Secretory production of a camelid single-domain antibody (VHH, nanobody) by the Serratia marcescens Lip system in Escherichia coli.

Escherichia coli is one of the most popularly used hosts to produce recombinant proteins. Most recombinant proteins are produced in the cytoplasm and periplasm, requiring multiple steps to extract and purify recombinant proteins. The Serratia marcescens Lip system (LipB-LipC-LipD) is a type 1 secretion system that selectively secretes LipA from the intracellular to extracellular space in a single step. This study aimed to establish a secretory production system for nanobodies, camelid-derived small molecule antibody fragments, using the S. marcescens Lip system. Surprisingly, E. coli harboring only LipC, a membrane fusion protein of the Lip system, could secrete an anti-green fluorescent protein (GFP)-Nb, a nanobody against GFP, without the addition of a long amino acid sequence. The LipC-based secretion system recognized the Val-Thr-Val sequence at the C-terminus of the nanobody. Finally, Strep-tagged anti-GFP-Nb was purified from culture supernatants of E. coli harboring LipC by Strep-affinity chromatography at a final yield of >5 mg per liter of culture supernatant. These results potently supported that the S. marcescens LipC-based secretion system has the potential to establish an efficient secretory production system for nanobodies.

Natriuretic peptide receptor-C releases and activates guanine nucleotide-exchange factor H1 in a ligand-dependent manner.

Although natriuretic peptide receptor-C (NPR-C) is involved in the clearance of natriuretic peptides from plasma, it also possesses other physiological functions, such as inhibition of adenylyl cyclase activity through Gαi. However, the physiological roles and intracellular signaling pathways of NPR-C have yet been not fully elucidated. In this study, we identified a RhoA-specific guanine nucleotide-exchange factor, GEF-H1, as a novel binding protein of NPR-C. We demonstrated that endogenous NPR-C interacted with GEF-H1 in HeLa cells, and that the interaction between NPR-C and GEF-H1 was dependent on a 37-amino acid cytoplasmic region of NPR-C. In contrast, another natriuretic peptide receptor, NPR-A, which includes the kinase homology and guanylyl cyclase domains in the intracellular region, did not interact with GEF-H1. We also revealed that the ligands of NPR-C (i.e., ANP, CNP, and osteocrin) caused dissociation of GEF-H1 from NPR-C. Furthermore, osteocrin treatment induced phosphorylation of GEF-H1 at Ser-886, enhanced the interaction of GEF-H1 with 14-3-3, and increased the amount of activated GEF-H1. These findings strongly supported that NPR-C may be involved in diverse physiological roles by regulating GEF-H1 signaling.

Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells.

Although we recently reported that sudachitin (5,7,4'-trihydroxy-6,8,3'-trimethoxyflavone), a polymethoxyflavone isolated from the peel of Citrus sudachi, can induce apoptosis in human keratinocyte HaCaT cells, the mechanism underlying its action remains unclear. In this study, we explored the mechanisms underlying sudachitin-induced apoptosis in HaCaT cells. Sudachitin activated p38MAPK and inhibited ERK1/2, whereas another polymethoxyflavone, nobiletin (5,6,7,8,3',4'-hexamethoxyflavone), activated ERK1/2. The p38MAPK inhibitor SB203580 significantly attenuated sudachitin-induced heat shock protein 27 phosphorylation, downstream of p38MAPK, and subsequent apoptosis, indicating that sudachitin induces apoptosis via the p38MAPK pathway. Additionally, sudachitin inhibited serum- and EGF-stimulated Raf-1-ERK1/2 activation, and blocked EGF-induced cell migration and proliferation in HaCaT cells. These results suggest that small structural differences in polymethoxyflavones can induce different cellular responses by altering the regulation of MAPK activities and that sudachitin may be a potential candidate for developing new drugs for skin diseases such as psoriasis.

Intracellular localization and binding partners of death associated protein kinase-related apoptosis-inducing protein kinase 1.

Death associated protein kinase (DAPK)-related apoptosis-inducing protein kinase (DRAK)-1 is a positive apoptosis regulator. However, the molecular mechanisms underlying the DRAK1-mediated apoptotic pathway remain unclear. In this study, we demonstrated the intracellular localization and binding partners of DRAK1. In human osteosarcoma cell line U2OS cells, DRAK1 was mainly localized in the nucleus and translocated outside the nucleus through Ser395 phosphorylation by protein kinase C. In the nucleus, DRAK1 associated with tumor suppressor p53 and positively regulated p53 transcriptional activity in response to DNA-damaging agent cisplatin. On the other hand, DRAK1 interacted with the mitochondrial inner-membrane protein, adenine nucleotide translocase (ANT)-2, an anti-apoptotic oncoprotein, outside the nucleus. These findings suggest that DRAK1 translocates in response to stimuli and induces apoptosis through its interaction with specific binding partners, p53 and/or ANT2.

