Amino-terminal Half Research Articles

Role of Amino-terminal Half of the S4-S5 Linker in Type 1 Ryanodine Receptor (RyR1) Channel Gating

The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.

Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP(i)). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP(i), a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP(i) complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn(2+), larger than Mg(2+), fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.

EspR, a key regulator of Mycobacterium tuberculosis virulence, adopts a unique dimeric structure among helix-turn-helix proteins

EspR is a transcriptional regulator that activates the ESX-1 secretion system during Mycobacterium tuberculosis infection and is critical for pathogenesis. It is unique among DNA-binding proteins as it is secreted as part of a feedback regulatory loop that serves to mitigate transcriptional activity. Here we report the crystal structure of a functional EspR dimer at 2.5-Å resolution. The amino-terminal half of EspR is a helix-turn-helix (HTH) DNA-binding domain and the carboxy terminus consists of a dimerization domain with similarity to the SinR:SinI sporulation regulator of Bacillus subtilis. Surprisingly, the HTH domains of EspR are arranged in an unusual conformation in which they are splayed at an oblique angle to each other, suggesting that EspR binds DNA in a profoundly different way than most other known HTH regulators. By mapping the EspR binding sites in the espACD promoter, using both in vivo and in vitro binding assays, we show that the EspR operators are located unusually far from the promoter. The EspR dimer binds to these sites cooperatively, but the two "half-sites" contacted by each DNA recognition motif are separated by 177 base pairs. The distinctive structure of EspR and the exceptional arrangement of its operator contacts suggest that it could promote DNA looping in its target promoter. We hypothesize that direct DNA looping mediated by single-site binding of each EspR monomer may facilitate transcriptional control of this important virulence system.

Cold Adaptation of Tropomyosin

The conformational stability of unphosphorylated and phosphorylated α,α-striated tropomyosins from rabbit and shark (95% identical sequences) has been investigated. Three additional core positions are occupied by atypical amino acids in the protein from shark: Thr179(d), Ser190(a), and Ser211(a). These changes are thought to have further destabilized most, if not all, of the carboxyl-terminal half of the molecule. Heat-induced unfolding of shark tropomyosin (2 mg/mL, 0.1 M salt, pH 7) as monitored by far-UV circular dichroism is biphasic [T(m1) ∼ 33 °C (main), and T(m2) ∼ 54 °C] and takes place over a wider temperature span than that of the mammalian protein. The relationship between ellipticity (and excess heat) and temperature is insensitive to the presence in either tropomyosin of covalently bound phosphate. At ∼10 mg/mL, the minor endotherm of shark tropomyosin is shifted to ∼60 °C and T(m2) - T(m1) is increased to 25 °C; otherwise, the results of calorimetry are in agreement with those of circular dichroism. Analyses of cyanogen bromide fragments by far-UV circular dichroism and intact protein by near-UV circular dichroism (T(m) ∼ 32 °C) show that the most stable sizable portion of shark tropomyosin is located within the amino-terminal half of the molecule. These findings illuminate those regions in tropomyosin where flexibility is critical and show that substitutions predicted to be unfavorable in one temperature regime are desirable in another.

Crystal structure of a phosphorylation-coupled saccharide transporter.

Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

William Boal Rodgers

The ligand binding pocket of biogenic amine G protein-coupled receptors is embedded in the membrane-spanning regions of these receptors, whereas the extracellular domains of the peptidergic receptors play a key...

