Translational control of long-term synaptic plasticity and memory storage by eIF2alpha

Both long-lasting changes in synaptic function and long-term memory require gene expression. However, the molecular mechanisms by which gene expression is turned on are not fully understood. In this review, we highlight the role of the eukaryotic initiation factor 2 alpha (eIF2alpha) signalling pathway in long-term synaptic plasticity and memory.     

Translational resistance of late alphavirus mRNA to eIF2alpha phosphorylation: a strategy to overcome the antiviral effect of protein kinase PKR

The double-stranded RNA-dependent protein kinase (PKR) is one of the four mammalian kinases that phosphorylates the translation initiation factor 2alpha in response to virus infection. This kinase is induced by interferon and activated by double-stranded RNA (dsRNA). Phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) blocks translation initiation of both cellular and viral mRNA, inhibiting virus replication. To counteract this effect, most viruses express inhibitors that prevent PKR activation in infected cells. Here we report that PKR is highly activated following infection with alphaviruses Sindbis (SV) and Semliki Forest virus (SFV), leading to the almost complete phosphorylation of eIF2alpha. Notably, subgenomic SV 26S mRNA is translated efficiently in the presence of phosphorylated eIF2alpha. This modification of eIF2 does not restrict viral replication; SV 26S mRNA initiates translation with canonical methionine in the presence of high levels of phosphorylated eIF2alpha. Genetic and biochemical data showed a highly stable RNA hairpin loop located downstream of the AUG initiator codon that is necessary to provide translational resistance to eIF2alpha phosphorylation. This structure can stall the ribosomes on the correct site to initiate translation of SV 26S mRNA, thus bypassing the requirement for a functional eIF2. Our findings show the existence of an alternative way to locate the ribosomes on the initiation codon of mRNA that is exploited by a family of viruses to counteract the antiviral effect of PKR.     

Interactions formed by individually expressed TAP1 and TAP2 polypeptide subunits

The transporter associated with antigen processing (TAP) supplies peptides into the lumen of the endoplasmic reticulum (ER) for binding by major histocompatibility complex (MHC) class I molecules. TAP comprises two polypeptides, TAP1 and TAP2, each a 'half-transporter' encoding a transmembrane domain and a nucleotide-binding domain. Immunoprecipitation of rat TAP1 and TAP2 expressed individually in the human TAP-deficient cell line, T2, revealed that both bound the endogenously expressed HLA-A2 and -B51 class I molecules. Using HLA-encoding recombinant vaccinia viruses HLA-A*2501, -B*2704, -B*3501 and -B*4402, alleles also associated with both TAP1 and TAP2. Thus, TAP1 and TAP2 do not appear to differ in their ability to interact with MHC class I alleles. Single TAP polypeptide subunits also formed MHC class I peptide-loading complexes, and their nucleotide-binding domains retained the ability to interact with ATP, and may permit the release of peptide-loaded MHC class I molecules in the absence of a peptide transport cycle. It is also demonstrated by chemical cross-linking that TAP2, but not TAP1, has the ability to form a homodimer complex both in whole cells and in detergent lysates. Together these data indicate that single TAP polypeptide subunits possess many of the features of the TAP heterodimer, demonstrating them to be useful models in the study of ATP-binding cassette (ABC) transporters.     

ATP-induced mitogenesis is modulated by phospholipase D2 through extracellular signal regulated protein kinase dephosphorylation in rat pheochromocytoma PC12 cells.