Citrus peel polymethoxyflavones, sudachitin and nobiletin, induce distinct cellular responses in human keratinocyte HaCaT cells.

A variety of polyphenols have been isolated from plants, and their biological activities have been examined. Sudachitin (5,7,4'-trihydroxy-6,8,3'-trimethoxyflavone) is a polymethoxyflavone that is isolated from the peel of Citrus sudachi. Although we previously reported that sudachitin possesses an anti-inflammatory activity, its other biological activities are not yet understood. In this study, we report a novel biological activity of sudachitin, which selectively induced apoptosis in human keratinocyte HaCaT cells. Another polymethoxyflavone, nobiletin (5,6,7,8,3',4'-hexamethoxyflavone), promoted autophagy but not apoptosis in HaCaT cells. On the other hand, 3'-demethoxysudachitin (5,7,4'-trihydroxy-6,8-dimethoxyflavone) failed to induce apoptosis and autophagy. These results show that three polymethoxyflavones have different effects on apoptosis and autophagy in HaCaT cells. Understanding the structure and biological activity of polymethoxyflavones may lead to the discovery of potential candidates for cancer drug development without significant toxic side effects. Abbreviations: ROS: reactive oxygen species; DMSO: dimethyl sulfoxide; MTT: 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PARP: poly(ADP-ribose) polymerase; PI: propidium iodide; MAPK: mitogen-activated protein kinase.

Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II.

Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.

Type II cGMP-dependent protein kinase negatively regulates fibroblast growth factor signaling by phosphorylating Raf-1 at serine 43 in rat chondrosarcoma cells.

Although type II cGMP-dependent protein kinase (PKGII) is a major downstream effector of cGMP in chondrocytes and attenuates the FGF receptor 3/ERK signaling pathway, its direct target proteins have not been fully explored. In the present study, we attempted to identify PKGII-targeted proteins, which are associated with the inhibition of FGF-induced MAPK activation. Although FGF2 stimulation induced the phosphorylation of ERK1/2, MEK1/2, and Raf-1 at Ser-338 in rat chondrosarcoma cells, pretreatment with a cell-permeable cGMP analog strongly inhibited their phosphorylation. On the other hand, Ser-43 of Raf-1 was phosphorylated by cGMP in a dose-dependent manner. Therefore, we examined the direct phosphorylation of Raf-1 by PKGII. Wild-type PKGII phosphorylated Raf-1 at Ser-43 in a cGMP-dependent manner, but a PKGII D412A/R415A mutant, which has a low affinity for cGMP, did not. Finally, we found that a phospho-mimic mutant, Raf-1 S43D, suppressed FGF2-induced MAPK pathway. These results suggest that PKGII counters FGF-induced MEK/ERK activation through the phosphorylation of Raf-1 at Ser-43 in chondrocytes.

Alisol B, a triterpene from Alismatis rhizoma (dried rhizome of Alisma orientale), inhibits melanin production in murine B16 melanoma cells.

To develop new whitening agents from natural products, we screened 80 compounds derived from crude drugs in Kampo medicine in a melanin synthesis inhibition assay using murine B16 melanoma cells. The screen revealed that treatment with alisol B, a triterpene from Alismatis rhizoma, significantly decreased both melanin content and cellular tyrosinase activity in B16 cells. However, alisol B did not directly inhibit mushroom tyrosinase activity in vitro. Therefore, we investigated the mechanism underlying the inhibitory effect of alisol B on melanogenesis. Alisol B suppressed mRNA induction of tyrosinase and its transcription factor, microphthalmia-associated transcription factor (MITF). Furthermore, alisol B reduced the phosphorylation of CREB and maintained the activation of ERK1/2. These results suggest that the reduction in melanin production by alisol B is due to the downregulation of MITF through the suppression of CREB and activation of ERK and that alisol B may be useful as a new whitening agent.

PCTAIRE kinase 3/cyclin-dependent kinase 18 is activated through association with cyclin A and/or phosphorylation by protein kinase A.

