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Article ; Online: Hyperosmotic Stress Induces Phosphorylation of CERT and Enhances Its Tethering throughout the Endoplasmic Reticulum

Kentaro Shimasaki / Keigo Kumagai / Shota Sakai / Toshiyuki Yamaji / Kentaro Hanada

International Journal of Molecular Sciences, Vol 23, Iss 4025, p

2022 Volume 4025

Abstract: The ceramide transport protein (CERT) delivers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, where ceramide is converted to sphingomyelin (SM). The function of CERT is regulated in two distinct phosphorylation-dependent events: ... ...

Abstract	The ceramide transport protein (CERT) delivers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, where ceramide is converted to sphingomyelin (SM). The function of CERT is regulated in two distinct phosphorylation-dependent events: multiple phosphorylations in a serine-repeat motif (SRM) and phosphorylation of serine 315 residue (S315). Pharmacological inhibition of SM biosynthesis results in an increase in SRM-dephosphorylated CERT, which serves as an activated form, and an enhanced phosphorylation of S315, which augments the binding of CERT to ER-resident VAMP-associated protein (VAP), inducing the full activation of CERT to operate at the ER–Golgi membrane contact sites (MCSs). However, it remains unclear whether the two phosphorylation-dependent regulatory events always occur coordinately. Here, we describe that hyperosmotic stress induces S315 phosphorylation without affecting the SRM-phosphorylation state. Under hyperosmotic conditions, the binding of CERT with VAP-A is enhanced in an S315 phosphorylation-dependent manner, and this increased binding occurs throughout the ER rather than restrictedly at the ER–Golgi MCSs. Moreover, we found that de novo synthesis of SM with very-long acyl chains preferentially increases via a CERT-independent mechanism under hyperosmotic-stressed cells, providing an insight into a CERT-independent ceramide transport pathway for de novo synthesis of SM.
Keywords	lipid transfer protein ; regulation ; sphingomyelin ; VAP ; membrane contact sites ; very-long-chain ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	500
Language	English
Publishing date	2022-04-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Compartmentalization of casein kinase 1 γ CSNK1G controls the intracellular trafficking of ceramide

Asako Goto / Shota Sakai / Aya Mizuike / Toshiyuki Yamaji / Kentaro Hanada

iScience, Vol 25, Iss 7, Pp 104624- (2022)

2022

Abstract: Summary: Casein kinase 1 γ (CK1G) is involved in the regulation of various cellular functions. For instance, the ceramide transport protein (CERT), which delivers ceramide to the Golgi apparatus for the synthesis of sphingomyelin (SM), is inactivated ... ...

Abstract	Summary: Casein kinase 1 γ (CK1G) is involved in the regulation of various cellular functions. For instance, the ceramide transport protein (CERT), which delivers ceramide to the Golgi apparatus for the synthesis of sphingomyelin (SM), is inactivated when it receives multiple phosphorylation by CK1G. Using human genome-wide gene disruption screening with an SM-binding cytolysin, we found that loss of the C-terminal region of CK1G3 rendered the kinase hyperactive in cells. Deletion of the C-terminal 20 amino acids or mutation of cysteine residues expected to be palmitoylated sites redistributed CK1G3 from cytoplasmic punctate compartments to the nucleocytoplasm. Wild-type CK1G3 exhibited a similar redistribution in the presence of 2-bromopalmitate, a protein palmitoylation inhibitor. Expression of C-terminal mutated CK1G1/2/3 similarly induced the multiple phosphorylation of the CERT SRM, thereby down-regulating de novo SM synthesis. These findings revealed that CK1Gs are regulated by a compartmentalization-based mechanism to access substrates present in specific intracellular organelles.
Keywords	Biological sciences ; Cell biology ; Functional aspects of cell biology ; Science ; Q
Language	English
Publishing date	2022-07-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Direct Inhibition of SARS-CoV-2 Spike Protein by Peracetic Acid

