Immobilon-P Membrane Research Articles

An N-Linked Glycan Modulates the Interaction between the CD1d Heavy Chain and β2-Microglobulin

Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with beta(2)-microglobulin (beta(2)m). Assembly occurs in the endoplasmic reticulum (ER) and involves the initial glycan-dependent association of the free heavy chain with calreticulin and calnexin and the thiol oxidoreductase ERp57. Folding and disulfide bond formation within the heavy chain occurs prior to beta(2)m binding. There are four N-linked glycans on the CD1d heavy chain, and we mutated them individually to ascertain their importance for the assembly and function of CD1d-beta(2)m heterodimers. None of the four were indispensable for assembly or the ability to bind alpha-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present alpha-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(alpha1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d-beta(2)m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with beta(2)m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from beta(2)m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from beta(2)m upon exposure to the detergent Triton X-100, whereas a mutant expressing only glycan 2 at Asn-42 was more stable. In addition, glycan 2 was not processed efficiently to the complex form in mature wild-type CD1d molecules. Modeling the glycans on the published structure indicated that glycan 2 interacts significantly with both the CD1d heavy chain and beta(2)m, which may explain these unusual properties.

Insoluble Aggregates and Protease-resistant Conformers of Prion Protein in Uninfected Human Brains

Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an alpha-helix to beta-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.

Decorin Modulates Fibrin Assembly and Structure

Emerging evidence indicates that fibrin clotting is regulated by different external factors. We demonstrated recently that decorin, a regulator of collagen fibrillogenesis and transforming growth factor-beta activity, binds to the D regions of fibrinogen (Dugan, T.A., Yang, V. W.-C., McQuillan, D.J., and Höök, M. (2003) J. Biol. Chem. 278, 13655-13662). We now report that the decorin-fibrinogen interaction alters the assembly, structure, and clearance of fibrin fibers. Relative to fibrinogen, substoichiometric amounts of decorin core protein modulated clotting, whereas an excess of an active decorin peptide was necessary for similar activity. These concentration-dependent effects suggest that decorin bound to the D regions sterically modulates fibrin assembly. Scanning electron microscopy images of fibrin clotted in the presence of increasing concentrations of decorin core protein showed progressively decreasing fiber diameter. The sequestration of Zn(2+) ions from the N-terminal fibrinogen-binding region abrogated decorin incorporation into the fibrin network. Compared with linear thicker fibrin fibers, the curving thin fibers formed with decorin underwent accelerated tissue-type plasminogen activator-dependent fibrinolysis. Collectively, these data demonstrate that decorin can regulate fibrin organization and reveal a novel mechanism by which extracellular matrix components can participate in hemostasis, thrombosis, and wound repair.

Proline-rich Motifs in the Parathyroid Hormone (PTH)/PTH-related Protein Receptor C Terminus Mediate Scaffolding of c-Src with β-Arrestin2 for ERK1/2 Activation

Parathyroid hormone (PTH) stimulates ERK1/2 through both G-protein signaling and beta-arrestin2-mediated internalization. Beta-arrestin may serve as a scaffold for c-Src. However, the molecular mechanisms for ERK1/2 activation by PTH remain unclear. By using a targeted mutagenesis approach, we investigated the PTH/PTH-related protein receptor (PTH1R) structural determinants for ERK1/2 activation and transcriptional activity in HEK-293 cells. First, ERK1/2 activation was inhibited by PTH1R mutations that specifically abrogate G(q)-protein kinase C signaling without a decrease in cAMP-protein kinase A. Second, PTH1R C-terminal mutations and/or deletions that prevent interaction with beta-arrestin inhibited ERK1/2 activation. Similar results were obtained in HEK-293 cells co-expressing wild-type PTH1R and a dominant-negative beta-arrestin2. Third, the c-Src inhibitor PP2 and a kinase-dead c-SrcK295M mutant co-expressed with wild-type PTH1R both inhibited ERK1/2 activation. Furthermore, c-Src co-precipitated with both PTH1R and beta-arrestin2 in response to PTH. Deleting the PTH1R-proximal C terminus abolished these interactions. However, the need for receptor interaction with beta-arrestin to co-precipitate Src and activate ERK1/2 was obviated by expressing a constitutively active c-SrcY527A mutant, suggesting direct binding of activated Src to PTH1R. Subsequently, we identified and mutated to alanine four proline-rich motifs in the PTH1R distal C terminus, which resulted in loss of both c-Src and arrestin co-precipitation and significantly decreased ERK1/2 activation. These data delineate the multiple PTH1R structural determinants for ERK1/2 activation and newly identify a unique mechanism involving proline-rich motifs in the receptor C terminus for reciprocal scaffolding of c-Src and beta-arrestin2 with a class II G-protein-coupled receptor.