PCTAIRE kinase 3 (PCTK3)/cyclin-dependent kinase 18 (CDK18) is an uncharacterized member of the CDK family because its activator(s) remains unidentified. Here we describe the mechanisms of catalytic activation of PCTK3 by cyclin A2 and cAMP-dependent protein kinase (PKA). Using a pulldown experiment with HEK293T cells, cyclin A2 and cyclin E1 were identified as proteins that interacted with PCTK3. An in vitro kinase assay using retinoblastoma protein as the substrate showed that PCTK3 was specifically activated by cyclin A2 but not by cyclin E1, although its activity was lower than that of CDK2. Furthermore, immunocytochemistry analysis showed that PCTK3 colocalized with cyclin A2 in the cytoplasm and regulated cyclin A2 stability. Amino acid sequence analysis revealed that PCTK3 contained four putative PKA phosphorylation sites. In vitro and in vivo kinase assays showed that PCTK3 was phosphorylated by PKA at Ser(12), Ser(66), and Ser(109) and that PCTK3 activity significantly increased via phosphorylation at Ser(12) by PKA even in the absence of cyclin A2. In the presence of cyclin A2, PCTK3 activity was comparable to CDK2 activity. We also found that PCTK3 knockdown in HEK293T cells induced polymerized actin accumulation in peripheral areas and cofilin phosphorylation. Taken together, our results provide the first evidence for the mechanisms of catalytic activation of PCTK3 by cyclin A2 and PKA and a physiological function of PCTK3.

Crystal structure of the cGMP-dependent protein kinase II leucine zipper and Rab11b protein complex reveals molecular details of G-kinase-specific interactions.

cGMP-dependent protein kinase (PKG)-interacting proteins (GKIPs) mediate cellular targeting of PKG isoforms by interacting with their leucine zipper (LZ) domains. These interactions prevent aberrant signaling cross-talk between different PKG isotypes. To gain detailed insight into isotype-specific GKIP recognition by PKG, we analyzed the type II PKG leucine zipper domain and found that residues 40-83 dimerized and specifically interacted with Rab11b. Next, we determined a crystal structure of the PKG II LZ-Rab11b complex. The PKG II LZ domain presents a mostly nonpolar surface onto which Rab11b docks, through van der Waals interactions. Contact surfaces in Rab11b are found in switch I and II, interswitch, and the ß1/N-terminal regions. This binding surface dramatically differs from that seen in the Rab11 family of interacting protein complex structures. Structural comparison with PKG Iα and Iß LZs combined with mutagenic analysis reveals that GKIP recognition is mediated through surface charge interactions.

Death-associated protein kinase 2 mediates nocodazole-induced apoptosis through interaction with tubulin.

Death-associated protein kinase 2 (DAPK2) is a positive regulator of apoptosis. Although we recently reported that 14-3-3 proteins inhibit DAPK2 activity and its subsequent apoptotic effects via binding to DAPK2, the molecular mechanisms underlying the DAPK2-mediated apoptotic pathway remain unclear. Therefore, we attempted to further identify DAPK2-interacting proteins using pull-down assays and mass spectrometry. The microtubule ß-tubulin was identified as a novel DAPK2-binding protein in HeLa cells. Pull-down assays revealed that DAPK2 interacted with the α/ß-tubulin heterodimer, and that the C-terminal region of DAPK2, which differs from that of other DAPK family members, was sufficient for the association with ß-tubulin. Although the microtubule-depolymerizing agent nocodazole induced apoptosis in HeLa cells, the level of apoptosis was significantly decreased in the DAPK2 knockdown cells. Furthermore, we found that treatment with nocodazole resulted in an increased binding of DAPK2 to ß-tubulin. These findings indicate that DAPK2 mediates nocodazole-induced apoptosis via binding to tubulin.

Suppression of death-associated protein kinase 2 by interaction with 14-3-3 proteins.

Death-associated protein kinase 2 (DAPK2), a Ca(2+)/calmodulin-regulated serine/threonine kinase, induces apoptosis. However, the signaling mechanisms involved in this process are unknown. Using a proteomic approach, we identified 14-3-3 proteins as novel DAPK2-interacting proteins. The 14-3-3 family has the ability to bind to phosphorylated proteins via recognition of three conserved amino acid motifs (mode 1-3 motifs), and DAPK2 contains the mode 3 motif ((pS/pT)X1-2-COOH). The interaction of 14-3-3 proteins with DAPK2 was dependent on the phosphorylation of Thr(369), and effectively suppressed DAPK2 kinase activity and DAPK2-induced apoptosis. Furthermore, we revealed that the 14-3-3 binding site Thr(369) of DAPK2 was phosphorylated by the survival kinase Akt. Our findings suggest that DAPK2-induced apoptosis is negatively regulated by Akt and 14-3-3 proteins.

