Hyunmi Lee

Sphingolipids and cell death

Sphingolipids (SLs) have been considered for many years as predominant building blocks of biological membranes with key structural functions and little relevance in cellular signaling. However, this view has changed dramatically in recent years with the recognition that certain SLs such as ceramide, sphingosine 1-phosphate and gangliosides, participate actively in signal transduction pathways, regulating many different cell functions such as proliferation, differentiation, adhesion and cell death. In particular, ceramide has attracted considerable attention in cell biology and biophysics due to its key role in the modulation of membrane physical properties, signaling and cell death regulation. This latter function is largely exerted by the ability of ceramide to activate the major pathways governing cell death such as the endoplasmic reticulum and mitochondria. Overall, the evidence so far indicates a key function of SLs in disease pathogenesis and hence their regulation may be of potential therapeutic relevance in different pathologies including liver diseases, neurodegeneration and cancer biology and therapy.

Ceramide synthases 2, 5, and 6 confer distinct roles in radiation-induced apoptosis in HeLa cells.

The role of ceramide neo-genesis in cellular stress response signaling is gaining increasing attention with recent progress in elucidating the novel roles and biochemical properties of the ceramide synthase (CerS) enzymes. Selective tissue and subcellular distribution of the six mammalian CerS isoforms, combined with distinct fatty acyl chain length substrate preferences, implicate differential functions of specific ceramide species in cellular signaling. We report here that ionizing radiation (IR) induces de novo synthesis of ceramide to influence HeLa cell apoptosis by specifically activating CerS isoforms 2, 5, and 6 that generate opposing anti- and pro-apoptotic ceramides in mitochondrial membranes. Overexpression of CerS2 resulted in partial protection from IR-induced apoptosis whereas overexpression of CerS5 increased apoptosis in HeLa cells. Knockdown studies determined that CerS2 is responsible for all observable IR-induced C(24:0) CerS activity, and while CerS5 and CerS6 each confer approximately 50% of the C(16:0) CerS baseline synthetic activity, both are required for IR-induced activity. Additionally, co-immunoprecipitation studies suggest that CerS2, 5, and 6 might exist as heterocomplexes in HeLa cells, providing further insight into the regulation of CerS proteins. These data add to the growing body of evidence demonstrating interplay among the CerS proteins in a stress stimulus-, cell type- and subcellular compartment-specific manner.

A role for the signal transduction protein PII in the control of nitrate/nitrite uptake in a cyanobacterium

In the cyanobacterium Synechococcus sp. strain PCC 7942, ammonium exerts a rapid and reversible inhibition of the nitrate and nitrite uptake, and the PII protein (GlnB) is differentially phosphorylated depending on the intracellular N/C balance. RNA/DNA hybridizations, as well as nitrate and nitrite uptake experiments, were carried out with the wild‐type strain and a PII‐null mutant. The transcriptional control by ammonium of the expression of the nir‐nrtABCD‐narB operon remained operative in the mutant but, in contrast to the wild‐type strain, the mutant took up nitrate and nitrite even in the presence of ammonium. Moreover, the wild‐type phenotype was restored by insertion of a copy of the wild‐type glnB gene in the genome of the PII‐null mutant. These results indicate that the unphosphorylated form of PII is involved in the short‐term inhibition by ammonium of the nitrate and nitrite uptake in Synechococcus sp. strain PCC 7942.

Kinetic characterization of mammalian ceramide synthases: Determination of Km values towards sphinganine

Ceramide is a key metabolite in the pathway of sphingolipid biosynthesis. In mammals, ceramide is synthesized by N‐acylation of a sphingoid long‐chain base by a family of ceramide synthases (CerS), each of which displays a high specificity towards acyl CoAs of different chain lengths. We now optimize a previously‐described assay for measuring CerS activity for use upon over‐expression of mammalian CerS, and using these conditions, establish the K m value of each CerS towards sphinganine. Remarkably, the K m values towards sphinganine are all similar, ranging from 2 to 5 μM, even for CerS proteins that are able to use more than one acyl CoA for ceramide synthesis (i.e. CerS4). The availability of this assay will permit further accurate characterization of the kinetic parameters of mammalian CerS proteins.

