAi Arabidopsis plants, while chromatin bridges were repeatedly observed in Tap46-silenced tobacco BY2 and Arabidopsis root-tip cells. Although an ancient Tap42/TOR paradigm observed in yeast appears to be conserved in plants, new TOR-independent functions of Tap46 might have evolved, the abrogation of which can cause massive PCD activation and anaphase bridge formation. Tap46 is a major regulator of cellular PP2A activity in plant cells by interacting with multiple phosphatase partners. Unraveling the molecular networks of Tap46 activity and interactions is essential for understanding its TOR-dependent and -independent functions in plants.     

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.     

Protein Misfolding and the Serpinopathies

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

Cerebral Amyloid Angiopathy in an Aged Sooty Mangabey (Cercocebus atys)

A 26-y-old male sooty mangabey (Cercocebus atys) was found at necropsy to have a moderate degree of cerebral amyloid β (Aβ) angiopathy in superficial and parenchymal blood vessels of the brain. Senile (Aβ) plaques were absent, as were neurofibrillary tangles and other signs of neurodegeneration. Affected blood vessels were arterial, capillary, and, less frequently, venous in nature. Histologically, the Aβ40 isoform was more prevalent than was Aβ42. As in humans but unlike in squirrel monkeys, the density of lesions in this mangabey increased along a rostral-to-caudal gradient. Therefore mangabeys appear to conform to the general tendency of nonhuman primates by developing cerebral Aβ angiopathy in the absence of other indices of Alzheimer-type neuropathology.Abbreviations: Aβ, amyloid β, CAA, cerebral amyloid angiopathy, GFAP, glial fibrillary acidic protein, Iba 1, microglia-expressed calcium-binding proteinOne of the most common microvasculopathies in the aging human brain is cerebral amyloid angiopathy (CAA), a disorder in which various aggregation-prone proteins accumulate in the walls of parenchymal and meningeal blood vessels.^4,9 Most often, the amyloidogenic protein is amyloid β (Aβ), a cleavage product of the Aβ precursor protein and the essential component of senile plaques in Alzheimer disease.^13,43 In the brain vasculature, the basal lamina is a primary site of Aβ deposition.^25,35 Severely affected arterioles show a loss of smooth muscle cells in the tunica media, a weakening of the vascular wall and a propensity to rupture.^3,34 CAA thus increases the risk of intracerebral bleeding and may be responsible for as much as 20% of nontraumatic hemorrhagic stroke in elderly humans.^15,18,35 CAA is present to various degrees in virtually all cases of Alzheimer disease,^15,16,21 but it also occurs independently.²⁴ As is the case for other proteopathies, advancing age is a significant risk factor for CAA.^8,19In humans, CAA most often affects the arteries and arterioles of the brain, particularly those in the leptomeninges and cortex.^2,25 CAA is less frequent in veins and capillaries,²⁵ but capillary CAA can be prominent in some cases.^26,33 The occipital lobe is affected most often^1,32,37 but all cortical regions are vulnerable. CAA is variable in occurrence in the cerebellum and uncommon in deep telencephalic gray structures, white matter, and the brainstem,³⁶ except in severely affected cases.³²Although its specific role in the pathogenesis of Alzheimer disease remains uncertain, there is now strong evidence that dementia is exacerbated by CAA.¹⁴ Furthermore, CAA is independently linked to cognitive decline both in rare familial cases²⁰ and in older humans with idiopathic CAA.^2,20 Despite the prevalence of cerebrovascular amyloidosis in elderly humans, surprisingly little is known about its effect on the brain, in part because of a paucity of natural animal models that closely mimic the human disorder.^17,38Nonhuman primates offer a unique opportunity to view CAA from a comparative perspective, given that they normally generate human-sequence Aβ and develop severe cerebral Aβ amyloidosis in old age, generally in the absence of other changes that characterize Alzheimer disease.¹² Nonhuman primates have the additional advantage that, compared with humans, their relatively small brains enable exhaustive regional analysis of microscopic lesions, something that, for practical reasons, is seldom undertaken in the human brain. Here we present the first investigation of age-associated brain changes in sooty mangabeys, focusing in particular on Aβ deposition and related abnormalities. One of the 2 aged mangabeys analyzed had Aβ deposition in the brain which was almost exclusively in the form of CAA. Remarkably, the vessel types affected and the regional distribution of CAA more closely resembled the pattern seen in humans than that in other nonhuman primates, particularly squirrel monkeys.⁶ Differences and similarities in CAA among primate species could provide fresh insights into the development of cerebral amyloidosis and related disorders in older humans.     

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     

Cell Migration from Baby to Mother

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