NVL2, a nucleolar AAA-ATPase, is associated with the nuclear exosome and is involved in pre-rRNA processing.

Nuclear VCP-like 2 (NVL2) is a member of the chaperone-like AAA-ATPase family and is involved in the biosynthesis of 60S ribosomal subunits in mammalian cells. We previously showed the interaction of NVL2 with a DExD/H-box RNA helicase MTR4/DOB1, which is a known cofactor for an exoribonuclease complex, the exosome. This finding implicated NVL2 in RNA metabolic processes during ribosome biogenesis. In the present study, we found that a series of mutations within the ATPase domain of NVL2 causes a defect in pre-rRNA processing into mature 28S and 5.8S rRNAs. Co-immunoprecipitation analysis showed that NVL2 was associated with the nuclear exosome complex, which includes RRP6 as a nucleus-specific catalytic subunit. This interaction was prevented by depleting either MTR4 or RRP6, indicating their essential role in mediating this interaction with NVL2. Additionally, knockdown of MPP6, another cofactor for the nuclear exosome, also prevented the interaction by causing MTR4 to dissociate from the nuclear exosome. These results suggest that NVL2 is involved in pre-rRNA processing by associating with the nuclear exosome complex and that MPP6 is required for maintaining the integrity of this rRNA processing complex.

Estrogen stimuli promote osteoblastic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells.

Estrogenic compounds include endogenous estrogens such as estradiol as well as soybean isoflavones, such as daidzein and its metabolite equol, which are known phytoestrogens that prevent osteoporosis in postmenopausal women. Indeed, mineralization of MC3T3-E1 cells, a murine osteoblastic cell line, was significantly decreased in medium containing fetal bovine serum treated with charcoal-dextran to deplete endogenous estrogens, but estradiol and these soybean isoflavones dose-dependently restored the differentiation of MC3T3-E1 cells; equol was tenfold more effective than daidzein. These differentiation-promoting effects were inhibited by the addition of fulvestrant, which is a selective downregulator of estrogen receptors. Analysis of the expression pattern of bone-related genes by reverse transcription PCR (RT-PCR)/quantitative real-time PCR (qRT-PCR), which focused on responsiveness to the estrogen stimuli, revealed that the transcription of PACE4, a subtilisin-like proprotein convertase, was tightly linked with the differentiation of MC3T3-E1 cells induced by estrogen stimuli. Moreover, treatment with RNAi of PACE4 in MC3T3-E1 cells resulted in a drastic decrease of mineralization in the presence of estrogen stimuli. These results strongly suggest that PACE4 participates in bone formation at least in osteoblast differentiation, and estrogen receptor-mediated stimuli induce osteoblast differentiation through the upregulation of PACE4 expression.

Structural basis of EHEP-mediated offense against phlorotannin-induced defense from brown algae to protect akuBGL activity.

The defensive-offensive associations between algae and herbivores determine marine ecology. Brown algae utilize phlorotannin as their chemical defense against the predator Aplysia kurodai, which uses ß-glucosidase (akuBGL) to digest the laminarin in algae into glucose. Moreover, A. kurodai employs Eisenia hydrolysis-enhancing protein (EHEP) as an offense to protect akuBGL activity from phlorotannin inhibition by precipitating phlorotannin. To underpin the molecular mechanism of this digestive-defensive-offensive system, we determined the structures of the apo and tannic acid (TNA, a phlorotannin analog) bound forms of EHEP, as well as the apo akuBGL. EHEP consisted of three peritrophin-A domains arranged in a triangular shape and bound TNA in the center without significant conformational changes. Structural comparison between EHEP and EHEP-TNA led us to find that EHEP can be resolubilized from phlorotannin precipitation at an alkaline pH, which reflects a requirement in the digestive tract. akuBGL contained two GH1 domains, only one of which conserved the active site. Combining docking analysis, we propose the mechanisms by which phlorotannin inhibits akuBGL by occupying the substrate-binding pocket, and EHEP protects akuBGL against this inhibition by binding with phlorotannin to free the akuBGL pocket.