Basic fibroblast growth factor promotes glial cell-derived neurotrophic factor gene expression mediated by activation of ERK5 in rat C6 glioma cells

Extracellular signal-regulated kinases (ERKs) play important physiological roles including proliferation, differentiation and gene expression. ERK5 contains kinase domain that shares homology with ERK1/2 and the T-E-Y activation motif at amino-terminal half, whereas the extended carboxy-terminal half is unique. Because the physiological role of ERK5 in glial cells remains unclear, we examined the involvement of ERK5 in expression of neurotrophic factors and cytokines in rat C6 glioma cells, comparing it with ERK1/2. Basic fibroblast growth factor (bFGF) induced both ERK5 and ERK1/2 phosphorylation in a time- and concentration-dependent manner. Among the neurotrophic factors and cytokines, bFGF induced significant gene expression of glial cell-derived neurotrophic factor (GDNF). The GDNF gene expression and protein synthesis induced by bFGF were blocked by BIX02189 and PD98059 that selectively inhibit ERK5 and ERK1/2 signaling, respectively. The effect was also blocked by overexpression of a dominant-negative MEK5 mutant, indicating that GDNF expression induced by bFGF requires both ERK5 and ERK1/2. Because GDNF gene expression is regulated by various transcription factors, we examined the activity of these factors. We demonstrated that phosphorylation of cAMP-response element-binding protein at Ser 133 was induced by bFGF, which was blocked by BIX02189 and PD98059. Expression of c-fos, a major component of activator protein-1, and early growth response-1 was enhanced by bFGF, and expression of these genes was blocked by BIX02189, PD98059 and overexpression of dominant-negative MEK5. Taking these results together, bFGF promotes GDNF expression accompanied by the activation of ERK5, ERK1/2 and their downstream transcription factors in C6 glioma cells.

Functional Analysis of a Peptide Sequence (Thr323 - Cys337) In ADAMTS13 Disintegrin-Like Domain Revealed Two Discontinuous Sequences Involved In the Interaction with VWF

AbstractAbstract 2200ADAMTS13 specifically cleaves multimeric von Willebrand factor (VWF) into smaller molecules to reduce its high reactivity with platelets. The disintegrin-like (D) domain, adjacent to the catalytic domain of ADAMTS13, plays an important role in the process of VWF cleavage. In this study, we aimed to elucidate critical peptide sequences in D-domain involved in the interaction with VWF. A series of partially overlapping peptide sequences, approximately 20 amino acids in length, covering the D-domain, were synthesized and the inhibitory effects on the catalytic activity of plasma ADAMTS13 was examined using FRETS-VWF73 assay. Consequently, some synthetic peptides were selected and the minimal length necessary for the inhibitory effect was determined as TFAREHLDMCQALSC (peptide323-337). Removal of the amino-terminal threonine diminished the inhibitory effect moderately, although deletion of the carboxyl-terminal cysteine abolished it completely. According to the amino acids alignment of ADAMTS family, this peptide sequence is not conserved, highlighting the specific role in the interaction with its substrate. From the recent analysis of crystal structure, amino-terminal half of the peptide323-337, TFAREHL (323-329), was disordered and designated as the variable (V) loop, which creates one of VWF-binding exosites (Akiyama, et al. Proc Natl Acad Sci USA. 2009; 106:19274-9). We hypothesized that the amino-terminal amino acids of the peptide323-337 contribute to VWF binding, whereas the carboxyl-terminal amino acids allow the structural stability of the peptide conformation. To evaluate the effect of carboxyl-terminal cysteine at 337, other synthetic peptides with alanine, serine, glycine or phenylalanine instead of the cysteine (C337A, C337S, C337G, or C337F) were tested about their inhibitory effects on the catalytic activity. Interestingly, C337A, C337S, C337G peptides exhibited slightly weaker inhibitory effects on VWF73 catalysis, although C337F peptide showed stronger inhibition than wild-type sequence, suggesting that the residue 337 regulates the characteristics of the peptide323-337. From the results of peptide screening, the amino- and carboxyl-terminal amino acids of the peptide323-337, TFAREHLDMCQALSC, likely play key roles in the inhibitory effects; therefore, the middle part of the sequence, HLDMC, was replaced by 5 alanines (AAAAA) or reversed sequence CMDLH. Surprisingly, the converted peptides still retained the equivalent level of inhibitory effects, indicating both sides of the amino- and carboxyl-terminal amino acids were especially significant in the interaction with VWF. In conclusion, we characterized the peptide sequence, TFAREHLDMCQALSC (323-337), in D-domain. The peptide clearly inhibited the cleavage of VWF73 and the both sides of amino- and carboxyl-terminal amino acids seemed especially important. The peptide sequence is supposed to bind to VWF for the precise cleavage in the process of proteolysis. By modifying this peptide sequence, such variant ADAMTS13 as gain-of-function recombinants might be developed, leading to an alternative anti-thrombotic drug. Disclosures:No relevant conflicts of interest to declare.