Extracellular ATP has been known to have many functions as a fast transmitter, and a co-transmitter, and to have morphogenic and mitogenic activity in neuronal cells. Although it was reported that ATP activates phospholipase D (PLD), the role of PLD versus the ATP function was unclear in neuronal cells. In this study, we investigated the role of PLD on the ATP-induced extracellular signal regulated protein kinase (ERK) activation and mitogenic effect in rat pheochromocytoma PC12 cells. In these cells ATP caused PLD2 activation and ERK phosphorylation, which was dramatically reduced by wild-type PLD2-overexpression but not by lipase-inactive-mutant PLD2-overexpression. The accumulation of phosphatidic acid (PA) by preincubating PC12 cells with propranolol (an inhibitor of PA phosphohydrolase) also decreased the ERK phosphorylation. Inhibition of phosphatases by okadaic acid or pervanadate completely blocked PLD2-dependent ERK dephosphorylation. In addition, ATP-stimulated thymidine incorporation was reduced by the overexpression of wild-type PLD2, but not by the overexpression of lipase-inactive-mutant PLD2. Okadaic acid pretreatment overcame the decrease of ATP-induced thymidine incorporation by PLD2 overexpression. Taken together, we suggest that PLD2 activity might play a negative role in ATP-induced ERK phosphorylation and mitogenic signal possibly through phosphatases.     

Polycystin-2 down-regulates cell proliferation via promoting PERK-dependent phosphorylation of eIF2alpha

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of renal, hepatic and pancreatic cysts and by non-cystic manifestations such as abnormal vasculature and embryo left-right asymmetry development. Polycystin-2 (PC2), in which mutations account for 10-15% of ADPKD, was previously shown to down-regulate cell proliferation, but the underlying mechanism was not elucidated. Here, we demonstrate that PC2, but not pathogenic mutants E837X and R872X, represses cell proliferation through promoting the phosphorylation of eukaryotic translation initiation factor eIF2alpha by pancreatic ER-resident eIF2alpha kinase (PERK). ER stress is known to enhance eIF2alpha phosphorylation through up-regulating PERK kinase activity (assessed by phosphorylated PERK). During ER stress, PC2 knockdown also repressed eIF2alpha phosphorylation but did not alter PERK phosphorylation, indicating that PC2 facilitates the eIF2alpha phosphorylation by PERK. PC2 was found to be in the same complex as PERK and eIF2alpha. Together, we demonstrate that PC2 negatively controls cell growth by promoting PERK-mediated eIF2alpha phosphorylation, presumably through physical interaction, which may underlie a pathogenesis mechanism of ADPKD and indicates that PC2 is an important regulator of the translation machinery.     

Semaphorin controls epidermal morphogenesis by stimulating mRNA translation via eIF2alpha in Caenorhabditis elegans

Conserved semaphorin-plexin signaling systems govern various aspects of animal development, including axonal guidance in vertebrates and epidermal morphogenesis in Caenorhabditis elegans. Here we provide in vivo evidence that stimulation of mRNA translation via eukaryotic initiation factor 2alpha (eIF2alpha) is an essential downstream event of semaphorin signaling in C. elegans. In semaphorin/plexin mutants, a marked elevation in the phosphorylation of eIF2alpha is observed, which causes translation repression and is causally related to the morphological epidermal phenotype in the mutants. Conversely, removal of constraints on translation by genetically reducing the eIF2alpha phosphorylation largely bypasses requirement for the semaphorin signal in epidermal morphogenesis. We also identify an actin-depolymerizing factor/cofilin, whose expression in the mutants is predominantly repressed, as a major translational target of semaphorin signaling. Thus, our results reveal a physiological significance for translation of mRNAs for cytoskeletal regulators, linking environmental cues to cytoskeletal rearrangement during cellular morphogenesis in vivo.     

