Specific Amino Acid Sequences Research Articles

The Specific Amino Acid Sequence between Helices 7 and 8 Influences the Binding Specificity of Human Apolipoprotein A-I for High Density Lipoprotein (HDL) Subclasses: A POTENTIAL FOR HDL PREFERENTIAL GENERATION

Humans have two major high density lipoprotein (HDL) sub-fractions, HDL(2) and HDL(3), whereas mice have a monodisperse HDL profile. Epidemiological evidence has suggested that HDL(2) is more atheroprotective; however, currently there is no direct experimental evidence to support this postulate. The amino acid sequence of apoA-I is a primary determinant of HDL subclass formation. The majority of the alpha-helical repeats in human apoA-I are proline-punctuated. A notable exception is the boundary between helices 7 and 8, which is located in the transitional segment between the stable N-terminal domain and the C-terminal hydrophobic domain. In this study we ask whether the substitution of a proline-containing sequence (PCS) separating other helices in human apoA-I for the non-proline-containing sequence (NPCS) between helices 7 and 8 (residues 184-190) influences HDL subclass association. The human apoA-I mutant with PCS2 replacing NPCS preferentially bound to HDL(2). In contrast, the mutant where PCS3 replaced NPCS preferentially associated with HDL(3). Thus, the specific amino acid sequence between helices 7 and 8 influences HDL subclass association. The wild-type and mutant proteins exhibited similar physicochemical properties except that the two mutants displayed greater lipid-associated stability versus wild-type human apoA-I. These results focus new attention on the influence of the boundary between helices 7 and 8 on the properties of apoA-I. The expression of these mutants in mice may result in the preferential generation of HDL(2) or HDL(3) and allow us to examine experimentally the anti-atherogenicity of the HDL subclasses.

Competitive Interactions of Collagen and a Jararhagin-derived Disintegrin Peptide with the Integrin α2-I Domain

Integrin alpha2beta1 is a major receptor required for activation and adhesion of platelets, through the specific recognition of collagen by the alpha2-I domain (alpha2-I), which binds fibrillar collagen via Mg(2+)-bridged interactions. The crystal structure of a truncated form of the alpha2-I domain, bound to a triple helical collagen peptide, revealed conformational changes suggestive of a mechanism where the ligand-bound I domain can initiate and propagate conformational change to the full integrin complex. Collagen binding by alpha2-I and fibrinogen-dependent platelet activity can be inhibited by snake venom polypeptides. Here we describe the inhibitory effect of a short cyclic peptide derived from the snake toxin metalloprotease jararhagin, with specific amino acid sequence RKKH, on the ability of alpha2-I to bind triple helical collagen. Isothermal titration calorimetry measurements showed that the interactions of alpha2-I with collagen or RKKH peptide have similar affinities, and NMR chemical shift mapping experiments with (15)N-labeled alpha2-I, and unlabeled RKKH peptide, indicate that the peptide competes for the collagen-binding site of alpha2-I but does not induce a large scale conformational rearrangement of the I domain.

Context-dependent regulation of Hox protein functions by CK2 phosphorylation sites.

Variations in Hox protein sequences and functions have been proposed to contribute to evolutionary changes in appendage shape and number in crustaceans and insects. One model is that insect Hox proteins of the Ultrabithorax (UBX) ortholog class evolved increased abilities to repress Distal-less (Dll) transcription and appendage development in part through the loss of serine and threonine residues in casein kinase 2 (CK2) phosphorylation sites. To explore this possibility, we constructed and tested the appendage repression function of chimeric proteins with insertions of different CK2 consensus sites or phosphomimetics of CK2 sites in C-terminal regions of Drosophila melanogaster UBX. Our results indicate that CK2 sites C-terminal to the homeodomain can inhibit the appendage repression functions of UBX proteins, but only in the context of specific amino acid sequences. Our results, combined with previous findings on evolutionary changes in Hox protein, suggest how intra-protein regulatory changes can diversify Hox protein function, and thus animal morphology.