Site-specific Phosphorylation Differentiates Active from Inactive Forms of the Human T-cell Leukemia Virus Type 1 Tax Oncoprotein

Site-specific Phosphorylation Differentiates Active from Inactive Forms of the Human T-cell Leukemia Virus Type 1 Tax Oncoprotein

Cox-2 Is Regulated by Toll-Like Receptor-4 (TLR4) Signaling: Role in Proliferation and Apoptosis in the Intestine

We recently showed that mice deficient in Toll-like receptor 4 (TLR4) or its adapter molecule MyD88 have increased signs of colitis compared with wild-type (WT) mice after dextran sodium sulfate (DSS)-induced injury. We wished to test the hypothesis that cyclooxygenase 2 (Cox-2)-derived prostaglandin E2 (PGE2) is important in TLR4-related mucosal repair. Cox-2 expression was analyzed by real-time polymerase chain reaction, immunohistochemistry, Western blotting, and luciferase reporter constructs. Small interfering RNA was used to inhibit expression of MyD88. TLR4-/- or WT mice were given 2.5% DSS for 7 days. Proliferation and apoptosis were assessed using bromodeoxyuridine staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays, respectively. PGE2 was given orally to DSS-treated mice. Intestinal epithelial cell lines up-regulated Cox-2 expression in a TLR4- and MyD88-dependent fashion. Lipopolysaccharide-mediated stimulation of PGE2 production was blocked by a selective Cox-2 inhibitor or small interfering RNA against MyD88. After DSS injury, Cox-2 expression increased only in WT mice. TLR4-/- mice have significantly reduced proliferation and increased apoptosis after DSS injury compared with WT mice. PGE2 supplementation of TLR4-/- mice resulted in improvement in clinical signs of colitis and restoration of proliferation and apoptosis to WT values. The mechanism for improved epithelial repair may be through PGE2-dependent activation of the epidermal growth factor receptor. We describe an important link between TLR4 signaling and Cox-2 expression in the gut. TLR4 and MyD88 signaling are required for optimal proliferation and protection against apoptosis in the injured intestine. Although TLR4 signaling is beneficial in the short term, chronic signaling through TLR4 may lower the threshold for colitis-associated cancer.

GCP60 Preferentially Interacts with a Caspase-generated Golgin-160 Fragment

Golgin-160, a ubiquitous protein in vertebrates, localizes to the cytoplasmic face of the Golgi complex. Golgin-160 has a large coiled-coil C-terminal domain and a non-coiled-coil N-terminal ("head") domain. The head domain contains important motifs, including a nuclear localization signal, a Golgi targeting domain, and three aspartates that are recognized by caspases during apoptosis. Some of the caspase cleavage products accumulate in the nucleus when overexpressed. Expression of a non-cleavable form of golgin-160 impairs apoptosis induced by some pro-apoptotic stimuli; thus cleavage of golgin-160 appears to play a role in apoptotic signaling. We used a yeast two-hybrid assay to screen for interactors of the golgin-160 head and identified GCP60 (Golgi complex-associated protein of 60 kDa). Further analysis demonstrated that GCP60 interacts preferentially with one of the golgin-160 caspase cleavage fragments (residues 140-311). This strong interaction prevented the golgin-160 fragment from accumulating in the nucleus when this fragment and GCP60 were overexpressed. In addition, cells overexpressing GCP60 were more sensitive to apoptosis induced by staurosporine, suggesting that nuclear-localized golgin-160-(140-311) might promote cell survival. Our results suggest a potential mechanism for regulating the nuclear translocation and potential functions of golgin-160 fragments.

C-Jun N-terminal Kinase Is Necessary for Platelet-derived Growth Factor-mediated Chemotaxis in Primary Fibroblasts

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family. It has become clear that JNK does not only have a role in induction of stress responses but also in processes such as cell movement. In this report we demonstrate that JNK activity is necessary for platelet-derived growth factor (PDGF)-BB-induced chemotaxis of primary foreskin fibroblasts and in other cell types. PDGF-BB stimulation was found to lead to activation of JNK with a maximum after 30 min. Inhibition of JNK reduced Ser178 phosphorylation of the focal adhesion component paxillin. Paxillin phosphorylation at this site has been shown to be involved in the dynamics of focal adhesions and consequently cell migration. Moreover, we observed localization of JNK to the actin-dense membrane ruffles induced by PDGF-BB stimulation both using immunofluorescence staining and green fluorescent protein-tagged JNK. This suggests a role for JNK at the leading edge of the cell compatible with a function in cell migration. Furthermore, we show that phosphatidylinositol 3-kinase (PI 3-kinase), which has an established role in PDGF-stimulated cell migration, is necessary for PDGF-induced activation of JNK. In conclusion, JNK is a critical component downstream of PI 3-kinase that may be involved in PDGF-stimulated chemotaxis presumably by modulating the integrity of focal adhesions by phosphorylating its components.

