Ceramide Generated by Sphingomyelin Hydrolysis and the Salvage Pathway Is Involved in Hypoxia/Reoxygenation-induced Bax Redistribution to Mitochondria

J Biol Chem. 2008 Sep 26;283(39):26509-17. Epub 2008 Aug 1. Links

Ceramide Generated by Sphingomyelin Hydrolysis and the Salvage Pathway Is Involved in Hypoxia/Reoxygenation-induced Bax Redistribution to Mitochondria in NT-2 Cells.

Jin J, Hou Q, Mullen TD, Zeidan YH, Bielawski J, Kraveka JM, Bielawska A, Obeid LM, Hannun YA, Hsu YT.

Departments of Biochemistry and Molecular Biology, Medicine, and Pediatrics, Division of Hematology/Oncology, Medical University of South Carolina, Charleston, South Carolina 29425.

Ceramide functions as an important second messenger in apoptosis signaling pathways. In this report, we show that treatment of NT-2 neuronal precursor cells with hypoxia/reoxygenation (H/R) resulted in ceramide up-regulation. This elevation in ceramide was primarily due to the actions of acid sphingomyelinase and ceramide synthase LASS 5, demonstrating the action of the salvage pathway. Hypoxia/reoxygenation treatment led to Bax translocation from the cytoplasm to mitochondria and cytochrome c release from mitochondria. Down-regulation of either acid sphingomyelinase or LASS 5-attenuated ceramide accumulation and H/R-induced Bax translocation to mitochondria. Overall, we have demonstrated that ceramide up-regulation following H/R is pertinent to Bax activation to promote cell death.

PMID: 18676372 [PubMed - in process]

NT-2 cells, 12-hour hypoxia, 0, 12, 24h reoxygenation
HPLC-MS, qPCR (quantitative PCR = realtime PCR??)
H/R results in ceramide accumulation, sphingomyelin decrease
C14, C16 ceramides most changed
C18:1, C20 also altered
C24 no change
suggests involvement of LASS5 or LASS6
LASS5 (by aPCR and siRNA) siRNA attenuates Bax activation, cyt c release, caspase3 activation
aSMase but not nSMase activity up, and mRNA up, siRNA attenuates Bax activation, therefore ceramide signal from salvage pathway

fumonisin B1 inhibits both de novo and salvage pathway generation of ceramide
how does aSMase get activated? coordinated with LASS5 activation? probably
how does increased short(er) chain ceramides activate Bax? not through Bid

Higashio, Traffic, 2007

Traffic. 2007 Nov 27 [Epub ahead of print] Links

Smy2p Participates in COPII Vesicle Formation Through the Interaction with Sec23p/Sec24p Subcomplex.

Higashio H, Sato K, Nakano A.

Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

The coat protein complex II (COPII) is essential for vesicle formation from the endoplasmic reticulum (ER) and is composed of two heterodimeric subcomplexes, Sec23p/Sec24p and Sec13p/Sec31p, and the small guanosine triphosphatase Sar1p. In an effort to identify novel factors that may participate in COPII vesicle formation, we isolated SMY2, a yeast gene encoding a protein of unknown function, as a multicopy suppressor of the temperature-sensitive sec24-20 mutant. We found that even a low-copy expression of SMY2 was sufficient for the suppression of the sec24-20 phenotypes, and the chromosomal deletion of SMY2 led to a severe growth defect in the sec24-20 background. In addition, SMY2 exhibited genetic interactions with several other genes involved in the ER-to-Golgi transport. Subcellular fractionation analysis showed that Smy2p was a peripheral membrane protein fractionating together with COPII components. However, Smy2p was not loaded onto COPII vesicles generated in vitro. Interestingly, coimmunoprecipitation between Smy2p and the Sec23p/Sec24p subcomplex was specifically observed in sec23-1 and sec24-20 backgrounds, suggesting that this interaction was a prerequisite for the suppression of the sec24-20 phenotypes by overexpression of SMY2. We propose that Smy2p is located on the surface of the ER and facilitates COPII vesicle formation through the interaction with Sec23p/Sec24p subcomplex.

PMID: 17973654 [PubMed - as supplied by publisher]

Smy2p - suppressor of myosin 2, temperature sensitive mutant
peripheral membrane protein, 100% in pellet of 100k rcf (no cytosolic pool)
790aa, 87kDa predicted, 100kDa apparent (hi pI of 8.97)
GYF domain (with C-term req'd for suppression), coiled-coil domain
ER - sucrose gradient, IF microscopy (perinuclear ER)
suppressor of ts sec24-20, alsosec16-2, sec22-3, bet1-1, sec34-1, sec35-1
deletion synthetically lethal with sec24-20
co-IPs sec23/24 only in sec24-20, sec23-1, requires coiled coil +carboxy terminal portion
yeast 2-hybrid interaction with Sec23p, but not Sec24p (also Msl5p, Mud2p, mRNA splicing)
Smy2p required to survive sec24-20 defect, scaffold protein?


Background:
sec24p A-site - SNARE Sed5p
B-site - SNAREs Sed5p, Bet1p, Golgi protein Sys1p
C-site - SNARE Sec22p
sec24-20 lacks c-term 30 aa (W897stop) in A site

Sec24p homologues Sfb2p(Iss1p), Sfb3p (Lst1p)
Sfb3p specialized for Pma1p packaging
Sfb2p functionally redundant for sec24p

Li, FEBS Letters, 2007

FEBS Lett. 2007 Dec 11;581(29):5698-702. Epub 2007 Nov 26.

Related Articles, Links

Mutations of the SM protein Sly1 resulting in bypass of GTPase requirement in vesicular transport are confined to a short helical region.

Li Y, Schmitt HD, Gallwitz D, Peng RW.

Department of Molecular Genetics, Max Planck Institute for Biophysical Chemistry, 37070 Göttingen, Germany.

SLY1-20 is a suppressor of Ypt1p lethality. It is essential for membrane fusion and appears to coordinate the function of Ypt1 and SNAREs. Mutations in the alpha helix 20 (T531 or E532) or complete deletion of E532 or the whole helix result in bypass of Ypt1 or Ypt6. Hypothesis that this helix is normally closed, protecting a conserved helical region (helices 13 and 14) of sly1p domain, these mutations are postulated to keep the "lid" constitutively open. Helix 20 and the TE/TD motif are conserved in fungi, but not in multi-cellular organisms