Glycolytic enzyme expression in human granulosa cells

To examine the differences in specific protein expression between mural and cumulus granulosa cells following 24-hour in vitro culture. Laboratory study. University Hospital. Human granulosa cells were collected at the time of egg collection during assisted reproduction. Cumulus cells associated with the oocyte were separated from mural cells from the periphery of the follicle before in vitro culture for 24 hours. Cells were then lysed and subjected to two-dimensional gel electrophoresis. Given that cumulus (cGC) and mural granulosa cells (mGC) differentiate from a single layer, it is likely that phenotypic differences between them may reflect specific molecular processes and structural adaptations. Computer-assisted analysis using dedicated software enabled the presence, absence, or relative volume of each individual protein spot to be estimated. Differentially expressed spots were identified using tandem mass spectrometry. The mean number of separate protein spots detected in mGC gels was 1,105 +/- 146, and in cGC it was 887 +/- 236, although there was no statistically significant difference between the two. Five enzymes of the glycolytic pathway were never expressed in cGC after 24 hours in vitro; these were triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase 1, and two isoforms of alpha enolase. These are the first data collected in humans consistent with a recent demonstration that isolated murine cGC cultured in vitro exhibit decreased expression of mRNA encoding glycolytic enzymes, and support the suggestion that some factor or factors secreted by the oocyte may be responsible for the maintenance of glycolysis in the adjacent cGC.

Podophyllotoxin directly binds a hinge domain in E2 of HPV and inhibits an E2/E7 interaction in vitro

Podophyllotoxin (PT), a strong cytotoxic agent from berberidaceae, has been known to inhibit tubulin polymerization. Although PT has been used for developing anticancer drugs as one of seed compounds, clinical treatment by itself has been unsuccessful because of the side effects, except one example in the treatments of warts. In this study, we screened peptides binding to PT with T7 phage display clonings in order to obtain more information about molecular mechanism of the action. A selected phage clone has a specific amino acid sequence to be SVPSRRRPDGRTHRSSRG. A homology search by protein database BLAST showed that this sequence had a similarity to a hinge domain (HD) of E2 protein in human papillomavirus (HPV) type 1a which is known to cause plantar warts. Surface plasmon resonance (SPR) analysis showed that PT bound to a recombinant HPV 1a E2 protein giving a K D = 24.1 μM which has compared with those of other domains of E2 protein. Also we demonstrated whether PT inhibited HD interaction or not. E7 protein of HPV has been known to be an oncoprotein and was reported to interact with HD of E2 protein. We demonstrated that an E2/E7 interaction was inhibited by the addition of PT in this report. And we showed the bindings of PT to other types of HPV. Our results suggest that PT is potential as a tool for clarifying the molecular mechanism of HPV.

First-Principles Free-Energy Analysis of Helix Stability: The Origin of the Low Entropy in π Helices

The temperature dependence of the stability of infinite poly-L-alanine alpha, pi, and 310 helices with respect to the fully extended structure (FES) is calculated using density functional theory and the harmonic approximation. We find that the vibrational entropy strongly reduces the stability of the helical conformations with respect to the FES. By mapping the ab initio data on an approximate mechanical model, we show that this effect is exclusively due to the formation of hydrogen bonds, whereas changes in the backbone stiffness are practically negligible. We furthermore observe that the temperature dependence is largest for the pi helix and smallest for the 310 helix and demonstrate that these trends are a generic behavior related to the geometric peculiarities of the respective helical conformations and independent of the specific amino acid sequence.