Use of monoclonal antibodies against Hendra and Nipah viruses in an antigen capture ELISA

BackgroundOutbreaks of Hendra (HeV) and Nipah (NiV) viruses have been reported starting in 1994 and 1998, respectively. Both viruses are capable of causing fatal disease in humans and effecting great economical loss in the livestock industry.ResultsThrough screening of hybridomas derived from mice immunized with γ-irradiated Nipah virus, we identified two secreted antibodies; one reactive with the nucleocapsid (N) protein and the other, the phosphoprotein (P) of henipaviruses. Epitope mapping and protein sequence alignments between NiV and HeV suggest the last 14 amino acids of the carboxyl terminus of the N protein is the target of the anti-N antibody. The anti-P antibody recognizes an epitope in the amino-terminal half of P protein. These monoclonal antibodies were used to develop two antigen capture ELISAs, one for virus detection and the other for differentiation between NiV and HeV. The lower limit of detection of the capture assay with both monoclonal antibodies was 400 pfu. The anti-N antibody was used to successfully detect NiV in a lung tissue suspension from an infected pig.ConclusionThe antigen capture ELISA developed is potentially affordable tool to provide rapid detection and differentiation between the henipaviruses.

Differential Effects of Sumoylation on Transcription and Alternative Splicing by Transcription Elongation Regulator 1 (TCERG1)

Modification of proteins by small ubiquitin-like modifier (SUMO) is emerging as an important control of transcription and RNA processing. The human factor TCERG1 (also known as CA150) participates in transcriptional elongation and alternative splicing of pre-mRNAs. Here, we report that SUMO family proteins modify TCERG1. Furthermore, TCERG1 binds to the E2 SUMO-conjugating enzyme Ubc9. Two lysines (Lys-503 and Lys-608) of TCERG1 are the major sumoylation sites. Sumoylation does not affect localization of TCERG1 to the splicing factor-rich nuclear speckles or the alternative splicing function of TCERG1. However, mutation of the SUMO acceptor lysine residues enhanced TCERG1 transcriptional activity, indicating that SUMO modification negatively regulates TCERG1 transcriptional activity. These results reveal a regulatory role for sumoylation in controlling the activity of a transcription factor that modulates RNA polymerase II elongation and mRNA alternative processing, which are discriminated differently by this post-translational modification.

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

Amino terminal hydrophobic import signals target the p14ARF tumor suppressor to the mitochondria

The p14ARF tumour suppressor is frequently targeted for inactivation in many human cancers and in individuals predisposed to cutaneous melanoma. The functions of p14ARF are closely linked with its subcellular distribution. Nucleolar p14ARF dampens ribosome biosynthesis and nucleoplasmic forms of p14ARF activate the p53 pathway and induce cell cycle arrest. p14ARF can also be recruited to mitochondria where it interacts with many mitochondrial proteins, including Bcl-xL and p32 to induce cell death. It has been suggested that the movement of p14ARF to mitochondria requires its interaction with p32, but we now show that the ARF-p32 interaction is not necessary for the accumulation of p14ARF in mitochondria. Instead, highly hydrophobic domains within the amino-terminal half of p14ARF act as mitochondrial import sequences. We suggest that once this hydrophobic pocket is exposed, possibly in a stimulus-dependent manner, it accelerates the mitochondrial import of p14ARF. This allows the interaction of p14ARF with mitochondrial proteins, including p32 and enables p53-independent cell death.

Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers.

