Structural and functional analysis of the apoptosis-associated tyrosine kinase (AATYK) family

Apoptosis-associated tyrosine kinase (AATYK) is a protein kinase that is predominantly expressed in the nervous system and is involved in apoptosis and neurite growth of cerebellar granule cells. In this study, we cloned three new members of the mouse AATYK family, AATYK1B, AATYK2 and AATYK3. AATYK1B is a splicing variant of the previously reported AATYK1 (referred to as AATYK1A hereafter). In comparison with AATYK1A, these three AATYK members were characterized by having an extra N-terminal region that consists of a signal peptide-like sequence and a predicted transmembrane (TM) region, which is followed by a kinase domain and a long C-terminal domain. Both TM-containing AATYK isoforms (AATYK(+)TM: AATYK1B, 2, and 3) and TM-lacking isoform (AATYK(-)TM: AATYK1A) were recovered in membrane fractions, suggesting that AATYK(+)TM and AATYK(-)TM are transmembrane- and peripheral-membrane protein kinases, respectively. AATYK1A was recovered in the soluble fraction when the cells were treated with 2-bromo palmitate, suggesting that AATYK1A associates with membrane via palmitoylation. The kinase domain was highly conserved among all AATYK members and was shown to be catalytically active. Three AATYK family members were predominantly expressed in adult mouse brains with almost similar expression profiles: widespread distribution over the various brain regions, especially in the cerebellum and hippocampus, and up-regulated expression during development of the cerebellum. In cultured cerebellar granule cells, AATYK1 was abundantly localized in both soma and axons, AATYK2 distribution was restricted to soma, and AATYK3 was punctately present over the cells. AATYK1 was concentrated in the central domain of growth cones of dorsal root ganglion neurons. Our results indicate that AATYK family members are brain-dominant and membrane-associated kinases with slightly different distribution patterns in the developing and adult mouse brain, which may be involved in fine regulation of neuronal functions including neurite extension and apoptosis.     

Differential effects of ceramide species on exocytosis in rat PC12 cells

Increases in several ceramide species have been shown by non-targeted lipid profiling (lipidomics) of the rat hippocampus after kainate lesions (Guan et al. FASEB J 20:1152–1161, 2006). This study was carried out to examine possible effects of ceramide species on exocytosis. Significant increase in membrane capacitance in voltage-clamped rat pheochromocytoma (PC12) cells, an indication of exocytosis, was detected immediately after external application of C2, C6, and C18 ceramide. In contrast, no increase in capacitance was found after addition of C16 and C20 ceramide, or DMSO vehicle. The effect of ceramide on exocytosis was dependent on the integrity of lipid rafts. Treatment of cells with the cholesterol binding agent/disruptor of lipid rafts, methyl β cyclodextrin, prior to addition of C18 ceramide suppressed the increase in capacitance induced by this lipid species. The ability of C2, C6 and C18 ceramide to trigger exocytosis was confirmed using total internal reflection fluorescence microscopy (TIRFM) experiments. External application of these species caused an exponential decrease in the number of subplasmalemmal neuropeptide Y (NPY)-enhanced green fluorescence protein (EGFP) labeled vesicles, indicating exocytosis. Interestingly, C18 is also the ceramide species that showed the greatest increase in the rat hippocampus after kainate excitotoxicity. It is postulated that C18 ceramide might facilitate exocytosis of glutamate from damaged neurons, thus propagating neuronal injury.     

Proteomic characterisation of neuronal sphingolipid-cholesterol microdomains: role in plasminogen activation

Sorting of certain membrane proteins requires a mechanism involving rafts, protein-lipid complexes enriched in glycosphingolipids and cholesterol. These microdomains remain at the plasma membrane of different cell types and play a role in signal transduction. Although recent reports have begun to describe molecules associated with rafts, their protein composition remains largely unknown, especially in neuronal cells. To address this question, we have purified detergent-insoluble raft fractions (DRMs) from primary cultures of hippocampal neurons. Bidimensional gel analysis and pharmacological raft lipid manipulation allowed the identification of neuronal raft proteins and their characterisation by MALDI-TOF analysis. Enolases were found among the proteins identified and functional studies demonstrate their participation in plasminogen binding. We also show the specific enrichment in rafts of several other plasminogen binding molecules and the exclusive activation of plasminogen to the protease plasmin in these microdomains. These observations suggest that neuronal rafts may play, in addition to intracellular signaling, a role in extracellular/membrane protein proteolysis.     

Differential regulation of ceramide in lipid-rich microdomains (rafts): antagonistic role of palmitoyl:protein thioesterase and neutral sphingomyelinase 2

Cell differentiation and myelination involve a fine balance between stasis and programmed cell death, yet the genes that regulate this have not been clearly defined. We therefore studied two key gene products involved in oligodendrocyte plasma membrane lipid metabolism and their antagonistic role in ceramide-mediated cell death signaling. Overexpression of palmitoyl:protein thioesterase (PPT1; verified by Western blot of the V5-tagged protein and increased enzyme activity) resulted in decreased ceramide in the detergent-resistant microdomain (DRM, or raft) relative to cholesterol and sphingomyelin (SM). This PPT1 overexpression also resulted in protection against cell death induced by either staurosporine or C(2)-ceramide. In contrast, overexpression of neutral sphingomyelinase 2 (NSMase2; verified by Western blot of the FLAG-tagged protein and increased enzyme activity) resulted in increased membrane NSMase and increased ceramide in rafts relative to cholesterol and SM. The difference in SM and ceramide turnover was quantitated by [(3)H]palmitate pulse-chase labeling. Furthermore, when NBD-SM was added to cells, it was hydrolyzed by NSMase-transfected cells at more than twofold the rate in untransfected cells. NSMase2 overexpression enhanced cell death induced by staurosporine or C(2)-ceramide, in contrast to the protective effect of PPT1 overexpression. The presence of a fraction of both PPT1 and NSMase2 in rafts together with their substrates (palmitoylated proteins and SM, respectively) suggests a mechanism for dynamic palmitoylation/depalmitoylation of certain proteins in controlling cell death via NSMase activation.     

