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Biomedical subjects

Suzanne Jackowski

Publications and source records attributed to Suzanne Jackowski.

26 records · Page 2Linked to original sources
PubMed2003

Gene structure, expression and identification of a new CTP:phosphocholine cytidylyltransferase beta isoform.

CTP:phosphocholine cytidylyltransferase (CCT) is a key regulatory enzyme in phosphatidylcholine (PtdCho) biosynthesis, and in mammals, there are two distinct genes that encode enzymes that catalyze this reaction. This work defines the structures of both the murine CCT genes (Pcyt1a and Pcyt1b) and identifies a new CCT protein, CCTbeta3, with a unique amino terminus that arises from an alternate initiation exon. CCTalpha is expressed in all tissues, and is most abundant in liver, kidney and heart. A second CCTalpha transcript is described that initiates from a separate untranslated exon that is most highly expressed in testis. The CCTbeta isoforms are most highly expressed in brain and reproductive tissues. CCTbeta3 is not expressed in embryonic brain tissues, but is a significant transcript in the adult. These data suggest unique roles for the CCT protein isoforms in the differential regulation of PtdCho biosynthesis in specific tissues.

Alternative Splicing↗
PubMed2003

Prevalence of necrosis in C2-ceramide-induced cytotoxicity in NB16 neuroblastoma cells.

The mechanism of cell death triggered by C2-ceramide was investigated using the NB16 neuroblastoma cell line. Treatment of NB16 cells with 20 microM C2-ceramide for 20 h resulted in approximately 75% loss of cell viability, but only 25% of cells were scored as apoptotic based on terminal deoxynucleotidyl transferase nick-end labeling. Ultrastructural analysis revealed early development of necrotic cytoplasmic vacuolization. After 20 h of treatment with C2-ceramide, the majority of cells possessed necrotic morphology with pronounced cytoplasmic vacuolization and without any nuclear changes, although a quarter of the cell population also exhibited clear perinuclear chromatin condensation characteristic of apoptosis. Flow cytometric analysis of cells labeled with both annexin V and propidium iodide showed the rapid accumulation of C2-ceramide-treated cells in the necrotic/late apoptotic fraction. In contrast, cells treated with tumor necrosis factor alpha plus cycloheximide (TNFalpha + CHX) first appeared in the early apoptotic fraction and then accumulated in the necrotic/late apoptotic fraction. Both C2-ceramide and TNFalpha + CHX increased caspase 8- and 3-like activities in cytosolic extracts; however, treatment of cells with the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected NB16 cells from TNFalpha + CHX-induced cell death but did not prevent C2-ceramide cytotoxicity. Although C2-ceramide triggered apoptosis in a fraction of the cells, cell death in the population was primarily caused by necrosis. Thus, C2-ceramide does not faithfully mimic the effects of apoptotic ligands such as TNFalpha, which are thought to be mediated by an accumulation of endogenous ceramide. The inhibition of phosphatidylcholine synthesis is a target for C2-ceramide-mediated cytotoxicity, and this work suggests that other agents that kill cells by inhibiting this pathway may also use a mixture of mechanisms, including necrosis as well as apoptosis.

Antineoplastic Agents↗
PubMed2003

Role of feedback regulation of pantothenate kinase (CoaA) in control of coenzyme A levels in Escherichia coli.

Pantothenate kinase (CoaA) is a key regulator of coenzyme A (CoA) biosynthesis in Escherichia coli, and its activity is controlled by feedback inhibition by CoA and its thioesters. The importance of feedback inhibition in the control of the intracellular CoA levels was tested by constructing three site-directed mutants of CoaA that were predicted to be feedback resistant based on the crystal structure of the CoaA-CoA binary complex. CoaA[R106A], CoaA[H177Q], and CoaA[F247V] were purified and shown to retain significant catalytic activity and be refractory to inhibition by CoA. CoaA[R106A] retained 50% of the catalytic activity of CoaA, whereas the CoaA[H177Q] and CoaA[F247V] mutants were less active. The importance of feedback control of CoaA to the intracellular CoA levels was assessed by expressing either CoaA or CoaA[R106A] in strain ANS3 [coaA15(Ts) panD2]. Cells expressing CoaA[R106A] had significantly higher levels of phosphorylated pantothenate-derived metabolites and CoA in vivo and excreted significantly more 4'-phosphopantetheine into the medium compared to cells expressing the wild-type protein. These data illustrate the key role of feedback regulation of pantothenate kinase in the control of intracellular CoA levels.

Binding Sites↗
PubMed2002

The murine pantothenate kinase (Pank1) gene encodes two differentially regulated pantothenate kinase isozymes.