ERK and p38 MAPK‐regulated peroxiredoxin 6 localization to lamellar bodies in MLE 12 cells

Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A2 (aiPLA2) activities, participates in lung surfactant phospholipid turnover and protects lung epithelium against oxidative stress. Prdx6 has been localized by immunocytochemistry and sub‐cellular fractionation to cytosol and acidic compartments (lamellar bodies and lysosomes) in lung alveolar epithelium. We postulate that Prdx6 subcellular localization determines the balance between the 2 activities. Using a GFP‐Prdx6 fusion protein, we show that ERK and p38 MAPK regulate Prdx6 localization to organellar structures compatible with lamellar bodies in the MLE12 mouse type II alveolar epithelial cell line. Treatment of GFP‐Prdx6 transfected MLE12 cells with PD98059 and SB202190, inhibitors of ERK and p38 MAPK, respectively, abolished Prdx6 organellar targeting; a JNK kinase inhibitor had no effect. Using mutagenesis, we show that Prdx6 organellar localization is determined by the 31–40 amino acid region which contains a phospholipid binding sequence GDSWG. Serine to alanine (S32A) mutation within this sequence abolished targeting and decreased protein binding to phospholipids as determined by FRET analysis. These studies suggest that Prdx6 targeting to acidic organelles and its consequent PLA2 activity may be determined by MAPK‐regulated lipid binding to a specific Prdx6 amino acid sequence. [HL019737]

Histochemische Identifizierung endogener Lektine durch markierte Neoglykoproteine bei humanen Kopf-Hals-Plattenepithelkarzinomen*

According to the "Population-based cancer register" of the Federal Republic of Germany only malignant neoplasms of the buccal cavity, the pharynx and larynx as well as cancers of the respiratory tract show an increasing rate of incidence and mortality. The molecular mechanisms and etiological factors causing this phenomenon are still little understood despite intensive research work. Recognition between receptors on a cellular level may be mediated by specific amino acid sequences on the level of protein-protein recognition. Additionally, the interactions between cell sugars and the corresponding protein receptor may play a decisive role in development, regeneration and organisation of cells and tissue. The high specificity of the binding of biotinylated neoglycoproteins in tissue sections enables to detect glycohistochemically binding sites for the carbohydrate ligands of the glycosylated carrier protein. The evidence of lectins in squamous cell cancer of the oral cavity, oropharynx, larynx, and hypopharynx has not been established so far. Squamous cell cancer tissue samples of twelve patients with different tumour locations were investigated by incubation of sections of paraffin-embedded samples and application of an avidin-biotin-peroxidase complex for visualisation with synthetic biotinylated neoglycoproteins. Altogether 168 stained sections were evaluated including controls. Pronounced cytoplasmatic staining was seen with the following neoglycoproteins: sialic acid-bovine serum albumin (BSA), glucuronic acid-BSA, N-acetylglucosamine (glcNAc)-BSA, N-acetylgalactosamine (beta-galNAc)-BSA, lactose-BSA, maltose-BSA, mannose-BSA, mannose-6-phosphate-BSA. No corresponding lectins seems to exist for the following investigated sugars: fucoidan, heparin, and the alpha-anomeric form of N-acetylgalactosamine, because no specific staining was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

IDENTIFICATION OF A STARCH-BRANCHING ENZYME AND COEXISTING STARCH BIOSYNTHETIC ENZYMES FROM PARTIALLY PURIFIED MUNG BEAN (VIGNA RADIATA L.) FRACTIONS

Three starch-branching enzyme (BE) bands (I, II and III) were detected by zymogram in the soluble extract of developing mung bean (Vigna radiata L. cv KPS1), and radioactive method was used to trace BE during purification by liquid chromatography. Protein profiles of bands I and II were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by proteomic analysis with liquid chromatography–mass spectroscopy/mass spectrometry and bioinformatic searching. The 96 kDa BE possessing specific amino acid sequences of the A-type family of BEs was identified from band II. Mung bean 92 kDa sucrose synthase and the previously identified 105 kDa starch phosphorylase (SP) and its 55 kDa fragments were also found in both bands I and II. Western blotting with SP antibodies showed that SP was present in all of the purified BE fractions. In summary, with the aid of proteomic analysis, we have identified a major mung bean BE and coexisting starch biosynthetic enzymes from partially purified fractions. PRACTICAL APPLICATIONS Mung bean starch possesses a unique structure of 45% dry weight of branched amylose content and is expected to be synthesized by unusual biological machinery. Among the enzymes in the starch biosynthesis pathway of higher plants, branching enzyme (BE) is considered responsible for producing branched structure from amylose and amylopectin chains. We utilized zymogram and radioactive methods to trace BE during purification by liquid chromatography. Protein profiles of active bands were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by proteomic analysis with liquid chromatography–mass spectroscopy/mass spectrometry and bioinformatic searching. A mung bean 96 kDa A-type BE and coexisting starch biosynthetic enzymes, starch phosphorylase and sucrose synthase, able to synthesize starch in vitro, were identified from partially purified fractions. The internal sequences found in mung bean BE enabled the design of degenerate primers for the cloning and expression of BE by molecular techniques to further investigate its physiological role and biotechnology applications in the food industry.