T cell costimulation by the hepatitis C virus envelope protein E2 binding to CD81 is mediated by Lck

Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross-linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck-deficient Jurkat T cells. In wild-type Jurkat cells, Lck was activated by CD81 cross-linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl-beta-cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3-deficient Jurkat cells. CD81 cross-linking increased and prolonged the anti-CD3-induced tyrosine phosphorylation of TCR1 and of other proteins, indicating that the CD81-mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross-linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling.     

植物固醇在脂质体中的应用研究进展

脂质体作为药物载体,由于其具有靶向性、可降低药物毒性等优点而备受关注。本文在阐述固醇分子在脂质体中的作用、脂质体液态有序相的特征、脂筏定义的基础上综述了植物细胞中脂筏的发现及植物固醇代替胆固醇作为脂质体膜材的必要性和可行性等相关问题的研究进展。     

Leishmania donovani glycosphingolipid facilitates antigen presentation by inducing relocation of CD1d into lipid rafts in infected macrophages

NKT cells respond to presentation of specific glycolipids with release of both Th1‐ and Th2‐type cytokines. Leishmania donovani (LD)‐infected splenic macrophages (sM?(I)) and bone marrow‐derived dendritic cells (BMDC(I)) failed to activate NKT cells in response to α‐galactosyl ceramide (α‐GalCer). The defective antigen presentation could be corrected by treating the cells with the immunostimulating glycosphingophospholipid (GSPL) of LD parasites. In vitro pulsing of BMDC(I) or sM?(I) with GSPL, caused the activation of the Vα14⁺ CD1d1‐specific NKT cell hybridoma DN32.D3. Localization of MHC II and CD1d molecules to membrane lipid rafts has been suggested to play an important role in antigen presentation. Confocal analysis clearly demonstrated that LD infection changed the pattern of CD1d distribution to the non‐lipid raft regions and this change could be reversed by GSPL treatment. Isoelectric focusing gel shift assay indicated that GSPL binds to CD1d. GSPL‐treated but not untreated BMDC(I) formed immune synapses with NKT cells and this was associated with calcium mobilization. In conclusion, GSPL treatment was associated with modification of BMDC(I)/sM?(I) lipid raft structure, which is a site for immune regulation.     

A new cytolytic protein from the sea anemone Urticina crassicornis that binds to cholesterol- and sphingomyelin-rich membranes

A new pore-forming cytolytic protein was isolated from the Northern red sea anemone, Urticina crassicornis. Its biochemical properties were characterized and partial N-terminal amino acid sequence was determined. The cytolysin, named UcI, has a molecular mass of around 30 kDa and lacks phospholipase A₂ activity. UcI lyses bovine erythrocytes at nanomolar concentrations. Hemolysis is a result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores and can be prevented by osmotic protectants of size >600 Da. The functional radius of an average pore was estimated to be about 0.66 nm. A more detailed study of the cytolytic activity of UcI was performed with lipid vesicles and monolayers. The toxin binds to monolayers and efficiently permeabilizes small lipid vesicles composed of sphingomyelin and cholesterol. However, the cytolytic activity is not prevented by preincubation with either pure cholesterol or sphingomyelin dispersions. We conclude that the presence of both sphingomyelin and cholesterol, key components of lipid rafts, greatly enhances toxin binding to membranes and probably facilitates pore formation. Alignment of the toxin partial amino acid sequence with sequences of cytolysins belonging to the actinoporin family reveals no sequence homology. We conclude that partial sequence of UcI resembles only the N-terminal part of UpI, a cytolytic protein isolated from a related sea anemone species, Urticina piscivora. The two proteins most probably belong to a separate family of sea anemone cytolysins that are worthy of further characterization.     

Palmitoylation regulates raft affinity for the majority of integral raft proteins

The physical basis for protein partitioning into lipid rafts remains an outstanding question in membrane biology that has previously been addressed only through indirect techniques involving differential solubilization by nonionic detergents. We have used giant plasma membrane vesicles, a plasma membrane model system that phase separates to include an ordered phase enriching for raft constituents, to measure the partitioning of the transmembrane linker for activation of T cells (LAT). LAT enrichment in the raft phase was dependent on palmitoylation at two juxtamembrane cysteines and could be enhanced by oligomerization. This palmitoylation requirement was also shown to regulate raft phase association for the majority of integral raft proteins. Because cysteine palmitoylation is the only lipid modification that has been shown to be reversibly regulated, our data suggest a role for palmitoylation as a dynamic raft targeting mechanism for transmembrane proteins.