PKC�� promotes a proliferation to differentiation switch in keratinocytes via upregulation of p27Kip1 mRNA through suppression of JNK/c-Jun signaling under stress conditions

To maintain epidermal homeostasis, the balance between keratinocyte proliferation and differentiation is tightly controlled. However, the molecular mechanisms underlying this balance remain unclear. In 3D organotypic coculture with mouse keratinocytes and fibroblasts, the thickness of stratified cell layers was prolonged, and growth arrest and terminal differentiation were delayed when PKCη-null keratinocytes were used. Re-expression of PKCη in PKCη-null keratinocytes restored stratified cell layer thickness, growth arrest and terminal differentiation. We show that in 3D cocultured PKCη-null keratinocytes, p27^Kip1 mRNA was downregulated, whereas JNK/c-Jun signaling was enhanced. Furthermore, inhibition of JNK/c-Jun signaling in PKCη-null keratinocytes led to upregulation of p27^Kip1 mRNA, and to thinner stratified cell layers. Collectively, our findings indicate that PKCη upregulates p27^Kip1 mRNA through suppression of JNK/c-Jun signaling. This results in promoting a proliferation to differentiation switch in keratinocytes.     

Loss of protein kinase C delta alters mammary gland development and apoptosis

As apoptotic pathways are commonly deregulated in breast cancer, exploring how mammary gland cell death is regulated is critical for understanding human disease. We show that primary mammary epithelial cells from protein kinase C delta (PKCδ) −/− mice have a suppressed response to apoptotic agents in vitro. In the mammary gland in vivo, apoptosis is critical for ductal morphogenesis during puberty and involution following lactation. We have explored mammary gland development in the PKCδ −/− mouse during these two critical windows. Branching morphogenesis was altered in 4- to 6-week-old PKCδ −/− mice as indicated by reduced ductal branching; however, apoptosis and proliferation in the terminal end buds was unaltered. Conversely, activation of caspase-3 during involution was delayed in PKCδ −/− mice, but involution proceeded normally. The thymus also undergoes apoptosis in response to physiological signals. A dramatic suppression of caspase-3 activation was observed in the thymus of PKCδ −/− mice treated with irradiation, but not mice treated with dexamethasone, suggesting that there are both target- and tissue-dependent differences in the execution of apoptotic pathways in vivo. These findings highlight a role for PKCδ in both apoptotic and nonapoptotic processes in the mammary gland and underscore the redundancy of apoptotic pathways in vivo.     

Protein kinase C�� is required for cardiomyocyte survival and cardiac remodeling

Protein kinase Cs (PKCs) constitute a family of serine/threonine kinases, which has distinguished and specific roles in regulating cardiac responses, including those associated with heart failure. We found that the PKCθ isoform is expressed at considerable levels in the cardiac muscle in mouse, and that it is rapidly activated after pressure overload. To investigate the role of PKCθ in cardiac remodeling, we used PKCθ^−/− mice. In vivo analyses of PKCθ^−/− hearts showed that the lack of PKCθ expression leads to left ventricular dilation and reduced function. Histological analyses showed a reduction in the number of cardiomyocytes, combined with hypertrophy of the remaining cardiomyocytes, cardiac fibrosis, myofibroblast hyper-proliferation and matrix deposition. We also observed p38 and JunK activation, known to promote cell death in response to stress, combined with upregulation of the fetal pattern of gene expression, considered to be a feature of the hemodynamically or metabolically stressed heart. In keeping with these observations, cultured PKCθ^−/− cardiomyocytes were less viable than wild-type cardiomyocytes, and, unlike wild-type cardiomyocytes, underwent programmed cell death upon stimulation with α1-adrenergic agonists and hypoxia. Taken together, these results show that PKCθ maintains the correct structure and function of the heart by preventing cardiomyocyte cell death in response to work demand and to neuro-hormonal signals, to which heart cells are continuously exposed.     