cGMP-dependent protein kinase I promotes cell apoptosis through hyperactivation of death-associated protein kinase 2.

cGMP-dependent protein kinase-I (cGK-I) induces apoptosis in various cancer cell lines. However, the signaling mechanisms involved remain unknown. Using protein microarray technology, we identified a novel cGK substrate, death-associated protein kinase 2 (DAPK2), which is a Ca(2+)/calmodulin-regulated serine/threonine kinase. cGK-I phosphorylated DAPK2 at Ser(299), Ser(367) and Ser(368). Interestingly, a phospho-mimic mutant, DAPK2 S299D, significantly enhanced its kinase activity in the absence of Ca(2+)/calmodulin, while a S367D/S368D mutant did not. Overexpression of DAPK2 S299D also resulted in a twofold increase in apoptosis of human breast cancer MCF-7 cells as compared with wild-type DAPK2. These results suggest that DAPK2 is one of the targets of cGK-I in apoptosis induction.

cGMP-dependent protein kinase I is involved in neurite outgrowth via a Rho effector, rhotekin, in Neuro2A neuroblastoma cells.

Although the cGMP/cGMP-dependent protein kinase (cGK) signaling is involved in the regulation of neurite outgrowth, its mechanism remains to be clarified. In this study, we identified a Rho effector, rhotekin, as a cGK-I-interacting protein. Rhotekin was also a substrate for cGK-Iα. In neurite-extended Neuro2A neuroblastoma cells, cGK-Iα and rhotekin were colocalized in the plasma membrane and extended neurites, while treatment with cGMP resulted in translocation of rhotekin to the cytoplasm. In addition, we found that cGK-Iα and rhotekin synergistically suppressed Rho-induced neurite retraction. Our findings suggest that cGK-Iα interacts with and phosphorylates rhotekin, thereby contributing to neurite outgrowth regulation.

Structure of ß-1,4-mannanase from the common sea hare Aplysia kurodai at 1.05†Šresolution.

ß-1,4-Mannanase (EC 3.2.1.78) catalyzes the hydrolysis of ß-1,4-glycosidic bonds within mannan, a major constituent group of the hemicelluloses. Bivalves and gastropods possess ß-1,4-mannanase and may degrade mannan in seaweed and/or phytoplankton to obtain carbon and energy using the secreted enzymes in their digestive systems. In the present study, the crystal structure of AkMan, a gastropod ß-1,4-mannanase prepared from the common sea hare Aplysia kurodai, was determined at 1.05 Šresolution. This is the first report of the three-dimensional structure of a gastropod ß-1,4-mannanase. The structure was compared with bivalve ß-1,4-mannanase and the roles of residues in the catalytic cleft were investigated. No obvious binding residue was found in subsite +1 and the substrate-binding site was exposed to the molecular surface, which may account for the enzymatic properties of mannanases that can digest complex substrates such as glucomannan and branched mannan.

Sudachitin, a polymethoxyflavone from Citrus sudachi, suppresses lipopolysaccharide-induced inflammatory responses in mouse macrophage-like RAW264 cells.

Although some polymethoxyflavones possess several important biological properties, including neuroprotective, anticancer, and anti-inflammatory ones, sudachitin, a polymethoxyflavone from Citrus sudachi, has been little studied. In this study, we found that sudachitin inhibited nitric oxide production by suppressing the expression of inducible nitric oxide synthase in lipopolysaccharide-stimulated macrophages, indicating that sudachitin has an anti-inflammatory effect.

Nonspecific phospholipase C3 of radish has phospholipase D activity towards glycosylinositol phosphoceramide.

Glycosylinositol phosphoceramide (GIPC) is a major sphingolipid in the plasma membranes of plants. Previously, we found an enzyme activity that produces phytoceramide 1-phosphate (PC1P) by hydrolysis of the D position of GIPC in cabbage and named this activity as GIPC-phospholipase D (PLD). Here, we purified GIPC-PLD by sequential chromatography from radish roots. Peptide mass fingerprinting analysis revealed that the potential candidate for GIPC-PLD protein was nonspecific phospholipase C3 (NPC3), which has not been characterized as a PLD. The recombinant NPC3 protein obtained by heterologous expression system in Escherichia coli produced PC1P from GIPC and showed essentially the same enzymatic properties as those we characterized as GIPC-PLD in cabbage, radish and Arabidopsis thaliana. From these results, we conclude that NPC3 is one of the enzymes that degrade GIPC.