Multi-laboratory evaluation of immunoaffinity LC-MS-based glucagon-like peptide-1 assay.

Background: Although the fit-for-purpose approach has been proposed for biomarker assay validation, practical data should be compiled to facilitate the predetermination of acceptance criteria. Methods: Immunoaffinity LC-MS was used to analyze glucagon-like peptide-1 as a model biomarker in six laboratories. Calibration curve, carryover, parallelism, precision, relative accuracy and processed sample stability were evaluated, and their robustness among laboratories was assessed. The rat glucagon-like peptide-1 concentrations in four blinded samples were also compared. Results: The obtained results and determined concentrations in the blinded samples at all laboratories were similar, with a few exceptions, and robust, despite the difference in optimization techniques among laboratories. Conclusion: The results provide insights into the predefinition of the acceptance criteria of immunoaffinity LC-MS-based biomarker assays.

Structural features of methionine aminopeptidase2-active core peptide essential for binding with S100A4.

Methionine aminopeptidase 2 (MetAP2) is one of the effector proteins of S100A4, a metastasis-associated calcium-binding protein. This interaction is involved in angiogenesis. The region of MetAP2 that interacts with S100A4 includes amino acids 170 to 208. A peptide corresponding to this region, named as NBD, has potent anti-angiogenic activity and suppresses tumor growth in a xenograft cancer model. However, the binding mode of NBD to S100A4 was totally unknown. Here we describe our analysis of the relationship between the inhibitory activity and the structure of NBD, which adopts a characteristic helix-turn-helix structure as shown by X-ray crystallographic analysis, and peptide fragments of NBD. We conducted physicochemical analyses of the interaction between S100A4 and the peptides, including surface plasmon resonance, microscale thermophoresis, and circular dichroism, and performed docking/molecular dynamics simulations. Active peptides had stable secondary structures, whereas inactive peptides had a little secondary structure. A computational analysis of the interaction mechanism led to the design of a peptide smaller than NBD, NBD-ΔN10, that possessed inhibitory activity. Our study provides a strategy for design for a specific peptide inhibitor against S100A4 that can be applied to the discovery of inhibitors of other protein-protein interactions.

Physiological function of phospholipase D2 in anti-tumor immunity: regulation of CD8+ T lymphocyte proliferation.

Two major phospholipase D (PLD) isozymes in mammals, PLD1 and PLD2, hydrolyze the membrane phospholipid phosphatidylcholine to choline and the lipid messenger phosphatidic acid. Although their roles in cancer cells have been well studied, their functions in tumor microenvironment have not yet been clarified. Here, we demonstrate that PLD2 in cytotoxic CD8+ T cells plays a crucial role in anti-tumor immunity by regulating their cell proliferation. We found that growth of tumors formed by subcutaneously transplanted cancer cells is enhanced in Pld2-knockout mice. Interestingly, this phenotype was found to be at least in part attributable to the ablation of Pld2 from bone marrow cells. The number of CD8+ T cells, which induce cancer cell death, significantly decreased in the tumor produced in Pld2-knockout mice. In addition, CD3/CD28-stimulated proliferation of primary cultured splenic CD8+ T cells is markedly suppressed by Pld2 ablation. Finally, CD3/CD28-dependent activation of Erk1/2 and Ras is inhibited in Pld2-deleted CD8+ T cells. Collectively, these results indicate that PLD2 in CD8+ T cells plays a key role in their proliferation through activation of the Ras/Erk signaling pathway, thereby regulating anti-tumor immunity.

Arf6 in lymphatic endothelial cells regulates lymphangiogenesis by controlling directional cell migration.