The M2 protein of influenza A virus is a membrane-spanning tetrameric proton channel targeted by the antiviral drugs amantadine and rimantadine 1. Resistance to these drugs has compromised their effectiveness against many influenza strains, including pandemic H1N1. A recent crystal structure of M2(22-46) showed electron densities attributed to a single amantadine in the N-terminal half of the pore 2, suggesting a physical occlusion mechanism for inhibition. However, a solution NMR structure of M2(18-60) showed four rimantadines bound to the C-terminal lipid-facing surface of the helices 3, suggesting an allosteric mechanism. Here we show by solid-state NMR spectroscopy that two amantadine-binding sites exist in M2 in phospholipid bilayers. The high-affinity site, occupied by a single amantadine, is located in the N-terminal channel lumen, surrounded by residues mutated in amantadine-resistant viruses. Quantification of the protein – amantadine distances resulted in a 0.3 Å-resolution structure of the high-affinity binding site. The second, low-affinity, site was observed on the C-terminal protein surface, but only when the drug reaches high concentrations in the bilayer. The orientation and dynamics of the drug are distinct in the two sites, as shown by 2H NMR. These results indicate that amantadine physically occludes the M2 channel, thus paving the way for developing new antiviral drugs against influenza viruses. The study demonstrates the ability of solid-state NMR to elucidate small-molecule interactions with membrane proteins and determine high-resolution structures of their complexes.

Comparison of the Conformational Stabilities of Striated Alpha-Tropomyosins Adapted to Different Temperature Regimes

The conformational stabilities of alpha-striated tropomyosins from warm and cold blooded sources have been compared using circular dichroism (CD) and differential scanning calorimetry. Alpha-tropomyosins from rabbit and shark share 95% sequence identity. There are three replacements in the core: residues Thr179Ala, Ser190Cys and Ser211Ala. At low temperature (pH 7) the two tropomyosins are equivalent in molar ellipticity at 222nm, however, shark tropomyosin unfolds over a wider temperature range than the mammalian counterpart. The dependence of this ellipticity on temperature (0.1M salt, pH 7, 2mg/mL protein + dithiothreitol in the case of rabbit tropomyosin) is characterized by the following transitions: shark, ∼33 (main, ∼two-thirds of total change in signal) and ∼54°C and rabbit, ∼41 (main) and ∼49°C. At ∼10mg/mL, the ΔTm for shark tropomyosin becomes larger on account of an upward shift in the minor transition, otherwise the results of calorimetry are in agreement with those of CD. Analysis of fragments CN1A (residues 11-128, Tm ∼59°C) and CN1B (residues 142 - 281, Tms ∼20 and 35°C) by far-UV CD and intact protein by near-UV CD (Tm ∼ 32°C), shows that the most stable section of shark tropomyosin is located in the amino-terminal half of the molecule, possibly the first ∼100 amino acids. The enhanced flexibility (lower stability) of the remainder of the molecule coincides with the presence of a row of destabilising ‘core’ amino acids between residues 179 - 221. A final point is that conformational stability is insensitive to the presence of a covalently bound phosphate group, consistent with the structural disordering of the corresponding region of each tropomyosin.

Roles of the intramolecular regions of FE65 in its trans-accumulation and in p53 stabilization in the nuclear matrix of osmotically stressed cells

The neural adaptor protein FE65 interacts with the amyloid β-protein precursor (APP). In osmotically stressed cells, the membrane APP-tethered FE65 is released into the cytoplasm and translocates to the nuclear matrix, where it stabilizes p53 via a non-canonical pathway. In this study, we found that the second phosphotyrosine interaction domain (PI2) of FE65 mediated its trans-accumulation in the nuclear matrix of osmotically stressed cells. The carboxyl-terminal half of FE65, which contains the PI2 domain, failed to stabilize p53, suggesting that the amino-terminal half of the protein plays an important role in the stabilization of p53 in osmotically stressed cells.