Selective inhibition of protein kinase C β2 attenuates the adaptor P66Shc-mediated intestinal ischemia–reperfusion injury

Apoptosis is a major mode of cell death occurring during ischemia–reperfusion (I/R) induced injury. The p66^Shc adaptor protein, which is mediated by PKCβ, has an essential role in apoptosis under oxidative stress. This study aimed to investigate the role of PKCβ₂/p66^Shc pathway in intestinal I/R injury. In vivo, ischemia was induced by superior mesenteric artery occlusion in mice. Ruboxistaurin (PKCβ inhibitor) or normal saline was administered before ischemia. Then blood and gut tissues were collected after reperfusion for various measurements. In vitro, Caco-2 cells were challenged with hypoxia–reoxygenation (H/R) to simulate intestinal I/R. Translocation and activation of PKCβ₂ were markedly induced in the I/R intestine. Ruboxistaurin significantly attenuated gut damage and decreased the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Pharmacological blockade of PKCβ₂ suppressed p66^Shc overexpression and phosphorylation in the I/R intestine. Gene knockdown of PKCβ₂ via small interfering RNA (siRNA) inhibited H/R-induced p66^Shc overexpression and phosphorylation in Caco-2 cells. Phorbol 12-myristate 13-acetate (PMA), which stimulates PKCs, induced p66^Shc phosphorylation and this was inhibited by ruboxistaurin and PKCβ₂ siRNA. Ruboxistaurin attenuated gut oxidative stress after I/R by suppressing the decreased expression of manganese superoxide dismutase (MnSOD), the exhaustion of the glutathione (GSH) system, and the overproduction of malondialdehyde (MDA). As a consequence, ruboxistaurin inhibited intestinal mucosa apoptosis after I/R. Therefore, PKCβ₂ inhibition protects mice from gut I/R injury by suppressing the adaptor p66^Shc-mediated oxidative stress and subsequent apoptosis. This may represent a novel therapeutic approach for the prevention of intestinal I/R injury.     

Simultaneous loss of phospholipase Cδ1 and phospholipase Cδ3 causes cardiomyocyte apoptosis and cardiomyopathy

Phospholipase C (PLC) is a key enzyme in phosphoinositide turnover. Among 13 PLC isozymes, PLCδ1 and PLCδ3 share high sequence homology and similar tissue distribution, and are expected to have functional redundancy in many tissues. We previously reported that the simultaneous loss of PLCδ1 and PLCδ3 caused embryonic lethality because of excessive apoptosis and impaired vascularization of the placenta. Prenatal death of PLCδ1/PLCδ3 double-knockout mice hampered our investigation of the roles of these genes in adult animals. Here, we generated PLCδ1/PLCδ3 double-knockout mice that expressed PLCδ1 in extra-embryonic tissues (cDKO mice) to escape embryonic lethality. The cDKO mice were born at the expected Mendelian ratio, which indicated that the simultaneous loss of PLCδ1 and PLCδ3 in the embryo proper did not impair embryonic development. However, half of the cDKO mice died prematurely. In addition, the surviving cDKO mice spontaneously showed cardiac abnormalities, such as increased heart weight/tibial length ratios, impaired cardiac function, cardiac fibrosis, dilation, and hypertrophy. Predating these abnormalities, excessive apoptosis of their cardiomyocytes was observed. In addition, siRNA-mediated simultaneous silencing of PLCδ1 and PLCδ3 increased apoptosis in differentiated-H9c2 cardiomyoblasts. Activation of Akt and protein kinase C (PKC) θ was impaired in the hearts of the cDKO mice. siRNA-mediated simultaneous silencing of PLCδ1 and PLCδ3 also decreased activated Akt and PKCθ in differentiated-H9c2 cardiomyoblasts. These results indicate that PLCδ1 and PLCδ3 are required for cardiomyocyte survival and normal cardiac function.     

��v��3 Integrin Limits the Contribution of Neuropilin-1 to Vascular Endothelial Growth Factor-induced Angiogenesis

Both vascular endothelial growth factor receptors (VEGFR) and integrins are major regulators of VEGF-induced angiogenesis. Previous work has shown that β3 integrin can regulate negatively VEGFR2 expression. Here we show that β3 integrin can regulate negatively VEGF-mediated angiogenesis by limiting the interaction of the co-receptor NRP1 (neuropilin-1) with VEGFR2. In the presence of αvβ3 integrin, NRP1 contributed minimally to VEGF-induced angiogenic processes in vivo, ex vivo, and in vitro. Conversely, when β3 integrin expression is absent or low or its function is blocked with RGD-mimetic inhibitors, VEGF-mediated responses became NRP1-dependent. Indeed, combined inhibition of β3 integrin and NRP1 decreased VEGF-mediated angiogenic responses further than individual inhibition of these receptors. We also show that αvβ3 integrin can associate with NRP1 in a VEGF-dependent fashion. Our data suggest that β3 integrin may, in part, negatively regulate VEGF signaling by sequestering NRP1 and preventing it from interacting with VEGFR2.     