Aberrant glycosylation modulates phosphorylation of tau by protein kinase A and dephosphorylation of tau by protein phosphatase 2A and 5

Microtubule-associated protein tau is abnormally hyperphosphorylated, glycosylated, and aggregated in affected neurons in Alzheimer's disease (AD). We recently found that the aberrant tau glycosylation precedes tau hyperphosphorylation in AD brain. In the present study, we developed assays to determine phosphorylation and dephosphorylation of tau at specific phosphorylation sites by using glycosylated tau purified from AD brain as a substrate. We then studied the effects of the aberrant glycosylation on phosphorylation and dephosphorylation of tau at each specific phosphorylation site. We found that deglycosylation of the aberrantly glycosylated tau decreased the subsequent phosphorylation of tau at Ser214, Ser262, and Ser356 in vitro by protein kinase A. On the other hand, deglycosylation of tau positively modulated the subsequent dephosphorylation by protein phosphatase 2A and protein phosphatase 5 in vitro at the phosphorylation sites Ser198, Ser199, and Ser202.Our results suggest that the aberrant glycosylation may modulate tau protein at a substrate level so that it is easier to be phosphorylated and more difficult to be dephosphorylated at some phosphorylation sites in AD brain. The combined impact of this modulation may be to make tau more susceptible to becoming abnormally hyperphosphorylated.     

Polymorphisms of TAP 1 and TAP2 genes in Graves' disease

Graves' disease is an autoimmune disorder in which HLA DQA1*0501 and DQB1*0201 confer predisposition. The genes for transporters associated with antigen processing (TAP1 and TAP2) locate near to HLA DQ coding regions and display only a limited degree of polymorphism. Since polymorphisms of TAP might influence susceptibility to Graves' disease by a possibly different selection of antigenic peptides, we investigated sequence variants of TAP1 and TAP2 genes in 235 patients with Graves' disease and 218 random healthy controls by polymerase chain reaction (PCR) followed by sequence specific oligonucleotide analysis (SSO), single strand conformational polymorphism (SSCP) analysis and amplification refractory mutation system (ARMS). TAP1*0301 (Val-333/Asp-637: 71% vs. 55% in controls. p< .0008, RR=2.05) and TAP2*0101 (Val-379/Ala-565/Thr-665/stop-687: 83% vs. 69% in controls, p< .003, RR=2.20) showed a positive association with Graves' disease whereas TAP1*0401 a negative (Ile-333/Gly-637: 4% vs. 13% in controls, p< .001, RR=0.25). After selection of patients and controls for HLA DQA1*0501 a similar association was found for TAP1*0301 (72% vs. 50% in controls, p< .002, RR=2.63) and TAP1*0401 (4% vs. 16% in controls, p< .004, RR=0.22), when matching for HLA DQB1*0201 as well as for TAP1*0401 (3% vs. 16% in controls, p< .005, RR=0.18). Our findings indicate that the positive association of TAP1*0301 and the negative of TAP1*0401 with Graves' disease cannot only be explained by linkage disequilibrium between TAP alleles and HLA DQ. Therefore, these TAP alleles contribute to genetic susceptibility in Graves' disease as additional permissive and protective factors.     

Frequencies of TAP1 and TAP2 gene polymorphisms in the Anatolian population

Anatolia has a long and complex record of immigration from various regions. Here, we have used TAP1 and TAP2 gene polymorphisms as genetic markers to study the relationship between the Anatolian population and other populations. A neighbour‐joining tree was constructed indicating the relatedness of European populations and the Anatolian population with respect to TAP1 and TAP2 allele frequencies.     

Polymorphisms of the TAP1 and TAP2 genes in human alveolar echinococcosis

We postulated that TAP genes may influence the susceptibility of some individuals to Echinococcus multilocularis infection. Six coding region variants (codons 333 and 637 in TAP1, and 379, 565, 651 and 665 in TAP2) were typed in 94 patients and 100 controls. Thr/Thr homozygosity at TAP2/665 was more prevalent in patients than in controls [64% vs. 45%, respectively; odds ratio (OR) = 2.1 (95% confidence interval (CI) 1.1; 2.7)] and Thr/Ala heterozygozity was less prevalent (32% vs. 50%, respectively) (P = 0.014). Of the 38 patients with progressive lesions, 76% were Thr/Thr, as compared with 55% of patients without progressive lesions and 45% of controls (P = 0.058 and 0.02, respectively), independent of HLA status. To determine whether this association is functionally relevant, functional analyses and/or confirmation in distinct populations of patients with alveolar echinococcosis would be required.     