Yuichiro Yamamoto / Yoshio Nakano / Mana Murae / Yoshimi Shimizu / Shota Sakai / Motohiko Ogawa / Tomoharu Mizukami / Tetsuya Inoue / Taishi Onodera / Yoshimasa Takahashi / Takaji Wakita / Masayoshi Fukasawa / Satoru Miyazaki / Kohji Noguchi

International Journal of Molecular Sciences, Vol 24, Iss 1, p

2022 Volume 20

Abstract: Peracetic acid (PAA) disinfectants are effective against a wide range of pathogenic microorganisms, including bacteria, fungi, and viruses. Several studies have shown the efficacy of PAA against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ...

Abstract	Peracetic acid (PAA) disinfectants are effective against a wide range of pathogenic microorganisms, including bacteria, fungi, and viruses. Several studies have shown the efficacy of PAA against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, its efficacy in SARS-CoV-2 variants and the molecular mechanism of action of PAA against SARS-CoV-2 have not been investigated. SARS-CoV-2 infection depends on the recognition and binding of the cell receptor angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) of the spike protein. Here, we demonstrated that PAA effectively suppressed pseudotyped virus infection in the Wuhan type and variants, including Delta and Omicron. Similarly, PAA reduced the authentic viral load of SARS-CoV-2. Computational analysis suggested that the hydroxyl radicals produced by PAA cleave the disulfide bridges in the RBD. Additionally, the PAA treatment decreased the abundance of the Wuhan- and variant-type spike proteins. Enzyme-linked immunosorbent assay showed direct inhibition of RBD-ACE2 interactions by PAA. In conclusion, the PAA treatment suppressed SARS-CoV-2 infection, which was dependent on the inhibition of the interaction between the spike RBD and ACE2 by inducing spike protein destabilization. Our findings provide evidence of a potent disinfection strategy against SARS-CoV-2.
Keywords	SARS-CoV-2 ; peracetic acid ; spike protein ; receptor-binding domain ; ACE2 ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	612
Language	English
Publishing date	2022-12-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Natural ligand-nonmimetic inhibitors of the lipid-transfer protein CERT

Naoki Nakao / Masaharu Ueno / Shota Sakai / Daichi Egawa / Hiroyuki Hanzawa / Shohei Kawasaki / Keigo Kumagai / Makoto Suzuki / Shu Kobayashi / Kentaro Hanada

Communications Chemistry, Vol 2, Iss 1, Pp 1-

2019 Volume 11

Abstract: The ceramide transport protein CERT is of potential therapeutic interest but is typically targeted using ceramide-derived ligands. Here the authors use virtual screening and a quantitative surface plasmon resonance assay to identify ceramide-nonmimetic ... ...

Abstract	The ceramide transport protein CERT is of potential therapeutic interest but is typically targeted using ceramide-derived ligands. Here the authors use virtual screening and a quantitative surface plasmon resonance assay to identify ceramide-nonmimetic inhibitors of a CERT subdomain and test their activity in live cells.
Keywords	Chemistry ; QD1-999
Language	English
Publishing date	2019-02-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Chemoenzymatically prepared konjac ceramide inhibits NGF-induced neurite outgrowth by a semaphorin 3A-like action

Seigo Usuki / Noriko Tamura / Shota Sakai / Tomohiro Tamura / Katsuyuki Mukai / Yasuyuki Igarashi

Biochemistry and Biophysics Reports, Vol 5, Iss C, Pp 160-

2016 Volume 167

Abstract: Dietary sphingolipids such as glucosylceramide (GlcCer) are potential nutritional factors associated with prevention of metabolic syndrome. Our current understanding is that dietary GlcCer is degraded to ceramide and further metabolized to sphingoid ... ...