Investigation of in vivo diferric tyrosyl radical formation in Saccharomyces cerevisiae Rnr2 protein: requirement of Rnr4 and contribution of Grx3/4 AND Dre2 proteins

The β₂ subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe₂^III-Tyr^•) that is essential for nucleotide reduction. The β₂ subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β′). Although only β is capable of iron binding and Tyr^• formation, cells lacking β′ are either dead or exhibit extremely low Tyr^• levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β′ is required for iron loading and Tyr^• formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr^•-reducing agent hydroxyurea. Transient induction of β′ in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr^• levels in β. Tyr^• can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β′ and is inhibited by adding an iron chelator prior to, but not after, β′ supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr^• levels and ββ′ activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr^• cofactor in RNR.     

Crystal Structure and Computational Analyses Provide Insights into the Catalytic Mechanism of 2,4-Diacetylphloroglucinol Hydrolase PhlG from Pseudomonas fluorescens

2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C–C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 Å resolution using x-ray crystallography and MAD methods. The overall structure includes a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet v1-like fold, which distinguishes PhlG from the classical α/β-fold hydrolases. A dumbbell-shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, and enzyme activity analysis revealed that cleavage of the 2,4-diacetylphloroglucinol C–C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. In addition, residues Tyr¹²¹, Tyr²²⁹, and Asn¹³², which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation. Taken together, these results revealed the active sites and zinc-dependent hydrolytic mechanism of PhlG and explained its substrate specificity as well.     

Environment of TyrZ in photosystem II from Thermosynechococcus elongatus in which PsbA2 is the D1 protein

The main cofactors that determine the photosystem II (PSII) oxygen evolution activity are borne by the D1 and D2 subunits. In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for D1. Among the 344 residues constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption spectroscopy, we show that: (i) the time-resolved EPR spectrum of Tyr_Z^• in the (S₃Tyr_Z^•)′ is slightly modified; (ii) the split EPR signal arising from Tyr_Z^• in the (S₂Tyr_Z^•)′ state induced by near-infrared illumination at 4.2 K of the S₃Tyr_Z state is significantly modified; and (iii) the slow phases of P₆₈₀^+⋅ reduction by Tyr_Z are slowed down from the hundreds of μs time range to the ms time range, whereas both the S₁Tyr_Z^• → S₂Tyr_Z and the S₃Tyr_Z^• → S₀Tyr_Z + O₂ transition kinetics remained similar to those in PsbA(1/3)-PSII. These results show that the geometry of the Tyr_Z phenol and its environment, likely the Tyr-O···H···Nϵ-His bonding, are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to the dynamics of the proton-coupled electron transfer processes associated with the oxidation of Tyr_Z being affected. From sequence comparison, we propose that the C144P and P173M substitutions in PsbA2-PSII versus PsbA(1/3)-PSII, respectively located upstream of the α-helix bearing Tyr_Z and between the two α-helices bearing Tyr_Z and its hydrogen-bonded partner, His-190, are responsible for these changes.     

Diversity in the C3b [corrected] contact residues and tertiary structures of the staphylococcal complement inhibitor (SCIN) protein family

To survive in immune-competent hosts, the pathogen Staphylococcus aureus expresses and secretes a sophisticated array of proteins that inhibit the complement system. Among these are the staphylococcal complement inhibitors (SCIN), which are composed of three active proteins (SCIN-A, -B, and -C) and one purportedly inactive member (SCIN-D or ORF-D). Because previous work has focused almost exclusively on SCIN-A, we sought to provide initial structure/function information on additional SCIN proteins. To this end we determined crystal structures of an active, N-terminal truncation mutant of SCIN-B (denoted SCIN-B^18–85) both free and bound to the C3c fragment of complement component C3 at 1.5 and 3.4 Å resolution, respectively. Comparison of the C3c/SCIN-B^18–85 structure with that of C3c/SCIN-A revealed that both proteins target the same functional hotspot on the C3b/C3c surface yet harbor diversity in both the type of residues and interactions formed at their C3b/C3c interfaces. Most importantly, these structures allowed identification of Arg⁴⁴ and Tyr⁵¹ as residues key for SCIN-B binding to C3b and subsequent inhibition of the AP C3 convertase. In addition, we also solved several crystal structures of SCIN-D to 1.3 Å limiting resolution. This revealed an unexpected structural deviation in the N-terminal α helix relative to SCIN-A and SCIN-B. Comparative analysis of both electrostatic potentials and surface complementarity suggest a physical explanation for the inability of SCIN-D to bind C3b/C3c. Together, these studies provide a more thorough understanding of immune evasion by S. aureus and enhance potential use of SCIN proteins as templates for design of complement targeted therapeutics.     