Arabidopsis CLV3 Peptide Directly Binds CLV1 Ectodomain

CLV1, which encodes a leucine-rich repeat receptor kinase, and CLV3, which encodes a secreted peptide, function in the same genetic pathway to maintain stem cell populations in Arabidopsis shoot apical meristem. Here, we show biochemical evidence, by ligand binding assay and photoaffinity labeling, that the CLV3 peptide directly binds the CLV1 ectodomain with a dissociation constant of 17.5 nM. The CLV1 ectodomain also interacts with the structurally related CLE peptides, with distinct affinities depending on the specific amino acid sequence. Our results provide direct evidence that CLV3 and CLV1 function as a ligand-receptor pair involved in stem cell maintenance.

Proteinase-activated Receptor-2 Induces Cyclooxygenase-2 Expression through β-Catenin and Cyclic AMP-response Element-binding Protein

Chronic inflammation of mucosae is associated with an increased cancer risk. Tumorigenesis in these tissues is associated with the activity of some proteinases, cyclooxygenase-2 (COX-2), and beta-catenin. Serine proteinases participate in both inflammation and tumorigenesis through the activation of proteinase-activated receptor-2 (PAR(2)), which up-regulates COX-2 by an unknown mechanism. We sought to determine whether beta-catenin participated in PAR(2)-induced COX-2 expression and through what cellular mechanism. In A549 epithelial cells, we showed that PAR(2) activation increased COX-2 expression through the beta-catenin/T cell factor transcription pathway. This effect was dependent upon ERK1/2 MAPK, which inhibited the beta-catenin-regulating protein, glycogen synthase kinase-3beta, and induced the activity of the cAMP-response element-binding protein (CREB). Knockdown of CREB by small interfering RNA revealed that PAR(2)-induced beta-catenin transcriptional activity and COX-2 expression were CREB-dependent. A co-immunoprecipitation assay revealed a physical interaction between CREB and beta-catenin. Thus, PAR(2) up-regulated COX-2 expression via an ERK1/2-mediated activation of the beta-catenin/Tcf-4 and CREB pathways. These findings reveal new cellular mechanisms by which serine proteinases may participate in tumor development and are particularly relevant to cancers associated with chronic mucosal inflammation, where serine proteinases are abundant and COX-2 overexpression is a common feature.

Morphology of Artificial Silica Matrices Formed via Autosilification of a Silaffin/Protein Polymer Chimera

Protein polymers (long-chain proteins in which a specific amino acid sequence "monomer" is repeated through the molecule) are found widely in nature, and these materials exhibit a diverse array of physical properties. One class of self-assembling proteins is hydrophobic-polar (HP) protein polymers capable of self-assembly under the appropriate solution conditions. We generated a chimeric protein consisting of an HP protein polymer monomer unit, EAK 1 (sequence n-AEAEAKAKAEAEAKAK-c), and a silaffin peptide, R5 (sequence: n-SSKKSGSYSGSKGSKRRIL-c). First identified in diatoms, silaffins represent a class of proteins and peptides capable of directing silica precipitation in vitro at neutral pH and ambient temperatures. The EAK 1-R5 chimera demonstrated self-assembly into hydrogels and the ability to direct silica precipitation in vitro. This chimera is capable of generating silica morphologies and feature sizes significantly different from those achievable with the R5 peptide alone, indicating that fusions of silaffins with self-assembling proteins may be a route to controlling the morphology of artificially produced silica matrices.