Heparin Potentiates the in Vivo Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists.

Cytoskeleton/Endoplasmic Reticulum Collapse Induced by Prostaglandin J2 Parallels Centrosomal Deposition of Ubiquitinated Protein Aggregates

Many neurodegenerative disorders, such as Parkinson disease, exhibit inclusion bodies containing ubiquitinated proteins. The mechanisms implicated in this aberrant protein deposition remain elusive. In these disorders signs of inflammation are also apparent in the affected central nervous system areas. We show that prostaglandin J2 (PGJ2), an endogenous product of inflammation, disrupts the cytoskeleton in neuronal cells. Furthermore, PGJ2 perturbed microtubule polymerization in vitro and decreased the number of free sulfhydryl groups on tubulin cysteines. A direct effect of PGJ2 on actin was not apparent, although actin filaments were altered in cells treated with PGJ2. This cyclopentenone prostaglandin triggered endoplasmic reticulum (ER) collapse and the redistribution of ER proteins, such as calnexin and catechol-O-methyltransferase, into a large centrosomal aggregate containing ubiquitinated proteins and alpha-synuclein. The PGJ2-dependent cytoskeletal rearrangement paralleled the development of the large centrosomal aggregate. Both of these events were replicated by treating cells with colchicine, which disrupts the microtubule/ER network, but not with brefeldin A, which impairs ER/Golgi transport. PGJ2 also perturbed 26 S proteasome assembly and activity, which preceded the accumulation of ubiquitinated proteins as detergent/salt-insoluble aggregates. Our data support a mechanism by which, upon PGJ2 treatment, cytoskeleton/ER collapse coincides with the relocation of ER proteins, other potentially neighboring proteins, and ubiquitinated proteins into centrosomal aggregates. Development of these large perinuclear aggregates is associated with disruption of the microtubule/ER network. This aberrant protein deposition, triggered by a product of inflammation, may be common to other compounds that disrupt microtubules and induce protein aggregation, such as MPP+ and rotenone, found to be associated with neurodegeneration.

Phosphorylation of Amphiphysin I by Minibrain Kinase/Dual-specificity Tyrosine Phosphorylation-regulated Kinase, a Kinase Implicated in Down Syndrome

Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/Dyrk1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/Dyrk1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/Dyrk1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/Dyrk1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/Dyrk1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.

Pro-NGF from Alzheimer's Disease and Normal Human Brain Displays Distinctive Abilities to Induce Processing and Nuclear Translocation of Intracellular Domain of p75NTR and Apoptosis

The pro form of neurotrophic growth factor (pro-NGF), purified by chromatography from human Alzheimer's disease (AD)-affected brains (ADhbi-pro-NGF), has been shown to induce apoptotic cell death in neuronal cell cultures through its interaction with the p75 neurotrophin receptor (p75NTR). In the present work, we report that ADhbi-pro-NGF stimulates processing of p75NTR with alpha- and gamma-secretases, yielding a 20-kd intracellular domain (p75(ICD)) that translocates to the nucleus. This process was accompanied by delayed apoptosis. In AD, p75(ICD) was significantly increased in human entorhinal cortex. Although human frontal cortex has been described as showing a higher pro-NGF increase in AD, the increase in the entorhinal cortex paralleled p75NTR processing in its intracellular domain. In addition, pro-NGF isolated from AD-affected brains differed functionally from pro-NGF isolated from comparably aged control brains, with pro-NGF isolated from control brains being unstable and undergoing degradation to NGF when added to cell culture. As p75(ICD) and pro-NGF are both mediators of apoptosis and are both found in increased levels in the cerebral cortex in AD, the present data have implications for understanding neuronal degeneration in AD.

The Early-Immediate Gene EGR-1 Is Induced by Transforming Growth Factor-β and Mediates Stimulation of Collagen Gene Expression

Transforming growth factor-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-beta and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.

Depletion of Hsp90β Induces Multiple Defects in B Cell Receptor Signaling

Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90beta-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90beta-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90beta gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90alpha gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90beta-specific function.