Abstract	Dietary sphingolipids such as glucosylceramide (GlcCer) are potential nutritional factors associated with prevention of metabolic syndrome. Our current understanding is that dietary GlcCer is degraded to ceramide and further metabolized to sphingoid bases in the intestine. However, ceramide is only found in trace amounts in food plants and thus is frequently taken as GlcCer in a health supplement. In the present study, we successfully prepared konjac ceramide (kCer) using endoglycoceramidase I (EGCase I). Konjac, a plant tuber, is an enriched source of GlcCer (kGlcCer), and has been commercialized as a dietary supplement to improve dry skin and itching that are caused by a deficiency of epidermal ceramide. Nerve growth factor (NGF) produced by skin cells is one of the itch factors in the stratum corneum of the skin. Semaphorin 3A (Sema 3A) has been known to inhibit NGF-induced neurite outgrowth of epidermal nerve fibers. It is well known that the itch sensation is regulated by the balance between NGF and Sema 3A. In the present study, while kGlcCer did not show an in vitro inhibitory effect on NGF-induced neurite outgrowth of PC12 cells, kCer was demonstrated to inhibit a remarkable neurite outgrowth. In addition, the effect of kCer was similar to that of Sema 3A in cell morphological changes and neurite retractions, but different from C2-Ceramide. kCer showed a Sema 3A-like action, causing CRMP2 phosphorylation, which results in a collapse of neurite growth cones. Thus, it is expected that kCer is an advanced konjac ceramide material that may have neurite outgrowth-specific action to relieve uncontrolled and serious itching, in particular, from atopic eczema.
Keywords	Ceramide ; Konjac ; NGF ; Semaphorin 3A ; Neurite outgrowth ; CRMP2 ; Biology (General) ; QH301-705.5 ; Biochemistry ; QD415-436
Subject code	571
Language	English
Publishing date	2016-03-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Inositol depletion restores vesicle transport in yeast phospholipid flippase mutants.

Kanako Yamagami / Takaharu Yamamoto / Shota Sakai / Tetsuo Mioka / Takamitsu Sano / Yasuyuki Igarashi / Kazuma Tanaka

PLoS ONE, Vol 10, Iss 3, p e

2015 Volume 0120108

Abstract: In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the ...

Abstract	In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases.
Keywords	Medicine ; R ; Science ; Q
Subject code	571
Language	English
Publishing date	2015-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: The fungal 9‐methyl‐sphingadiene is a novel ligand for both PPARγ and GPR120

Esaki, Shota / Tomotaka Nagasawa / Haruka Tanaka / Aoi Tominaga / Daisuke Mikami2 / Seigo Usuki2 / Hiroshi Hamajima / Hisatoshi Hanamatsu / Shota Sakai / Yoichiro Hama / Yasuyuki Igarashi / Hiroshi Kitagaki / Susumu Mitsutake

Journal of food biochemistry. 2018 Oct., v. 42, no. 5

2018

Abstract: 9‐Methyl‐sphingadiene has a characteristic structure containing a methyl group and is only found in fungi including mushrooms and mycota such as Aspergillus oryzae. The Japanese daily ingest it through traditional fermented foods, such like miso, soy ... ...