Highly Promiscuous Nature of Prion Polymerization

The primary structure of the prion protein (PrP) is believed to be the key factor in regulating the species barrier of prion transmission. Because the strength of the species barrier was found to be affected by the prion strain, the extent to which the barrier can indeed be attributed to differences in the PrP primary structures of either donor and acceptor species remains unclear. In this study, we exploited the intrinsic property of PrP to polymerize spontaneously into disease-related amyloid conformations in the absence of a strain-specified template and analyzed polymerization of mouse and hamster full-length recombinant PrPs. Unexpectedly, we found no evidence of species specificity in cross-seeding polymerization assays. Even when both recombinant PrP variants were present in mixtures, preformed mouse or hamster fibrils displayed no selectivity in elongation reactions and consumed equally well both homologous and heterologous substrates. Analysis of individual fibrils revealed that fibrils can elongate in a bidirectional or unidirectional manner. Our work revealed that, in the absence of a cellular environment, post-translational modifications, or strain-specified conformational constraints, PrP fibrils are intrinsically promiscuous and capable of utilizing heterologous PrP variants as a substrate in a highly efficient manner. This study suggests that amyloid structures are capable of accommodating local perturbations arising because of a mismatch in amino acid sequences and highlights the promiscuous nature of the self-propagating activity of amyloid fibrils.

A Novel Class of Activating Mutations in NOTCH1 in T-ALL.

Aberrant activation of NOTCH1 signaling induces transformation of T-cell progenitors and plays a prominent role in the pathogenesis of over 50% of human T-cell acute lymphoblastic leukemias (T-ALL), which harbor activating mutations in the heterodimerization (HD) and PEST domains of NOTCH1. Here we report a new class of activating mutations in NOTCH1 in human T-ALL. These so called JuxtaMembrane Expansion (JME) mutants consist of internal tandem duplications of exon 28 and adjacent intronic sequences in the NOTCH1 gene, which result in expansions of the extracellular juxtamembrane region of the NOTCH1 receptor. Western blot analysis of T-ALL cell lines lacking known NOTCH1 mutations demonstrated high levels of activated NOTCH1 protein in Jurkat T-ALL cells, suggesting the presence of an as yet unidentified activating NOTCH1 mutation in this cell line. Sequence analysis of Jurkat NOTCH1 transcripts revealed an internal tandem duplication in exon 28 of NOTCH1, resulting in the insertion of 17 amino acids at position 1740 in the extracelullar juxtamembrane region of the receptor. Subsequent PCR amplification of NOTCH1 exon 28 sequences from 194 primary T-ALL lymphoblast samples identified seven additional in frame insertion mutations ranging from 11 to 36 amino acids in length, all of which were located in the vicinity of codon 1740 in the extracelullar juxtamembrane region of the NOTCH1 receptor. Luciferase assays showed that expression of the NOTCH1 Jurkat JME17 mutant allele induced over 200 fold activation of a NOTCH1 reporter construct compared to controls. Activation of NOTCH1 signaling requires proteolytic cleavage of the receptor, first by an ADAM metalloprotease (S2 clevage) and subsequently by the gamma-secretase complex. NOTCH1 signaling induced by the Jurkat JME17 mutant was completely abrogated by incubation with CompE, a highly active gamma-secretase inhibitor. Consistently, treatment of Jurkat T-ALL cells with CompE resulted in rapid clearance of activated NOTCH1 protein and in marked downregulation of NOTCH1 target genes such as HES1 and DELTEX1. Interestingly, and in contrast with previously described HD mutations, JME NOTCH1 alleles retain an intact HD domain and a protected canonical S2 metalloprotease cleavage site. Thus, we hypothesized that activation of NOTCH1 by JME mutations could be mediated by aberrant metalloprotease cleavage at ectopic S2 sites within the JME insertion sequence. However, mutation of the canonical S2 cleavage abrogated the function of the NOTCH1 Jurkat JME17 mutant allele. Furthermore, analysis of artificially generated JME insertions containing sequences unrelated to the leukemia-derived JME alleles, showed that activation of NOTCH1 by JME mutations depends primarily on the length of the inserted peptides and not on their specific amino acid sequences. Thus, shorter insertions of 5 to 13 amino acids in length induced moderate (5–10 fold) activation of the NOTCH1 receptor, while insertions of 14 amino acids or longer induced marked (>70 fold) increases in NOTCH1 signaling. Overall, these results provide further insight in the mechanisms that control the activation of the NOTCH1 receptor in T-ALL.