[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and ��4��2 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α₂βγδ and expressed human α4β2 nAChRs with [³H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr¹⁹⁸ in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu¹⁰⁹ and γTyr¹¹⁷ in Segment E of the complementary (−) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, [³H]epibatidine photolabeled α4Tyr¹⁹⁵ (equivalent to Torpedo αTyr¹⁹⁰) in Segment C as well as β2Val¹¹¹ and β2Ser¹¹³ in Segment E (equivalent to Torpedo γLeu¹⁰⁹ and γTyr¹¹¹, respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the α-γ transmitter binding site, whereas it binds in two distinct orientations in the α4β2 nAChR.Nicotinic acetylcholine receptors (nAChRs)³ are prototypical members of the Cys loop superfamily of neurotransmitter-gated ion channels that mediate the actions of the neurotransmitter acetylcholine (1). nAChRs from vertebrate skeletal muscle and the electric organs of Torpedo rays are heteropentamers of homologous subunits with a stoichiometry of 2α:β:γ(ϵ):δ that are arranged pseudosymmetrically around central cation-selective ion channels (1, 2). There are 12 mammalian neuronal nAChR subunit genes: nine neuronal α subunits (α2–α10) and three neuronal β subunits (β2–β4). The α4β2 nAChR is the most abundant and widely distributed nAChR subtype expressed in the brain and is a major target for potential therapeutic agents for neurological diseases and conditions, including nicotine dependence and Alzheimer and Parkinson diseases (3, 4). Although the ratio of α4 to β2 subunit in vivo is uncertain, expressed receptors containing either three α4 or three β2 subunits have distinct pharmacological properties (5, 6).The agonist binding sites (ABS) of nAChRs are located within the amino-terminal extracellular domain at the interface of adjacent subunits (α-γ and α-δ in the Torpedo nAChR), and different nAChR subunit combinations form ABS with distinct physical and pharmacological properties (3, 7). Affinity labeling studies with Torpedo nAChR and site-directed mutational analyses of muscle and neuronal nAChRs identified key amino acids delineating the ABS from three noncontiguous stretches of the α subunit (Segments A-C, the principal component (+ face)) and three noncontiguous regions of the non-α subunit (Segments D–F, the complementary component (− face)) (8, 9). The three-dimensional structure of the ABS in the absence and presence of nAChR agonists or competitive antagonists has been determined for snail acetylcholine-binding proteins (AChBPs) that are soluble homopentamers homologous to the extracellular (amino-terminal) domain of a nAChR (10–12). In the AChBP, four aromatic amino acids from Segments A–C that are conserved within α subunits, along with a conserved Trp in Segment D, form a core aromatic “pocket” with a dimension optimal for accommodation of a trimethylammonium group. The other amino acids in the non-α subunits closest to the aromatic pocket, which are generally not conserved among γ, δ, or neuronal β subunits, are on three antiparallel β strands. The AChBP structure was used to refine the structure of the Torpedo nAChR in the absence of agonist to 4 Å resolution (13). In this structure, there is a reorientation of Segments A–C, resulting in the absence of a well defined core aromatic binding pocket.Analysis of agonist interactions with mutant nAChRs containing fluorine-substituted core aromatic residues provides evidence that cation-π interactions, particularly with αTrp¹⁴⁹ in Segment B, are important determinants of agonist binding affinity (14) and for the higher affinity binding of nicotine to α4β2 nAChRs compared with α₂βγδ nAChRs (15). Mutational analyses and molecular docking calculations have also provided evidence that two molecules of very similar structure may actually bind to a single receptor in very different orientations, as seen for two high affinity antagonists, d-tubocurarine and its quaternary ammonium analog metocurine, binding to the AChBP and to the muscle nAChR (16, 17).Photoaffinity labeling provides an alternative means to identify amino acids contributing to a drug binding site (18, 19) and has been used to determine the orientation of drugs bound in the ABS of Torpedo nAChR (20). Epibatidine binds with very high affinity (∼10 pm) to heteromeric neuronal nAChRs (e.g. α4β2) and with nanomolar affinity to α7 and muscle-type/Torpedo nAChRs (3). Utilizing a photoreactive analogue of epibatidine (azidoepibatidine; Fig. 1) and mass spectrometry, Tomizawa et al. (21) identified photolabeled amino acids in the Aplysia AChBP (Tyr¹⁹⁵ in Segment C and Met¹¹⁶ in Segment E), establishing an orientation for bound azidoepibatidine consistent with the orientation of epibatidine in an AChBP crystal structure (12).Open in a separate windowFIGURE 1.Structure of [³H]epibatidine (top) and azidoepibatidine (bottom).In this report, we use [³H]epibatidine as a photoaffinity reagent to identify the amino acids photolabeled in an expressed α4β2 nAChR and in the Torpedo α₂βγδ nAChR. Comparisons of the labeled amino acids seen in the Torpedo nAChR α-γ binding site and in the α4β2 nAChR, in conjunction with the results of docking calculations for epibatidine binding to homology models of the α₂βγδ and α4β2 nAChRs, suggests that epibatidine binds in a single orientation in the α-γ site but in two orientations in the α4β2 ABS.     