Abstract	9‐Methyl‐sphingadiene has a characteristic structure containing a methyl group and is only found in fungi including mushrooms and mycota such as Aspergillus oryzae. The Japanese daily ingest it through traditional fermented foods, such like miso, soy sauce, and sake. However, the food function of 9‐methyl‐sphingadiene is hardly elucidated. In this study, we first revealed that 9‐methyl‐sphingadiene is a novel ligand for peroxisome proliferator‐activated receptor γ (PPARγ) and GPR120, and induces differentiation in 3T3‐L1 cells. Synthetic ligands of PPARγ are widely used to treat type 2 diabetes, and are known to accelerate adipogenesis. GPR120 plays an important role in the browning of adipocytes and their differentiation into beige cells. An increase in beige cells and brown adipocytes is known to suppress obesity. Thus, ligands for PPARγ and/or GPR120 are thought to have important function in controlling diabetes and obesity. Our result indicates that fungal sphingoid bases in fermented foods have anti‐diabetic role. Practical applications One major characteristic of Japanese meal is to use various food fermented with the non‐pathogenic fungus Aspergillus oryzae, Koji. The Koji contains unique sphingoid base, 9‐methyl‐sphingadiene. In this study, we investigated a food function of the 9‐methyl‐sphingadiene, and found that 9‐methyl‐sphingadiene is a novel ligand for PPARγ and GPR120. Since the ligands of PPARγ and GPR120 are involved in anti‐obesity and anti‐diabetes, our result indicates that daily intake of Koji might prevent the development of obesity and diabetes. This study do not only reveals one of the health benefit of Japanese food containing Koji, but also the possibility in the application of 9‐methyl‐sphingadiene as functional food.
Keywords	Aspergillus oryzae ; adipogenesis ; brown adipocytes ; functional foods ; koji ; ligands ; miso ; moieties ; mushrooms ; noninsulin-dependent diabetes mellitus ; obesity ; peroxisome proliferator-activated receptor gamma ; sake ; soy sauce
Language	English
Dates of publication	2018-10
Size	p. e12624.
Publishing place	John Wiley & Sons, Ltd
Document type	Article
Note	JOURNAL ARTICLE
ZDB-ID	433846-7
ISSN	1745-4514 ; 0145-8884
ISSN (online)	1745-4514
ISSN	0145-8884
DOI	10.1111/jfbc.12624
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice.

Masayuki Sugimoto / Masato Wakabayashi / Yoichi Shimizu / Takeshi Yoshioka / Kenichi Higashino / Yoshito Numata / Tomohiko Okuda / Songji Zhao / Shota Sakai / Yasuyuki Igarashi / Yuji Kuge

PLoS ONE, Vol 11, Iss 3, p e

2016 Volume 0152191

Abstract: Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to ... ...

Abstract	Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18-C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18-C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys.
Keywords	Medicine ; R ; Science ; Q
Subject code	616
Language	English
Publishing date	2016-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Lard-based high-fat diet increases secretory leukocyte protease inhibitor expression and attenuates the inflammatory response of acute lung injury in endotoxemic rats

Fujiwara, Mayu / Aya Nishiokada / Makoto Miyoshi / Makoto Usami / Michiko Aoyama-Ishikawa / Michiko Takahashi / Noriaki Maeshige / Shota Sakai / Yasuhiro Hamada / Yu Usami

Clinical nutrition. 2015 Oct., v. 34, no. 5

2015

Abstract: Acute lung injury (ALI) is less severe in obese than in nonobese patients, but the mechanism is unclear. Secretory leukocyte protease inhibitor (SLPI) is the key anti-inflammatory protein in various lung diseases. We have previously reported changes of ... ...

Abstract	Acute lung injury (ALI) is less severe in obese than in nonobese patients, but the mechanism is unclear. Secretory leukocyte protease inhibitor (SLPI) is the key anti-inflammatory protein in various lung diseases. We have previously reported changes of the surgical stress in obese rats using lard-based high-fat diet (HFD). The purpose of this study was to elucidate the effect of lard-based HFD on the pathophysiology of lipopolysaccharide (LPS)-induced ALI, and the role of SLPI expression.Male Wistar rats were fed lard-based HFD (60 kcal% fat) or control diet (CD) for either 4 or 12 weeks and were killed after intraperitoneal LPS injection. Analyses included messenger RNA expression of TNF-α, macrophage inflammatory protein (MIP)-2, inducible nitric oxide synthase (iNOS), IL-10 and SLPI in the lung tissue and bronchoalveolar lavage fluid, and histology of the lungs.Rats fed HFD for 12 weeks showed suppression of the lung injury and oxidative stress after LPS injection, as indicated by reduction of pulmonary TNF-α, MIP-2 and iNOS mRNA expression and 8-hydroxy-2′-deoxyguanosine immunostaining. The increased pulmonary SLPI caused by lard was associated with decreased pro-inflammatory cytokines and oxidative stress, which eventually resulted in the prevention of ALI. Those effects of lard on LPS-induced ALI were greater after 12 weeks than after 4 weeks feeding, as indicated by the reduction of TNF-α, MIP-2 and iNOS levels.Feeding lard-based HFD for 12 weeks attenuated LPS-induced ALI with increased pulmonary SLPI expression in rats.
Keywords	gene expression ; high fat diet ; histology ; inducible nitric oxide synthase ; inflammation ; interleukin-10 ; lard ; leukocytes ; lipopolysaccharides ; lungs ; messenger RNA ; obesity ; oxidative stress ; pathophysiology ; patients ; proteinase inhibitors ; rats ; respiratory tract diseases ; tumor necrosis factor-alpha
Language	English
Dates of publication	2015-10
Size	p. 997-1009.
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	604812-2
ISSN	1532-1983 ; 0261-5614
ISSN (online)	1532-1983
ISSN	0261-5614
DOI	10.1016/j.clnu.2014.11.001
Database	NAL-Catalogue (AGRICOLA)