Polymorphism of TAP1 and TAP2 in Japanese patients with rheumatoid arthritis

Contribution of polymorphism of transporter associated with antigen processing 1 and 2 (TAP1 and 2) alleles to pathogenesis of Japanese rheumatoid arthritis (RA) was studied in 92 RA patients by PCR-RFLP. The allele frequency of TAP2A was slightly low (38.0%) and the frequencies of TAP2B and TAP2C were slightly high (39.7% and 17.9%) in RA, but these differences were not significant. These increases and decrease were due to the positive or negative associations with HLA-DRB1*0405. It was very likely that slight differences in TAP2A, TAP2B and TA2C in RA were secondary phenomenon reflecting an increase in HLA-DRB1*0405. The prevalence of TAP2E allele was low (3.3%, P <0.01, P_c=not significant) and not correlated with HLA-DRB1*0405.     

Determination of TAP1 and TAP2 polymorphism in the Chinese Han population by real-time TaqMan polymerase chain reaction

The heterodimeric transporter associated with antigen processing (TAP) complex plays a key role in immune surveillance. TAP1 and TAP2 typing was usually performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism and PCR-sequence-specific oligonucleotide probe. As an alternative to these methods, we have established TaqMan assays to determine the frequencies of the TAP1 and TAP2 alleles. We have used these new TaqMan assays to genotype the polymorphisms in 339 unrelated Chinese Hans residing in North and South China. We detected five TAP1 and four TAP2 alleles. All the loci conform to the Hardy-Weinberg expectations. The most frequent alleles in Chinese Hans were TAP1*0101 (79.79%) and TAP2*0101 (82.74%). The two-locus haplotype analysis showed highly significant positive linkage disequilibrium for one TAP1-TAP2 haplotype (TAP1*020101-TAP2*0102), three TAP1-DRB1 haplotypes (TAP1*020101-DRB1*03, TAP1*020102-DRB1*13, and TAP1*0301-DRB1*16), and three TAP2-DRB1 haplotypes (TAP2*0102-DRB1*09, TAP2*0103-DRB1*04, and TAP2*0201-DRB1*01). The three-locus haplotype analysis showed highly significant positive linkage disequilibrium for TAP1*0101-TAP2*0101-DRB1*07, TAP1*0101-TAP2*0103-DRB1*04, TAP1*020101-TAP2*0101-DRB1*03, and TAP1*020101-TAP2*0102-DRB1*13. Comparison of the allele frequencies with those of other populations showed that the TAP1 allele distribution was very similar in all the groups, except for the Guarani, Kaingang, and Anatolian populations, but TAP2 distribution was significantly different from that of the other populations. The new TaqMan method provides relatively accurate, high-resolution, simple, and fast assays for TAP genotyping.     

Polymorphisms of the TAP1 and TAP2 genes in human alveolar echinococcosis.

We postulated that TAP genes may influence the susceptibility of some individuals to Echinococcus multilocularis infection. Six coding region variants (codons 333 and 637 in TAP1, and 379, 565, 651 and 665 in TAP2) were typed in 94 patients and 100 controls. Thr/Thr homozygosity at TAP2/665 was more prevalent in patients than in controls [64% vs. 45%, respectively; odds ratio (OR) = 2.1 (95% confidence interval (CI) 1.1; 2.7)] and Thr/Ala heterozygozity was less prevalent (32% vs. 50%, respectively) (P = 0.014). Of the 38 patients with progressive lesions, 76% were Thr/Thr, as compared with 55% of patients without progressive lesions and 45% of controls (P = 0.058 and 0.02, respectively), independent of HLA status. To determine whether this association is functionally relevant, functional analyses and/or confirmation in distinct populations of patients with alveolar echinococcosis would be required.     