Antiviral effect of the mammalian translation initiation factor 2α kinase GCN2 against RNA viruses

In mammals, four different protein kinases, heme-regulated inhibitor, double-stranded RNA-dependent protein kinase (PKR), general control non-derepressible-2 (GCN2) and PKR-like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the alpha-subunit of the initiation factor 2 (eIF2alpha). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl-tRNA synthetase-related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2-/- mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild-type GCN2, but not the catalytically inactive GCN2-K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses.

Use of Substrate Analogs and Mutagenesis to Study Substrate Binding and Catalysis in the Sir2 Family of NAD-dependent Protein Deacetylases

The Sir2 family of enzymes is highly conserved throughout evolution and functions in silencing, control of life span, apoptosis, and many other cellular processes. Since the discovery of the NAD-dependent deacetylase activity of Sir2 proteins, there has been a flurry of activity aiming to uncover the mode of substrate binding and catalysis. Structural and biochemical studies have led to several proposed reaction mechanisms, yet the exact catalytic steps remain unclear. Here we present in vitro studies of yeast homolog Hst2 that shed light on the mechanism of Sir2 proteins. Using acetyl-lysine substrate analogs, we demonstrate that the Hst2 reaction proceeds via an initial SN2-type mechanism with the direct formation of an ADP-ribose-acetyl-lysine intermediate. Kinetic studies further suggest that ADP-ribose inhibits the Hst2 reaction in a biologically relevant manner. Through biochemical and kinetic analyses of point mutants, we also clarify the role of several conserved core domain residues in substrate binding, stabilization of the ADP-ribose-acetyl-lysine intermediate, and catalysis. These findings bring us a few steps closer to understanding Sir2 activity and may provide a useful platform for the design of Sir2-specific inhibitors for analysis of Sir2 function and possibly therapeutic applications.

Formation of Tau Inclusions in Knock-in Mice with Familial Alzheimer Disease (FAD) Mutation of Presenilin 1 (PS1)

Mutations in the presenilin 1 (PS1) gene are responsible for the early onset of familial Alzheimer disease (FAD). Accumulating evidence shows that PS1 is involved in gamma-secretase activity and that FAD-associated mutations of PS1 commonly accelerate Abeta(1-42) production, which causes Alzheimer disease (AD). Recent studies suggest, however, that PS1 is involved not only in Abeta production but also in other processes that lead to neurodegeneration. To better understand the causes of neurodegeneration linked to the PS1 mutation, we analyzed the development of tau pathology, another key feature of AD, in PS1 knock-in mice. Hippocampal samples taken from FAD mutant (I213T) PS1 knock-in mice contained hyperphosphorylated tau that reacted with various phosphodependent tau antibodies and with Alz50, which recognizes the conformational change of PHF tau. Some neurons exhibited Congo red birefringence and Thioflavin T reactivity, both of which are histological criteria for neurofibrillary tangles (NFTs). Biochemical analysis of the samples revealed SDS-insoluble tau, which under electron microscopy examination, resembled tau fibrils. These results indicate that our mutant PS1 knock-in mice exhibited NFT-like tau pathology in the absence of Abeta deposition, suggesting that PS1 mutations contribute to the onset of AD not only by enhancing Abeta(1-42) production but by also accelerating the formation and accumulation of filamentous tau.

Synthesis, Processing, and Composition of the Virion-associated HTLV-1 Reverse Transcriptase

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).

Chaperoning Checkpoint Kinase 1 (Chk1), an Hsp90 Client, with Purified Chaperones

Checkpoint kinase 1 (Chk1), a serine/threonine kinase that regulates DNA damage checkpoints, is destabilized when heat shock protein 90 (Hsp90) is inhibited, suggesting that Chk1 is an Hsp90 client. In the present work we examined the interplay between Chk1 and Hsp90 in intact cells, identified a source of unchaperoned Chk1, and report the in vitro chaperoning of Chk1 in reticulocyte lysates and with purified chaperones and co-chaperones. We find that bacterially expressed Chk1 is post-translationally chaperoned to an active kinase. This reaction minimally requires Hsp90, Hsp70, Hsp40, Cdc37, and the protein kinase CK2. The co-chaperone Hop, although not essential for the activation of Chk1 in vitro, enhanced the chaperoning process, whereas the co-chaperone p23 did not stimulate the chaperoning reaction. Additionally, we found that the C-terminal regulatory domain of Chk1 affects the association of Chk1 with Hsp90. Collectively these results provide new insights into Hsp90-dependent chaperoning of a client kinase and identify a novel, biochemically tractable model system that will be useful to further dissect the Hsp90-dependent chaperoning of this important and ubiquitous class of Hsp90 clients.

High Expression of Antioxidant Proteins in Dendritic Cells

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.