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Article: Histological analyses by matrix-assisted laser desorption/ionization-imaging mass spectrometry reveal differential localization of sphingomyelin molecular species regulated by particular ceramide synthase in mouse brains

Sugimoto, Masayuki / Akio Kihara / Kenichi Higashino / Masato Wakabayashi / Shota Sakai / Takeshi Yoshioka / Yasuyuki Igarashi / Yoichi Shimizu / Yoshito Numata / Yuji Kuge / Yukari Tanaka

Biochimica et biophysica acta. 2015 Dec., v. 1851, no. 12

2015

Abstract: Sphingomyelin (SM) is synthesized by SM synthase (SMS) from ceramide (Cer). SM regulates signaling pathways and maintains organ structure. SM comprises a sphingoid base and differing lengths of acyl-chains, but the importance of its various forms and ... ...

Abstract	Sphingomyelin (SM) is synthesized by SM synthase (SMS) from ceramide (Cer). SM regulates signaling pathways and maintains organ structure. SM comprises a sphingoid base and differing lengths of acyl-chains, but the importance of its various forms and regulatory synthases is not known. It has been reported that Cer synthase (CerS) has restricted substrate specificity, whereas SMS has no specificity for different lengths of acyl-chains. We hypothesized that the distribution of each SM molecular species was regulated by expression of the CerS family. Thus, we compared the distribution of SM species and CerS mRNA expression using molecular imaging. Spatial distribution of each SM molecular species was investigated using ultra-high-resolution imaging mass spectrometry (IMS). IMS revealed that distribution of SM molecular species varied according to the lengths of acyl-chains found in each brain section. Furthermore, a combination study using in situ hybridization and IMS revealed the spatial expression of CerS1 to be associated with the localization of SM (d18:1/18:0) in cell body-rich gray matter, and CerS2 to be associated with SM (d18:1/24:1) in myelin-rich white matter. Our study is the first comparison of spatial distribution between SM molecular species and CerS isoforms, and revealed their distinct association in the brain. These observations were demonstrated by suppression of CerS2 using siRNA in HepG2 cells; that is, siRNA for CerS2 specifically decreased C22 very long-chain fatty acid (VLCFA)- and C24 VLCFA-containing SMs. Thus, histological analyses of SM species by IMS could be a useful approach to consider their molecular function and regulative mechanism.
Keywords	brain ; ceramides ; gene expression ; human cell lines ; image analysis ; in situ hybridization ; mass spectrometry ; messenger RNA ; mice ; signal transduction ; small interfering RNA ; sphingomyelins ; sphingosine N-acyltransferase ; substrate specificity ; very long chain fatty acids
Language	English
Dates of publication	2015-12
Size	p. 1554-1565.
Publishing place	Elsevier B.V.
Document type	Article
ISSN	1388-1981
DOI	10.1016/j.bbalip.2015.09.004
Database	NAL-Catalogue (AGRICOLA)

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