Yukiko Sasazawa

Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition

Caffeine is one of the most frequently ingested neuroactive compounds. All known mechanisms of apoptosis induced by caffeine act through cell cycle modulation or p53 induction. It is currently unknown whether caffeine-induced apoptosis is associated with other cell death mechanisms, such as autophagy. Herein we show that caffeine increases both the levels of microtubule-associated protein 1 light chain 3-II and the number of autophagosomes, through the use of western blotting, electron microscopy and immunocytochemistry techniques. Phosphorylated p70 ribosomal protein S6 kinase (Thr389), S6 ribosomal protein (Ser235/236), 4E-BP1 (Thr37/46) and Akt (Ser473) were significantly decreased by caffeine. In contrast, ERK1/2 (Thr202/204) was increased by caffeine, suggesting an inhibition of the Akt/mTOR/p70S6K pathway and activation of the ERK1/2 pathway. Although insulin treatment phosphorylated Akt (Ser473) and led to autophagy suppression, the effect of insulin treatment was completely abolished by caffeine addition. Caffeine-induced autophagy was not completely blocked by inhibition of ERK1/2 by U0126. Caffeine induced reduction of mitochondrial membrane potentials and apoptosis in a dose-dependent manner, which was further attenuated by the inhibition of autophagy with 3-methyladenine or Atg7 siRNA knockdown. Furthermore, there was a reduced number of early apoptotic cells (annexin V positive, propidium iodide negative) among autophagy-deficient mouse embryonic fibroblasts treated with caffeine than their wild-type counterparts. These results support previous studies on the use of caffeine in the treatment of human tumors and indicate a potential new target in the regulation of apoptosis.

Xanthohumol Impairs Autophagosome Maturation through Direct Inhibition of Valosin-Containing Protein

Autophagy is a bulk, nonspecific protein degradation pathway that is involved in the pathogenesis of cancer and neurodegenerative disease. Here, we observed that xanthohumol (XN), a prenylated chalcone present in hops (Humulus lupulus L.) and beer, modulates autophagy. By using XN-immobilized beads, valosin-containing protein (VCP) was identified as a XN-binding protein. VCP has been reported to be an essential protein for autophagosome maturation. Using an in vitro pull down assay, we showed that XN bound directly to the N domain, which is known to mediate cofactor and substrate binding to VCP. These data indicated that XN inhibited the function of VCP, thereby allowing the impairment of autophagosome maturation and resulting in the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II). This is the first report demonstrating XN as a VCP inhibitor that binds directly to the N domain of VCP. Our finding that XN bound to and inactivated VCP not only reveals the molecular mechanism of XN-modulated autophagy but may also explain how XN exhibits various biological activities that have been reported previously.

Conophylline Protects Cells in Cellular Models of Neurodegenerative Diseases by Inducing Mammalian Target of Rapamycin (mTOR)-independent Autophagy

Background: Autophagy is essential for prevention of neurodegenerative diseases. Results: Conophylline induces mTOR-independent autophagy and protects against neurotoxicity. Conclusion: Conophylline protects cells by enhancement of autophagy in models of neurodegenerative diseases. Significance: Conophylline would be a therapeutic agent for neurodegenerative diseases. Macroautophagy is a cellular response that leads to the bulk, nonspecific degradation of cytosolic components, including organelles. In recent years, it has been recognized that autophagy is essential for prevention of neurodegenerative diseases, including Parkinson disease (PD) and Huntington disease (HD). Here, we show that conophylline (CNP), a vinca alkaloid, induces autophagy in an mammalian target of rapamycin-independent manner. Using a cellular model of PD, CNP suppressed protein aggregation and protected cells from cell death caused by treatment with 1-methyl-4-phenylpyridinium, a neurotoxin, by inducing autophagy. Moreover, in the HD model, CNP also eliminated mutant huntingtin aggregates. Our findings demonstrate the possible use of CNP as a therapeutic drug for neurodegenerative disorders, including PD and HD.

C-mannosylation of thrombopoietin receptor (c-Mpl) regulates thrombopoietin-dependent JAK-STAT signaling

The thrombopoietin receptor, also known as c-Mpl, is a member of the cytokine superfamily, which regulates the differentiation of megakaryocytes and formation of platelets by binding to its ligand, thrombopoietin (TPO), through Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. The loss-of-function mutations of c-Mpl cause severe thrombocytopenia due to impaired megakaryocytopoiesis, and gain-of-function mutations cause thrombocythemia. c-Mpl contains two Trp-Ser-Xaa-Trp-Ser (Xaa represents any amino acids) sequences, which are characteristic sequences of type I cytokine receptors, corresponding to C-mannosylation consensus sequences: Trp-Xaa-Xaa-Trp/Cys. C-mannosylation is a post-translational modification of tryptophan residue in which one mannose is attached to the first tryptophan residue in the consensus sequence via C-C linkage. Although c-Mpl contains some C-mannosylation sequences, whether c-Mpl is C-mannosylated or not has been uninvestigated. We identified that c-Mpl is C-mannosylated not only at Trp(269) and Trp(474), which are putative C-mannosylation site, but also at Trp(272), Trp(416), and Trp(477). Using C-mannosylation defective mutant of c-Mpl, the C-mannosylated tryptophan residues at four sites (Trp(269), Trp(272), Trp(474), and Trp(477)) are essential for c-Mpl-mediated JAK-STAT signaling. Our findings suggested that C-mannosylation of c-Mpl is a possible therapeutic target for platelet disorders.