Frequencies of TAP1 and TAP2 gene polymorphisms in the Anatolian population.

Anatolia has a long and complex record of immigration from various regions. Here, we have used TAP1 and TAP2 gene polymorphisms as genetic markers to study the relationship between the Anatolian population and other populations. A neighbour-joining tree was constructed indicating the relatedness of European populations and the Anatolian population with respect to TAP1 and TAP2 allele frequencies.     

Activity-dependent control of bulk endocytosis by protein dephosphorylation in central nerve terminals

Bulk endocytosis is the process by which nerve terminals retrieve large amounts of synaptic vesicle membrane during periods of strong stimulation intensity. The process is rapidly activated and is most probably calcium dependent in a similar manner to synaptic vesicle exocytosis. This article briefly summarizes the current knowledge of bulk endocytosis with respect to its activation, kinetics and molecular mechanism. It also presents recent data from our laboratory showing that the dephosphorylation of a group of endocytosis proteins called the dephosphins by the Ca²⁺-dependent protein phosphatase calcineurin is key to the activity-dependent stimulation of the process. Possible downstream effectors of calcineurin are discussed such as the large GTPase dynamin I and its phosphorylation-dependent interaction partner syndapin I.     

Analysis of TAP1 and TAP2 polymorphism of mother-infant in Chinese patients with pre-eclampsia

To analyze the polymorphism of TAP gene and the shared rates of alleles between mothers and their infants inChinese patients with pre-eclampsia,TAP1 and TAP2 genotyping was performed by the amplification refractorymutation system-polymerase chain reaction(ARMS-PCR)in 42 patients,106 normal pregnant women,and theirneonates.The allelic frequency of TAP and the alleles shared in maternal-fetus were compared and analyzed in thetwo groups.Our results showed that,with totally eight alleles of TAP1 and TAP2 examined in the samples,nosignificant difference was found in allelic frequencies between pre-eclampsia group and control group,as well asbetween mothers and their neonates.Similar finding was obtained in the comparison with shared alleles.Inconclusion,our results do not support a role for the polymorphisms of TAP in the etiology of pre-eclampsia.Cellular & Molecular Immunology.2005;2(2):141-144.     

Development and characterization of mouse anti-human LMP2, LMP7, TAP1 and TAP2 monoclonal antibodies

Abstract: Low molecular mass polypeptides (LMP) 2 and LMP7 and transporter associated with antigen processing (TAP) subunits TAP1 and TAP2 play a crucial role in antigen processing and cell surface expression of HLA class I molecules. Since monoclonal antibodies (mAb) to these molecules will facilitate the analysis of their expression, structure and function in normal and transformed cells, in the present study we have developed these reagents. Specifically anti-LMP2 and LMP7 mAb were generated from BALB/c mice immunized with specific peptides, and anti-TAPl and TAP2 mAb from BALB/c mice immunized with respective recombinant proteins. mAb VF101–39F7 and VF1O1–39G5 were shown to be specific for LMP2, mAb VF103–5D5 and VF103–8C2 for LMP7, mAb VF108–1B3 and VF108–12D6 for TAP1 and mAb VF118–1E4 and VF118–2C5 for TAP2, since they reacted specifically with the corresponding immunogens in ELISA and with the corresponding LMP and TAP subunits when tested in Western blotting with human lymphoid cell extracts. Furthermore, the mAb immunoprecipitated components with the characteristic electrophoretic mobility from lymphoid cells. Both anti-LMP and anti-TAP mAb stained keratinocytes and infiltrating lymphocytes in frozen and formalin-fixed, paraffin embedded sections of normal skin in indirect immunoperoxidase reactions. Furthermore, all the mAb except mAb VF103–5D5 stained the cytoplasm of lymphoid cells in an intracytoplasmic staining reaction. The specificity and reactivity pattern of the mAb we have characterized indicate that they will be valuable reagents to analyze the cellular expression and tissue distribution of LMP and TAP subunits.