In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ_1–42) is the most important pathophysiological event associated with Alzheimer''s disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α-helix to a β-sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu²⁺ and various drugs used for AD treatment, such as galanthamine (Reminyl®), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu²⁺ and galanthamine prevent the formation of Aβ_1–42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ_1–42 acquires a characteristic U-shape before the α-helix to β-sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ₁₋₄₂ in the presence of Cu²⁺ or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu²⁺) or to Lys 28 (galanthamine), which prevents Aβ₁₋₄₂ from adopting the U-characteristic conformation that allows the amino acids to transition to a β-sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu²⁺ and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.     

TRAIL promotes caspase-dependent pro-inflammatory responses via PKC�� activation by vascular smooth muscle cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is best known for its selective cytotoxicity against transformed tumor cells. Most non-transformed primary cells and several cancer cell lines are not only resistant to death receptor-induced apoptosis, but also subject to inflammatory responses in a nuclear factor-κB (NF-κB)-dependent manner. Although the involvement of TRAIL in a variety of vascular disorders has been proposed, the exact molecular mechanisms are unclear. Here, we aimed to delineate the role of TRAIL in inflammatory vascular response. We also sought possible molecular mechanisms to identify potential targets for the prevention and treatment of post-angioplastic restenosis and atherosclerosis. Treatment with TRAIL increased the expression of intercellular adhesion molecule-1 by primary human vascular smooth muscle cells via protein kinase C (PKC)δ and NF-κB activation. Following detailed analysis using various PKCδ mutants, we determined that PKCδ activation was mediated by caspase-dependent proteolysis. The protective role of PKCδ was further confirmed in post-traumatic vascular remodeling in vivo. We propose that the TRAIL/TRAIL receptor system has a critical role in the pathogenesis of inflammatory vascular disorders by transducing pro-inflammatory signals via caspase-mediated PKCδ cleavage and subsequent NF-κB activation.     

Novel dextranase catalyzing cycloisomaltooligosaccharide formation and identification of catalytic amino acids and their functions using chemical rescue approach

A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7–14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr⁴⁵¹–Val¹⁰⁸²), a portion of which shares identity (35% at Ala³⁹–Ser¹³⁰⁴ of PsDex) with Pro³²–Ala⁷⁵⁵ of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val⁸³⁷–Met⁹³² for PsDex and Tyr⁴⁰⁴–Tyr⁴⁹² for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala³⁹–Ser¹³⁰⁴) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp¹⁸⁹, Asp³⁴⁰, Glu⁴¹², and Asp¹²⁵⁴ of PsDex) of catalytic candidates. Their amide mutants decreased activity (1/1, 500 to 1/40, 000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN₃. D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp³⁴⁰ and Glu⁴¹² as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN₃.     

Identification of the PDZ3 domain of the adaptor protein PDZK1 as a second, physiologically functional binding site for the C terminus of the high density lipoprotein receptor scavenger receptor class B type I

The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1–PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI (“target peptide”) binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr²⁰ → Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the target peptide (K_d = 37.0 μm), albeit with ∼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr²⁵³ → Ala substitution. Comparison of the 1.5-Å resolution crystal structure of PDZ3 with its bound target peptide (⁵⁰⁵QEAKL⁵⁰⁹) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr²⁰ → Ala (PDZ1) + Tyr²⁵³ → Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr²⁵³ → Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr²⁰ → Ala (PDZ1) + Tyr²⁵³ → Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.     