N‐Glycosylation of extracellular matrix protein 1 (ECM1) regulates its secretion, which is unrelated to lipoid proteinosis

Extracellular matrix protein 1 (ECM1) is expressed in a wide variety of tissues and plays important roles in extracellular matrix formation. Additionally, ECM1 gene mutations cause lipoid proteinosis (LP), a rare skin condition of genetic origin. However, an effective therapeutic approach of LP is not established. Here, we showed that ECM1 gene mutation observed in LP patients significantly suppresses its secretion. As ECM1 has three putative N‐glycosylation sites and most of mutated ECM1 observed in LP patients are defective in these N‐glycosylation sites, we investigated the correlation between LP and N‐glycosylation of ECM1. We identified that the Asn354 and Asn444 residues in ECM1 were N‐glycosylated by mass spectrometry analysis. In addition, an N‐linked glycan at Asn354 negatively regulated secretion of ECM1, contrary to LP patient‐derived mutants. These results indicate that the defect of N‐glycosylation in ECM1 is not involved in the aberration of secretion of LP‐derived mutated ECM1.

Pirin regulates epithelial to mesenchymal transition independently of Bcl3-Slug signaling

Epithelial to mesenchymal transition (EMT) is an important mechanism for the initial step of metastasis. Proteomic analysis indicates that Pirin is involved in metastasis. However, there are no reports demonstrating its direct contribution. Here we investigated the involvement of Pirin in EMT. In HeLa cells, Pirin suppressed E‐cadherin expression and regulated the expression of other EMT markers. Furthermore, cells expressing Pirin exhibited a spindle‐like morphology, which is reminiscent of EMT. A Pirin mutant defective for Bcl3 binding decreased E‐cadherin expression similar to wild‐type, suggesting that Pirin regulates E‐cadherin independently of Bcl3‐Slug signaling. These data provide direct evidence that Pirin contributes to cancer metastasis.

Determination of topological structure of ARL6ip1 in cells: Identification of the essential binding region of ARL6ip1 for conophylline

Conophylline (CNP) has various biological activities, such as insulin production. A recent study identified ADP‐ribosylation factor‐like 6‐interacting protein 1 (ARL6ip1) as a direct target protein of CNP. In this study, we revealed that ARL6ip1 is a three‐spanning transmembrane protein and determined the CNP‐binding domain of ARL6ip1 by deletion mutation analysis of ARL6ip1 with biotinyl‐amino‐CNP. These results suggest that CNP is expected to be useful for future investigation of ARL6ip1 function in cells. Because of the anti‐apoptotic function of ARL6ip1, CNP may be an effective therapeutic drug and/or a novel chemosensitizer for human cancers and other diseases.

C-mannosylation of R-spondin3 regulates its secretion and activity of Wnt/β-catenin signaling in cells

R‐spondin3 (Rspo3) is a secreted protein, which acts as an agonist of canonical Wnt/β‐catenin signaling that plays an important role in embryonic development and homeostasis. In this study, we focused on C‐mannosylation, a unique type of glycosylation, of human Rspo3. Rspo3 has two putative C‐mannosylation sites at Trp153 and Trp156; however, it had been unclear whether these sites are C‐mannosylated or not. We demonstrated that Rspo3 was C‐mannosylated at both Trp153 and Trp156 by mass spectrometry. Using C‐mannosylation‐defective Rspo3 mutant‐overexpressing cell lines, we found that C‐mannosylation of Rspo3 promotes its secretion and activates Wnt/β‐catenin signaling.

Regulation of secretion and enzymatic activity of lipoprotein lipase by C-mannosylation

Lipoprotein lipase (LPL) is a crucial enzyme in lipid metabolism and transport, and its enzymatic deficiency causes metabolic disorders, such as hypertriglyceridemia. LPL has one predicted C-mannosylation site at Trp417. In this study, we demonstrated that LPL is C-mannosylated at Trp417 by mass spectrometry. Furthermore, by using wild-type and a C-mannosylation-defective mutant of LPL-overexpressing cell lines, we revealed that both secretion efficiency and enzymatic activity of C-mannosylation-defective mutant LPL were lower than those of wild-type. These data suggest the importance of C-mannosylation for LPL functions.

Involvement of conserved tryptophan residues for secretion of TIMP‑2

Tissue inhibitor of metalloproteinases (TIMPs) are endogenous inhibitor proteins of matrix metalloproteinases and contain 12 cysteine residues that are conserved among TIMPs, and which are important for their activity and structure. In the present study, three tryptophan residues conserved among TIMPs were revealed to be important for the secretion of TIMP-2. Replacement of conserved tryptophan residues in TIMP-2 with alanine led to a decrease in extracellular TIMP-2 levels and an increase in intracellular TIMP-2 levels. Furthermore, wild-type TIMP-2 and TIMP-2 mutated at unconserved tryptophan residues mainly localized in the Golgi apparatus, while TIMP-2 proteins mutated at conserved tryptophan were mainly observed in the endoplasmic reticulum (ER). This indicated that conserved tryptophan is essential for transporting TIMP-2 from the ER to Golgi apparatus. These observations suggested that conserved tryptophan residues among the TIMP family of proteins have critical roles for ER-Golgi transport and subsequent secretion of TIMP-2.