Zfp206, Oct4, and Sox2 Are Integrated Components of a Transcriptional Regulatory Network in Embryonic Stem Cells

Zfp206 (recently renamed Zscan10) encodes a zinc finger transcription factor specifically expressed in human and mouse embryonic stem cells (ESC). It has been shown that Zfp206 is required to maintain ESC in an undifferentiated, pluripotent state. Presented here are data showing that Zfp206 works together with two other transcription factors, Oct4 and Sox2, which are also essential regulators of ESC pluripotency. We show that Zfp206 binds to the Oct4 promoter and directly regulates Oct4 expression. Genome-wide mapping of Zfp206-binding sites in ESC identifies more than 3000 target genes, many of which encode transcription factors that are also targeted for regulation by Oct4 and Sox2. In addition, we show that Zfp206 physically interacts with both Oct4 and Sox2. These data demonstrate that Zfp206 is a key component of the core transcriptional regulatory network and together with Oct4 and Sox2 regulates differentiation of ESC.

Mycobacterium tuberculosis PhoP Recognizes Two Adjacent Direct-Repeat Sequences To Form Head-to-Head Dimers

Mycobacterium tuberculosis PhoP of the PhoP-PhoR two-component signaling system orchestrates a complex transcription program and is essential for the growth and virulence of the tubercle bacillus. PhoP comprises a phosphorylation domain at the amino-terminal half and a DNA-binding domain in the carboxy-terminal half of the protein. We show here that the protein recognizes a 23-bp sequence of the phoP upstream region comprising two adjacent direct repeat motifs believed to promote transcription regulation. DNA binding, which involves the recruitment of two monomeric PhoP molecules, was dependent on conserved adenines of the repeat sequences and the orientation of the repeat motifs relative to each other. Although response regulators such as PhoB and FixJ dimerize upon phosphorylation, we demonstrate here that PhoP dimerization can also be stimulated by DNA binding. Using the established asymmetric tandem binding model by members of the OmpR/PhoB protein family as a guide, we set out to examine intermolecular interactions between PhoP dimers by protein cross-linking. Our results are consistent with a model in which two PhoP protomers bind the duplex DNA with a symmetric head-to-head orientation to project their N termini toward one another, arguing against previously proposed head-to-tail tandem dimer formation for members of the OmpR/PhoB protein subfamily.

ERK5 Activity Is Required for Nerve Growth Factor-induced Neurite Outgrowth and Stabilization of Tyrosine Hydroxylase in PC12 Cells

Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation, and gene expression. ERK5 is approximately twice the size of ERK1/2, and its amino-terminal half contains the kinase domain that shares homology with ERK1/2 and TEY activation motif, whereas the carboxyl-terminal half is unique. In this study, we examined a physiological role of ERK5 in rat pheochromocytoma cells (PC12), comparing it with ERK1/2. Nerve growth factor (NGF) induced phosphorylation of both ERK5 and ERK1/2, whereas the cAMP analog dibutyryl cAMP (Bt(2)cAMP) caused only ERK1/2 phosphorylation. U0126, at 30 mum, that blocks ERK1/2 signaling selectively attenuated neurite outgrowth induced by NGF and Bt(2)cAMP, but BIX02188 and BIX02189, at 30 mum, that block ERK5 signaling and an ERK5 dominant-negative mutant suppressed only NGF-induced neurite outgrowth. Next, we examined the expression of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. Both NGF and Bt(2)cAMP increased tyrosine hydroxylase gene promoter activity in an ERK1/2-dependent manner but was ERK5-independent. However, when both ERK5 and ERK1/2 signalings were inhibited, tyrosine hydroxylase protein up-regulation by NGF and Bt(2)cAMP was abolished, because of the loss of stabilization of tyrosine hydroxylase protein by ERK5. Taking these results together, ERK5 is involved in neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells, and ERK5, along with ERK1/2, plays essential roles in the neural differentiation process.

