Toshiya Hayano

Proteomic analysis of immunogenic proteins from salivary glands of Aedes aegypti

Humans develop anti-salivary proteins after arthropod bites or exposure to insect salivary proteins. This reaction indicates that vector bites have a positive effect on the host immune response, which can be used as epidemiological markers of exposure to the vector. Our previous study identified two immunogenic proteins with molecular weights of 31 kDa and 56 kDa from salivary gland extract (SGE) of Aedes aegypti that cross-reacted with serum samples from Dengue Hemorrhagic Fever (DHF) patients and healthy people in an endemic area (Indonesia). Serum samples from individuals living in non-endemic area (sub-tropical country) and infants did not show the immunogenic reactions. The objective of this research was to identify two immunogenic proteins, i.e., 31 and 56 kDa by using proteomic analysis. In this study, proteomic analysis resulted in identification of 13 proteins and 7 proteins from the 31 kDa- and 56 kDa-immunogenic protein bands, respectively. Among those proteins, the D7 protein (Arthropode Odorant-Binding Protein, AOBP) was the most abundant in 31-kDa band, and apyrase was the major protein of the 56-kDa band.

Comparative proteomic analysis reveals differentially expressed proteins in Caenorhabditis elegans pgl-1 mutants grown at 20 °C and 25 °C

PGL-1 is an RNA-binding protein component of germ granules and essential for fertility in Caenorhabditis elegans. To clarify the molecular function of PGL-1, we performed comparative proteomic analysis using 2-D DIGE and LC-MS/MS. Five groups of synchronized adult hermaphrodites were analyzed: (1) wild-type N2 grown at 20°C, (2) pgl-1(bn101) mutants grown at 20°C, (3) pgl-1(bn101) mutants grown at 20°C then upshifted to 25°C after the L1 stage, (4) pgl-1(ct131) mutants grown at 20°C, and (5) pgl-1(ct131) mutants grown at 20°C then upshifted to 25°C after the L1 stage. The five groups were divided into two experimental sets for 2-D DIGE: set A included N2 and pgl-1(bn101) mutants, and set B included N2 and pgl-1(ct131) mutants. Dunnetts test indicated 90 and 100 specific spots, respectively, with significantly different expression levels from the rest of the experimental set (q≤0.1). Among them, 69 and 58 spots, respectively, were analyzed by LC-MS/MS. Finally, we identified 19 proteins from 24 specific spots common to both the experimental sets. RNAi analysis indicated that decreased eef-1G expression is strongly associated with the temperature-sensitive sterile phenotype of pgl-1. Our results suggest that PGL-1 is closely involved in translational processes during C. elegans germline development.

Hyperactive mTOR induces neuroendocrine differentiation in prostate cancer cell with concurrent up-regulation of IRF1

Neuroendocrine‐differentiated prostate cancer (NEPCa) is refractory to androgen deprivation therapy and shows a poor prognosis. The underlying mechanisms responsible for neuroendocrine differentiation (NED) are yet to be clarified. In this study, we investigated the role of mammalian target of rapamycin (mTOR) in NEPCa.

Abstract A04: Hyperactive mTOR induces neuroendocrine differentiation in LNCaP prostate cancer cell with concurrent upregulation of interferon regulatory factor 1

The PI3K-Akt-mTOR pathway is frequently activated in prostate cancer. However, the precise function of mTOR in prostate cancer progression is yet to be elucidated. Thus, in this study, we performed a gain-of-function analysis by establishing human prostate cancer LNCaP stable line that expresses hyperactive mTOR (LNCaP-mTOR). Hyperactive mTOR induced neuroendocrine differentiation (NED) that can be suppressed by an mTOR inhibitor, rapamycin. Subsequent comprehensive mass spectrometric analysis in conjunction with cascade analysis indicates that the members of interferon regulator factor (IRF) family, especially IRF1, are key transcription factors in NED of LNCaP-mTOR. Aside from its major role in immune response, IRF1 is known to be a tumor suppressor. To further investigate the function of IRF1 in NED induced by hyperactive mTOR, we disrupted the IRF1 gene in LNCaP-mTOR by CRISPR/Cas system. The disruption of the IRF1 gene resulted in an acceleration of an NED-associated morphological change, elevation of neuroendocrine cell marker, neuron specific enolase, and a partial recovery of growth arrest. These results suggest that IRF1 partially suppresses NED at downstream of hyperactive mTOR. Together, our results indicate that two seemingly contradictory events, NED and the induction of IRF1, are induced by hyperactive mTOR, giving rise to a notion that pharmacological inhibition of mTOR can be a “double-edged sword” for prostate cancer treatments. Citation Format: Mayuko Kanayama, Toshiya Hayano, Tatsuya Maeda, Shigeo Horie, Atsu Aiba. Hyperactive mTOR induces neuroendocrine differentiation in LNCaP prostate cancer cell with concurrent upregulation of interferon regulatory factor 1. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A04.