Synthetic 15-mer Peptide (PCK3145) Derived from Prostate Secretory Protein with In Vitro and In Vivo Activity Against Non-Hodgkin's Lymphoma and Other Hematologic Malignancies.

Prostate secretory protein 94 (PSP-94) has been shown to exert anti-tumor activity against prostate cancer cells, particularly in the form of PCK3145, a synthetic peptide corresponding to amino acids 31–45 of PSP-94. Indeed, when tested in a murine model, this peptide could reduce experimental prostate tumour growth. In addition, when evaluated in a Phase I clinical study, this peptide demonstrated a particularly interesting safety profile, with almost complete lack of toxicity. In order to determine whether PCK3145 could exert cytotoxic activity against other marrow infiltrating cancers, we tested its activity both in vitro and in vivo against non-Hodgkin's lymphoma (NHL) and other hematologic cancers. Interestingly, PCK3145 inhibited the proliferation of human NHL (SR) and myeloma (RPMI-8226) cell lines in vitro. To explore its anti-tumor activity in vivo, the impact of PCK3145 was also measured by inoculating P815 malignant cells into syngeneic DBA mice. First, four groups of 6 DBA mice were injected subcutaneously with 2x104 P815 cells and then treated with subcutaneous injections of PCK3145, and compared to1.a peptide with the scrambled amino acid sequence,2.PCK5266 (peptide derived from amino acids 52 to 66 of PSP-94), and3.phosphate-buffered saline (PBS).Treatment with PCK3145 significantly decreased the growth of P815 tumours in comparison to PBS (p<0.001), scrambled peptide (p<0.05) and PCK5266 (p<0.01), confirming in vivo anti-tumor activity and suggesting that tumour growth inhibition is due to the specific amino acid sequence of PCK3145. The same model was used to determine the effect of PCK3145 on metastatic dissemination following intraperitoneal administration of the peptide. PCK3145 treatment led to a decreased number of liver metastasis compared to PBS (p<0.05) and scrambled peptide (p<0.05) controls. In order to determine whether PCK3145 exerted its activity by altering metalloproteinase release, metalloproteinase MMP-9 levels were measured 3 weeks post-tumor cell exposure. MMP-9 levels, measured by ELISA, in the peripheral blood of treated P815 bearing mice were similar to those obtained with healthy animals (12.83±1.890 (mean±SD) ng/ml and 7.183±0.4070 ng/ml, respectively), while MMP-9 levels were elevated in mice treated with PBS and scrambled peptide (35.12±8.559 ng/ml and 22.60±3.944 ng/ml, respectively; p<0.05). We next tested PCK3145 treatment on human SR lymphoma cell line grown subcutaneously in NOD/SCID mice. Similarly to results obtained with murine tumors, treatment with PKC3145 resulted in significant inhibition of SR non-Hodgkin's lymphoma growth compared to treatment with PBS (p<0.001) and scrambled peptide (p<0.01). These results demonstrate that in vivo treatment with PCK3145 can reduce tumor cell proliferation of both murine and human hematologic cancers. In addition, PCK3145 has the potential to inhibit tumor cells dissemination by lowering MMP-9 secretion. Thus, PCK3145 represents a unique peptide demonstrating sequence-specific anti-tumor activity against NHL and other hematologic malignancies. Based on these results, clinical studies are being designed to evaluate its therapeutic activity in humans.