A Ceramide-binding C1 Domain Mediates Kinase Suppressor of Ras Membrane Translocation

Genetic and biochemical data support Kinase Suppressor of Ras 1 (KSR1) as a positive regulator of the Ras-Raf-MAPK pathway, functioning as a kinase and/or scaffold to regulate c-Raf-1 activation. Membrane translocation mediated by the KSR1 CA3 domain, which is homologous to the atypical PKC C1 lipid-binding domain, is a critical step of KSR1-mediated c-Raf-1 activation. In this study, we used an ELISA to characterize the KSR1 CA3 domain as a lipid-binding moiety. Purified GST-KSR1-CA3 protein effectively binds ceramide but not other lipids including 1,2-diacylglyceol, dihydroceramide, ganglioside GM1, sphingomyelin and phosphatidylcholine. Upon epidermal growth factor stimulation of COS-7 cells, KSR1 translocates into and is activated within glycosphingolipid-enriched plasma membrane platforms. Pharmacologic inhibition of ceramide generation attenuates KSR1 translocation and KSR1 kinase activation in COS-7 cells. Disruption of two cysteines, which are indispensable for maintaining ternary structure of all C1 domains and their lipid binding capability, mitigates ceramide-binding capacity of purified GST-KSR1-CA3 protein, and inhibits full length KSR1 membrane translocation and kinase activation. These studies provide evidence for a mechanism by which the second messenger ceramide can target proteins to subcellular compartments in the process of transmembrane signal transduction.

Control of nitrogen and carbon metabolism in cyanobacteria

In the unicellular cyanobacteria that do not fix molecular nitrogen, interactions between N assimilation and C metabolism occur through the signal transducer PII and the global nitrogen regulator NtcA. Under high CO2 concentration, PII liganded to ATP and boundto 2-oxoglutarate becomes phosphorylated and negatively controls the high affinity transport for bicarbonate. In contrast, under low CO2, PII being only liganded to ATP becomes dephosphorylated and negatively controls the nitrate/nitrite active transport system. The redox state of the cells together with NtcA also modulate the phosphorylation state of PII. Moreover, the regulation of the expression of the gene encoding PII is at least in part NthA-dependent. This network of transcriptional and post-transcriptional regulations allows cells to rapidly acclimate by adjusting their carbon and nitrogen metabolism in response to changes in environmental coditions.

Regulation of Carbon and Nitrogen Metabolism in the Unicellular Cyanobacteria Synechococcus spp.

Cyanobacteria are photosynthetic prokaryotes that mainly use CO2 and bicarbonate as carbon sources, and ammonium and nitrate as nitrogen sources to fulfil their requirement for growth. Some strains can also fix molecular N2 either under aerobic or anaerobic conditions16,27. Under autotrophic conditions, the enzymatic reactions required for the utilization of any form of inorganic nitrogen depend upon the availability of energy (ATP) and reductant generated from photosynthesis, as well as upon CO2-fixation products, which in part act as amino acceptors to generate amino acids and other organic nitrogenous compounds. There might thus exist tight interactions between nitrogen and carbon metabolism to balance the intracellular N/C ratio.

Correction: Mitochondrial Ceramide-Rich Macrodomains Functionalize Bax upon Irradiation

The authors would like to correct Fig 5, as errors were introduced in the preparation of this figure for publication. In Fig 5D[b], the image for the Light fraction Bak Western blot and the numbering of the fractions are incorrect. The authors have provided a corrected version of Fig 5 here. Fig 5 Ceramide induces formation of a mitochondrial ceramide-rich macrodomain (MCRM). The authors confirm that these changes do not alter their findings. The authors have provided the original Western blot and additional underlying data as Supporting Information.