The small GTPase Arf6 plays pivotal roles in a wide variety of cellular events such as endocytosis, exocytosis, and actin cytoskeleton reorganization. However, the physiological functions of Arf6 at the whole animal level have not yet been thoroughly understood. Here, we show that Arf6 regulates developmental and tumor lymphangiogenesis in mice. Lymphatic endothelial cell (LEC)-specific Arf6 conditional knockout (LEC-Arf6 cKO) mouse embryos exhibit severe skin edema and impairment in the formation of lymphatic vessel network at the mid-gestation stage. Knockdown of Arf6 in human LECs inhibits in vitro capillary tube formation and directed cell migration induced by vascular endothelial growth factor-C (VEGF-C) by inhibiting VEGF-C-induced internalization of ß1 integrin. Finally, we found that LEC-Arf6 cKO mice transplanted with B16 melanoma cells attenuated tumor lymphangiogenesis and progression. Collectively, these results demonstrate that Arf6 in LECs plays a crucial role in physiological and pathological lymphangiogenesis.

Regulation of HGF-induced hepatocyte proliferation by the small GTPase Arf6 through the PIP2-producing enzyme PIP5K1A.

HGF and its receptor c-Met are critical molecules in various biological processes. Others and we have previously shown that the small GTPase Arf6 plays a pivotal role in HGF signaling in hepatocytes. However, the molecular mechanism of how Arf6 regulates HGF signaling is unclear. Here, we show that Arf6 plays an important role in HGF-stimulated hepatocyte proliferation and liver regeneration through the phosphatidylinositol 4,5-bisphosphate (PIP2)-producing enzyme PIP5K1A. We find that knockdown of Arf6 and PIP5K1A in HepG2 cells inhibits HGF-stimulated proliferation, Akt activation, and generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and its precursor PIP2. Interestingly, PIP5K1A is recruited to c-Met upon HGF stimulation in an Arf6 activity-dependent manner. Finally, we show that hepatocyte proliferation and liver regeneration after partial hepatectomy are suppressed in Pip5k1a knockout mice. These results provide insight into the molecular mechanism for HGF-stimulated hepatocyte proliferation and liver regeneration: Arf6 recruits PIP5K1A to c-Met and activates it upon HGF stimulation to produce PIP2 and subsequently PIP3, which in turn activates Akt to promote hepatocyte proliferation, thereby accelerating liver regeneration after liver injury.

The small G protein Arf6 expressed in keratinocytes by HGF stimulation is a regulator for skin wound healing.

The earlier step of cutaneous wound healing process, re-epithelialization of the wounded skin, is triggered by a variety of growth factors. However, molecular mechanisms through which growth factors trigger skin wound healing are less understood. Here, we demonstrate that hepatocyte growth factor (HGF)/c-Met signaling-induced expression of the small G protein Arf6 mRNA in keratinocytes is essential for the skin wound healing. Arf6 mRNA expression was dramatically induced in keratinocytes at the wounded skin, which was specifically suppressed by the c-Met inhibitor. Wound healing of the skin was significantly delayed in keratinocyte-specific Arf6 conditional knockout mice. Furthermore, Arf6 deletion from keratinocytes remarkably suppressed HGF-stimulated cell migration and peripheral membrane ruffle formation, but did not affect skin morphology and proliferation/differentiation of keratinocytes. These results are consistent with the notion that Arf6 expressed in skin keratinocytes through the HGF/c-Met signaling pathway in response to skin wounding plays an important role in skin wound healing by regulating membrane dynamics-based motogenic cellular function of keratinocytes.

ACAP3 regulates neurite outgrowth through its GAP activity specific to Arf6 in mouse hippocampal neurons.

ACAP3 (ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 3) belongs to the ACAP family of GAPs (GTPase-activating proteins) for the small GTPase Arf (ADP-ribosylation factor). However, its specificity to Arf isoforms and physiological functions remain unclear. In the present study, we demonstrate that ACAP3 plays an important role in neurite outgrowth of mouse hippocampal neurons through its GAP activity specific to Arf6. In primary cultured mouse hippocampal neurons, knockdown of ACAP3 abrogated neurite outgrowth, which was rescued by ectopically expressed wild-type ACAP3, but not by its GAP activity-deficient mutant. Ectopically expressed ACAP3 in HEK (human embryonic kidney)-293T cells showed the GAP activity specific to Arf6. In support of this observation, the level of GTP-bound Arf6 was significantly increased by knockdown of ACAP3 in hippocampal neurons. In addition, knockdown and knockout of Arf6 in mouse hippocampal neurons suppressed neurite outgrowth. These results demonstrate that ACAP3 positively regulates neurite outgrowth through its GAP activity specific to Arf6. Furthermore, neurite outgrowth suppressed by ACAP3 knockdown was rescued by expression of a fast cycle mutant of Arf6 that spontaneously exchanges guanine nucleotides on Arf6, but not by that of wild-type, GTP- or GDP-locked mutant Arf6. Thus cycling between active and inactive forms of Arf6, which is precisely regulated by ACAP3 in concert with a guanine-nucleotide-exchange factor(s), seems to be required for neurite outgrowth of hippocampal neurons.