Pantothenate kinase (PanK) is a rate-determining enzyme in coenzyme A (CoA) biosynthesis. The mouse murine pantothenate kinase (Pank1) gene consists of seven introns and eight exons and is located on chromosome 19 (19C2-3). Two biochemically distinct PanK1 protein isoforms, PanK1 alpha and PanK1 beta, are encoded by the Pank1 gene. Both proteins have the same 363 amino acid catalytic domain encoded by exons 2 through 7. The PanK1 beta transcript begins with exon 1 beta and translates into a ten-residue amino terminus plus the catalytic domain. The PanK1 alpha transcript initiates at an alternate upstream site at exon 1 alpha which is spliced with exon 2, excluding exon 1 beta. Exon 1 alpha encodes a 184-residue regulatory domain at the amino terminus of the PanK1 alpha protein that confers feedback inhibition by free CoA and long-chain acyl-CoA, and increases the regulation of PanK enzyme activity by acetyl-CoA and malonyl-CoA. Differential expression of the PanK1 alpha and PanK1 beta transcripts would alter the amount of CoA produced in cells as a function of the ratio of free CoA to acetyl-CoA, a reflection of the metabolic status of the tissue.

Acetyl Coenzyme A↗
PubMed2002

Inhibition of CTP:phosphocholine cytidylyltransferase by C(2)-ceramide and its relationship to apoptosis.

Apoptosis induced by antitumor phospholipid analogs takes place after the inhibition of the CTP:phosphocholine cytidylyltransferase (CCT; EC 2.7.7.15) catalyzed step of phosphatidylcholine (PtdCho) biosynthesis. Exposure of cells to synthetic short-chain ceramide analogs also triggers apoptosis concomitant with decreased PtdCho biosynthesis, and the present study was undertaken to ascertain whether C(2)-ceramide inhibition of PtdCho synthesis is direct or secondary to other ceramide-mediated cellular responses. The exposure of COS-7 cells to either C(2)-ceramide, ET-18-OCH(3), or farnesol resulted in time- and dose-dependent apoptotic cell death. Cells treated with C(2)-ceramide or ET-18-OCH(3) selectively and immediately accumulated phosphocholine, whereas CDP-choline increased with farnesol treatment. In vitro assays of CCT activity demonstrated that C(2)-ceramide directly inhibited CCT. Comparison of different N-linked sphingosine derivatives suggests an inverse relationship between the length of the N-linked carbon chain and the derivatives ability to trigger apoptosis and inhibit CCT. Taken together, our results suggest CCT as a primary target for C(2)-ceramide inhibition that accounts for its cytotoxic effects.

Animals↗
PubMed2002

A missense mutation in the fabB (beta-ketoacyl-acyl carrier protein synthase I) gene confers tiolactomycin resistance to Escherichia coli.

Thiolactomycin (TLM) is an antibiotic that inhibits bacterial type II fatty acid synthesis at the condensing enzyme step, and beta-ketoacyl-acyl carrier protein synthase I (FabB) is the relevant target in Escherichia coli. TLM resistance is associated with the upregulation of efflux pumps. Therefore, a tolC knockout mutant (strain ANS1) was constructed to eliminate the contribution of type I secretion systems to TLM resistance. Six independent TLM-resistant clones of strain ANS1 were isolated, and all possessed the same missense mutation in the fabB gene (T1168G) that directed the expression of a mutant protein, FabB(F390V). FabB(F390V) was resistant to TLM in vitro. Leucine is the only other amino acid found at position 390 in nature, and the Staphylococcus aureus FabF protein, which contains this substitution, was sensitive to TLM. Structural modeling predicted that the CG2 methyl group of the valine side chain interfered with the positioning of the C11 methyl on the isoprenoid side chain of TLM in the binary complex, whereas the absence of a bulky methyl group on the leucine side chain permitted TLM binding. These data illustrate that missense mutations that introduce valine at position 390 confer TLM resistance while maintaining the vital catalytic properties of FabB.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2001

Structure and mechanism of CTP:phosphocholine cytidylyltransferase (LicC) from Streptococcus pneumoniae.

Pneumococcal LicC is a member of the nucleoside triphosphate transferase superfamily and catalyzes the transfer of a cytidine monophosphate from CTP to phosphocholine to form CDP-choline. The structures of apo-LicC and the LicC-CDP-choline-Mg(2+) ternary complex were determined, and the comparison of these structures reveals a significant conformational change driven by the multivalent coordination of Mg(2+). The key event is breaking the Glu(216)-Arg(129) salt bridge, which triggers the coalescence of four individual beta-strands into two extended beta-sheets. These movements reorient the side chains of Trp(136) and Tyr(190) for the optimal binding and alignment of the phosphocholine moiety. Consistent with these conformational changes, LicC operates via a compulsory ordered kinetic mechanism. The structures explain the substrate specificity of LicC for CTP and phosphocholine and implicate a direct role for Mg(2+) in aligning phosphocholine for in-line nucleophilic attack and stabilizing the negative charge that develops in the pentacoordinate transition state. These results provide a structural basis for assigning a specific role for magnesium in the catalytic mechanism of pneumococcal LicC.

Catalysis↗