Role of protein kinase C δ in ER stress and apoptosis induced by oxidized LDL in human vascular smooth muscle cells

During atherogenesis, excess amounts of low-density lipoproteins (LDL) accumulate in the subendothelial space where they undergo oxidative modifications. Oxidized LDL (oxLDL) alter the fragile balance between survival and death of vascular smooth muscle cells (VSMC) thereby leading to plaque instability and finally to atherothrombotic events. As protein kinase C δ (PKCδ) is pro-apoptotic in many cell types, we investigated its potential role in the regulation of VSMC apoptosis induced by oxLDL. We found that human VSMC silenced for PKCδ exhibited a protection towards oxLDL-induced apoptosis. OxLDL triggered the activation of PKCδ as shown by its phosphorylation and nuclear translocation. PKCδ activation was dependent on the reactive oxygen species generated by oxLDL. Moreover, we demonstrated that PKCδ participates in oxLDL-induced endoplasmic reticulum (ER) stress-dependent apoptotic signaling mainly through the IRE1α/JNK pathway. Finally, the role of PKCδ in the development of atherosclerosis was supported by immunohistological analyses showing the colocalization of activated PKCδ with ER stress and lipid peroxidation markers in human atherosclerotic lesions. These findings highlight a role for PKCδ as a key regulator of oxLDL-induced ER stress-mediated apoptosis in VSMC, which may contribute to atherosclerotic plaque instability and rupture.     

PKC-dependent ERK phosphorylation is essential for P2X7 receptor-mediated neuronal differentiation of neural progenitor cells

Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X₇ receptors (P2X₇Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X₇R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X₇R might be expressed in neural progenitor cells (NPCs). P2X₇R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X₇R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X₇Rs induced Ca²⁺ influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X₇R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca²⁺ concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X₇R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca²⁺ and treatment with blockers for P2X₇R and PKC activity. Stimulation of P2X₇R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X₇R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway.     

Characterization of a New ��(1�C3)-Glucan Branching Activity of Aspergillus fumigatus

A new HPLC method was developed to separate linear from β(1–6)-branched β(1–3)-glucooligosaccharides. This methodology has permitted the isolation of the first fungal β(1–6)/β(1–3)-glucan branching transglycosidase using a cell wall autolysate of Aspergillus fumigatus (Af). The encoding gene, AfBGT2 is an ortholog of AfBGT1, another transglycosidase of A. fumigatus previously analyzed (Mouyna, I., Hartland, R. P., Fontaine, T., Diaquin, M., Simenel, C., Delepierre, M., Henrissat, B., and Latgé, J. P. (1998) Microbiology 144, 3171–3180). Both enzymes release laminaribiose from the reducing end of a β(1–3)-linked oligosaccharide and transfer the remaining chain to another molecule of the original substrate. The AfBgt1p transfer occurs at C-6 of the non-reducing end group of the acceptor, creating a kinked β(1–3;1–6) linear molecule. The AfBgt2p transfer takes place at the C-6 of an internal group of the acceptor, resulting in a β(1–3)-linked product with a β(1–6)-linked side branch. The single Afbgt2 mutant and the double Afbgt1/Afbgt2 mutant in A. fumigatus did not display any cell wall phenotype showing that these activities were not responsible for the construction of the branched β(1–3)-glucans of the cell wall.     

Diet-Induced Obesity Promotes Colon Tumor Development in Azoxymethane-Treated Mice

Obesity is an important risk factor for colon cancer in humans, and numerous studies have shown that a high fat diet enhances colon cancer development. As both increased adiposity and high fat diet can promote tumorigenesis, we examined the effect of diet-induced obesity, without ongoing high fat diet, on colon tumor development. C57BL/6J male mice were fed regular chow or high fat diet for 8 weeks. Diets were either maintained or switched resulting in four experimental groups: regular chow (R), high fat diet (H), regular chow switched to high fat diet (RH), and high fat diet switched to regular chow (HR). Mice were then administered azoxymethane to induce colon tumors. Tumor incidence and multiplicity were dramatically smaller in the R group relative to all groups that received high fat diet at any point. The effect of obesity on colon tumors could not be explained by differences in aberrant crypt foci number. Moreover, diet did not alter colonic expression of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-6, interleukin-1β, and interferon-γ, which were measured immediately after azoxymethane treatment. Crypt apoptosis and proliferation, which were measured at the same time, were increased in the HR relative to all other groups. Our results suggest that factors associated with obesity – independently of ongoing high fat diet and obesity – promote tumor development because HR group animals had significantly more tumors than R group, and these mice were fed the same regular chow throughout the entire carcinogenic period. Moreover, there was no difference in the number of aberrant crypt foci between these groups, and thus the effect of obesity appears to be on subsequent stages of tumor development when early preneoplastic lesions transition into adenomas.