Pathological functions of the small GTPase Arf6 in cancer progression: Tumor angiogenesis and metastasis.

Although several lines of evidence have shown that the small GTPase ADP-ribosylation factor 6 (Arf6) plays pivotal roles in cancer progression of several types of cancers, little is known about the functions of Arf6 in tumor microenvironment. We demonstrated that Arf6 in vascular endothelial cells (VECs) plays a crucial role in tumor angiogenesis and growth using endothelial cell-specific Arf6 conditional knockout mice into which B16 melanoma and Lewis lung carcinoma cells were implanted. It was also found that Arf6 in VECs positively regulates hepatocyte growth factor (HGF)-induced ß1 integrin recycling, which is a critical event for tumor angiogenesis by promoting cell migration. Importantly, pharmacological inhibition of HGF-induced Arf6 activation significantly suppresses tumor angiogenesis and growth in mice, suggesting that Arf6 signaling would be a potential target for anti-angiogenic therapy. In this manuscript, we summarize the multiple roles of Arf6 in cancer progression, particularly in cancer cell invasion/metastasis and our recent findings on tumor angiogenesis, and discuss a possible approach to develop innovative anti-cancer drugs.

In Vivo Linking of Membrane Lipids and the Anion Transporter Band 3 with Thiourea-modified Amphiphilic Lipid Probes.

Membrane proteins interact with membrane lipids for their structural stability and proper function. However, lipid-protein interactions are poorly understood at a molecular level especially in the live cell membrane, due to current limitations in methodology. Here, we report that amphiphilic lipid probes can be used to link membrane lipids and membrane proteins in vivo. Cholesterol and a phospholipid were both conjugated to a fluorescent tag through a linker containing thiourea. In the erythrocyte, the cholesterol probe fluorescently tagged the anion transporter band 3 via thiourea. Tagging by the cholesterol probe, but not by the phospholipid probe, was competitive with an anion transporter inhibitor, implying the presence of a specific binding pocket for cholesterol in this ~100 kDa protein. This method could prove an effective strategy for analyzing lipid-protein interactions in vivo in the live cell membrane.

Gradual reduction in rRNA transcription triggers p53 acetylation and apoptosis via MYBBP1A.

The nucleolus, whose primary function is ribosome biogenesis, plays an essential role in p53 activation. Ribosome biogenesis is inhibited in response to cellular stress and several nucleolar proteins translocate from the nucleolus to the nucleoplasm, where they activate p53. In this study, we analysed precisely how impaired ribosome biogenesis regulates the activation of p53 by depleting nucleolar factors involved in rRNA transcription or rRNA processing. Nucleolar RNA content decreased when rRNA transcription was inhibited. In parallel with the reduced levels of nucleolar RNA content, the nucleolar protein Myb-binding protein 1 A (MYBBP1A) translocated to the nucleoplasm and increased p53 acetylation. The acetylated p53 enhanced p21 and BAX expression and induced apoptosis. In contrast, when rRNA processing was inhibited, MYBBP1A remained in the nucleolus and nonacetylated p53 accumulated, causing cell cycle arrest at the G1 phase by inducing p21 but not BAX. We propose that the nucleolus functions as a stress sensor to modulate p53 protein levels and its acetylation status, determining cell fate between cell cycle arrest and apoptosis by regulating MYBBP1A translocation.

Perturbation of ribosome biogenesis drives cells into senescence through 5S RNP-mediated p53 activation.

The 5S ribonucleoprotein particle (RNP) complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses.