Regulation of the innate immune response by threonine-phosphatase of Eyes absent

Innate immunity is stimulated not only by viral or bacterial components, but also by non-microbial danger signals (damage-associated molecular patterns). One of the damage-associated molecular patterns is chromosomal DNA that escapes degradation. In programmed cell death and erythropoiesis, DNA from dead cells or nuclei expelled from erythroblasts is digested by DNase II in the macrophages after they are engulfed. DNase II(-/-) (also known as Dnase2a(-/-)) mice suffer from severe anaemia or chronic arthritis due to interferon-beta (IFN-beta) and tumour necrosis factor-alpha (TNF-alpha) produced from the macrophages carrying undigested DNA in a Toll-like receptor (TLR)-independent mechanism. Here we show that Eyes absent 4 (EYA4), originally identified as a co-transcription factor, stimulates the expression of IFN-beta and CXCL10 in response to the undigested DNA of apoptotic cells. EYA4 enhanced the innate immune response against viruses (Newcastle disease virus and vesicular stomatitis virus), and could associate with signalling molecules (IPS-1 (also known as MAVS), STING (TMEM173) and NLRX1). Three groups have previously shown that EYA has phosphatase activity. We found that mouse EYA family members act as a phosphatase for both phosphotyrosine and phosphothreonine. The haloacid dehalogenase domain at the carboxy terminus contained the tyrosine-phosphatase, and the amino-terminal half carried the threonine-phosphatase. Mutations of the threonine-phosphatase, but not the tyrosine-phosphatase, abolished the ability of EYA4 to enhance the innate immune response, suggesting that EYA regulates the innate immune response by modulating the phosphorylation state of signal transducers for the intracellular pathogens.

Overexpression of inhibitor-1 or -2 of protein phosphatase-2A causes abnormal hyperphosphorylation of tau and cognitive impairment in the rat

The activity of phosphoseryl/phosphothreonyl protein phosphatase (PP) -2A, the major human brain PP, is selectively decreased and is believed to be a cause of the abnormal hyperphosphorylation of tau in AD brain (Gong et al., J Neurochem 61:921-7, 1993; ibid J Neurochem 65:732-8, 1995). The activity of PP-2A is regulated by two inhibitor proteins, I1PP2A which is also known as PHAPI, mapmodulin, pp32, and LANP, and I2PP2A which is also known as PHAPII, Setα, and TAF-1β. Both the levels of mRNA and protein expression of I1PP2A and I2PP2A are elevated in the affected areas of AD brain, and are probably a cause of the decreased PP-2A activity and consequently the abnormal hyperphosphorylation of tau. Objective: To assess whether overexpression of I1PP2A- or I2PP2A-induced inhibition of PP-2A activity produces abnormal hyperphosphorylation of tau and behavioral impairment in the rat. We employed the adeno associated viral vector to induce expression of human brain I1PP2A or I2PP2A as GFP fusion proteins in rat brain. At various time periods from ∼ 9 weeks to ∼9 months after ICV injection with the virus, the effect of the overexpression of I1PP2A or I2PP2A on abnormal hyperphosphorylation, neurodegeneration, and cognition was studied. The infection of the newborn rat pups with AAV produced a long-lasting expression up to ∼9 months investigated. The expression of amino terminal half of I1PP2A which contains the inhibitory domain (Chen et al., J Biol Chem 283:10513-21, 2008) caused the abnormal hyperphosphorylation of tau at Thr231/Ser235, Ser262/356, and at Ser393/404 in the neocortex and a learning and memory impairment in Morris water maze. Expression of the carboxy terminal half of I2PP2A caused abnormal hyperphosphorylation of tau at Ser199, Ser262, and Ser396, a decrease in MAP 2, synaptophysin and synapsin 1, and impairment in learning in Morris water maze. This study suggests that overexpression of I1PP2A and/or I2PP2A previously observed in AD brain, causes abnormal hyperphosphorylation of tau and cognitive impairment. Thus, restoration of PP-2A activity by inhibiting the activity of I1PP2A and/or I2PP2A is a promising therapeutic approach for AD and related tauopathies.