Between Order and Disorder in Protein Structures: Analysis of “Dual Personality” Fragments in Proteins

In their natural environment, three-dimensional structures of proteins undergo significant fluctuations and are often partially or completely disordered. This phenomenon recently became the focus of much attention, as many proteins, especially from higher organisms, were shown to contain large intrinsically disordered regions. Such disordered regions may become ordered only under very specific circumstances, if at all, and can be recognized by specific amino acid composition and sequence signatures. Here, we suggest that the balance between order and disorder is much more subtle in that many regions are very close to the order/disorder boundary. Specifically, analysis of redundant sets of experimental models of protein structures, where emphasis is put on comparison of structures of identical proteins solved in different conditions and functional states, shows hundreds of fragments captured in two states: ordered and disordered. We show that such fragments, which we call here "dual personality" (DP) fragments, have distinctive features that differentiate them from both regularly folded and intrinsically disordered fragments. We hypothesize, and show on several examples, that such fragments are often targets of regulation, either by allostery or posttranslational modifications.

Suppression of Tubulin Polymerization by the LKB1-Microtubule-associated Protein/Microtubule Affinity-regulating Kinase Signaling

LKB1, a tumor suppressor gene mutated in the Peutz-Jeghers syndrome, encodes a serine/threonine protein kinase. Recent biochemical studies have shown that LKB1 activates 14 AMP-activated protein kinase-related kinases including MARKs (microtubule-associated protein/microtubule affinity-regulating kinases) that regulate microtubule dynamics. Here we show in vitro that LKB1 phosphorylates and activates MARK2, which in turn phosphorylates microtubule-associated protein Tau at the KXGS motif and suppresses tubulin polymerization. In cells, forced expression of LKB1 suppresses microtubule regrowth, whereas LKB1 knockdown accelerates it. We further show that the phosphorylation of Tau by the LKB1-MARK signaling triggers proteasome-mediated degradation of Tau. These results indicate that LKB1 is involved in the regulation of microtubule dynamics through the activation of MARKs.

Cloning and Expression of Partial Japanese Flounder (Paralichthys olivaceus) IgD

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7 d Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.

A potent immunosuppressive retroviral peptide: cytokine patterns and signaling pathways

A synthetic bioactive peptide composed of 17 amino acids (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane envelope proteins induces not only significant immunoregulatory functions but also exhibits Th1-inhibiting properties, as described by its ability to suppress cell-mediated immunity and inhibit the production of interleukin (IL) 12, IL-2, gamma interferon, and tumor necrosis factor alpha, while enhancing IL-10. An important molecular mechanism responsible for the observed cytokine profiles by CKS-17 is provided by our findings demonstrating that this small peptide activates several intracellular signaling molecules, i.e., elevates intracellular cyclic adenosine monophosphate (cAMP) levels, and induces phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, mitogen-activated protein kinase/ERK kinase (MEK), protein kinase D, Raf1, and phospholipase C gamma1 (PLCgamma1). The activation of ERK1/2 is via the PLCgamma1-protein kinase C-Raf1-MEK signaling cascade. The activation of both ERK1/2 and cAMP appears to be via a mechanism sensitive to AG879, a receptor tyrosine kinase inhibitor, but not to AG825, AG1296, or AG1478. Furthermore, phosphoinositide-3 kinase appears to mediate the CKS-17-induced activation of ERK1/2, but not of cAMP. A specific amino acid sequence as well as the dimerization of this peptide is required to confer these biological activities. The results obtained are compelling and reproducible. This highly conserved molecule may enable us to understand a basic mechanism(s) of intracellular signaling pathways, regulation of Th1/Th2 cytokines, immunosuppression, and immunologic tolerance.

Discriminating taste of prions

Prions are infectious proteins that are involved in brain-wasting disorders such as mad cow disease. In yeast, specific sequences of amino acids in prions seem to mediate prion propagation and cross-species transmissibility.