The nucleolar protein nucleophosmin is essential for autophagy induced by inhibiting Pol I transcription.

Various cellular stresses activate autophagy, which is involved in lysosomal degradation of cytoplasmic materials for maintaining nutrient homeostasis and eliminating harmful components. Here, we show that RNA polymerase I (Pol I) transcription inhibition induces nucleolar disruption and autophagy. Treatment with autophagy inhibitors or siRNA specific for autophagy-related (ATG) proteins inhibited autophagy but not nucleolar disruption induced by Pol I transcription inhibition, which suggested that nucleolar disruption was upstream of autophagy. Furthermore, treatment with siRNA specific for nucleolar protein nucleophosmin (NPM) inhibited this type of autophagy. This showed that NPM was involved in autophagy when the nucleolus was disrupted by Pol I inhibition. In contrast, NPM was not required for canonical autophagy induced by nutrient starvation, as it was not accompanied by nucleolar disruption. Thus, our results revealed that, in addition to canonical autophagy, there may be NPM-dependent autophagy associated with nucleolar disruption.

Novel nucleolar pathway connecting intracellular energy status with p53 activation.

In response to a shortage of intracellular energy, mammalian cells reduce energy consumption and induce cell cycle arrest, both of which contribute to cell survival. Here we report that a novel nucleolar pathway involving the energy-dependent nucleolar silencing complex (eNoSC) and Myb-binding protein 1a (MYBBP1A) is implicated in these processes. Namely, in response to glucose starvation, eNoSC suppresses rRNA transcription, which results in a reduction in nucleolar RNA content. As a consequence, MYBBP1A, which is anchored to the nucleolus via RNA, translocates from the nucleolus to the nucleoplasm. The translocated MYBBP1A induces acetylation and accumulation of p53 by enhancing the interaction between p300 and p53, which eventually leads to the cell cycle arrest (or apoptosis). Taken together, our results indicate that the nucleolus works as a sensor that transduces the intracellular energy status into the cell cycle machinery.

Critical role of the nucleolus in activation of the p53-dependent postmitotic checkpoint.

Cells eventually exit from mitosis during sustained arrest at the spindle checkpoint, without sister chromatid separation and cytokinesis. The resulting tetraploid cells are arrested in the subsequent G1 phase in a p53-dependent manner by the regulatory function of the postmitotic G1 checkpoint. Here we report how the nucleolus plays a critical role in activation of the postmitotic G1 checkpoint. During mitosis, the nucleolus is disrupted and many nucleolar proteins are translocated from the nucleolus into the cytoplasm. Among the nucleolar factors, Myb-binding protein 1a (MYBBP1A) induces the acetylation and accumulation of p53 by enhancing the interaction between p300 and p53 during prolonged mitosis. MYBBP1A-dependent p53 activation is essential for the postmitotic G1 checkpoint. Thus, our results demonstrate a novel nucleolar function that monitors the prolongation of mitosis and converts its signal into activation of the checkpoint machinery.

RNA content in the nucleolus alters p53 acetylation via MYBBP1A.

A number of external and internal insults disrupt nucleolar structure, and the resulting nucleolar stress stabilizes and activates p53. We show here that nucleolar disruption induces acetylation and accumulation of p53 without phosphorylation. We identified three nucleolar proteins, MYBBP1A, RPL5, and RPL11, involved in p53 acetylation and accumulation. MYBBP1A was tethered to the nucleolus through nucleolar RNA. When rRNA transcription was suppressed by nucleolar stress, MYBBP1A translocated to the nucleoplasm and facilitated p53-p300 interaction to enhance p53 acetylation. We also found that RPL5 and RPL11 were required for rRNA export from the nucleolus. Depletion of RPL5 or RPL11 blocked rRNA export and counteracted reduction of nucleolar RNA levels caused by inhibition of rRNA transcription. As a result, RPL5 or RPL11 depletion inhibited MYBBP1A translocation and p53 activation. Our observations indicated that a dynamic equilibrium between RNA generation and export regulated nucleolar RNA content. Perturbation of this balance by nucleolar stress altered the nucleolar RNA content and modulated p53 activity.