Joachim Schnier

Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae.

Translation intitiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond. One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A. To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae. Two genes named TIF51A and TIF51B were cloned and sequenced. The two yeast proteins are closely related, sharing 90% sequence identity, and each is ca. 63% identical to the human protein. The purified protein expressed from the TIF51A gene substitutes for HeLa eIF-5A in the mammalian methionyl-puromycin synthesis assay. Strains lacking the A form of eIF-5A, constructed by disruption of TIF51A with LEU2, grow slowly, whereas strains lacking the B form, in which HIS3 was used to disrupt TIF51B, show no growth rate phenotype. However, strains with both TIF51A and TIF51B disrupted are not viable, indicating that eIF-5a is essential for cell growth in yeast cells. Northern (RNA) blot analysis shows two mRNA species, a larger mRNA (0.9 kb) transcribed from TIF51A and a smaller mRNA (0.8 kb) encoded by TIF51B. Under the aerobic growth conditions of this study, the 0.8-kb TIF51B transcript is not detected in the wild-type strain and is expressed only when TIF51A is disrupted. The TIF51A gene was altered by site-directed mutagenesis at the site of hypusination by changing the Lys codon to that for Arg, thereby producing a stable protein that retains the positive charge but is not modified to the hypusine derivative. The plasmid shuffle technique was used to replace the wild-type gene with the mutant form, resulting in failure of the yeast cells to grow. This result indicates that hypusine very likely is required for the vital in vivo function of eIF-5A and suggests a precise, essential role for the polyamine spermidine in cell metabolism.

Flavopiridol Inhibits Glycogen Phosphorylase by Binding at the Inhibitor Site

Flavopiridol (L86–8275) ((−)-cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl]-4H-benzopyran-4-one), a potential antitumor drug, currently in phase II trials, has been shown to be an inhibitor of muscle glycogen phosphorylase (GP) and to cause glycogen accumulation in A549 non-small cell lung carcinoma cells (Kaiser, A., Nishi, K., Gorin, F.A., Walsh, D.A., Bradbury, E. M., and Schnier, J. B., unpublished data). Kinetic experiments reported here show that flavopiridol inhibits GPb with an IC50 = 15.5 μm. The inhibition is synergistic with glucose resulting in a reduction of IC50for flavopiridol to 2.3 μm and mimics the inhibition of caffeine. In order to elucidate the structural basis of inhibition, we determined the structures of GPb complexed with flavopiridol, GPb complexed with caffeine, and GPa complexed with both glucose and flavopiridol at 1.76-, 2.30-, and 2.23-Å resolution, and refined to crystallographic R values of 0.216 (Rfree = 0.247), 0.189 (Rfree = 0.219), and 0.195 (Rfree = 0.252), respectively. The structures provide a rational for flavopiridol potency and synergism with glucose inhibitory action. Flavopiridol binds at the allosteric inhibitor site, situated at the entrance to the catalytic site, the site where caffeine binds. Flavopiridol intercalates between the two aromatic rings of Phe285 and Tyr613. Both flavopiridol and glucose promote the less active T-state through localization of the closed position of the 280s loop which blocks access to the catalytic site, thereby explaining their synergistic inhibition. The mode of interactions of flavopiridol with GP is different from that ofdes-chloro-flavopiridol with CDK2, illustrating how different functional parts of the inhibitor can be used to provide specific and potent binding to two different enzymes.

Identification of cytosolic aldehyde dehydrogenase 1 from non-small cell lung carcinomas as a flavopiridol-binding protein

The synthetic flavone flavopiridol can be cytostatic or cytotoxic to mammalian cells, depending on the concentration of the drug and the duration of exposure. It has been shown to inhibit the cyclin‐dependent kinase (CDK) family of cell cycle regulatory enzymes. However, the existence of additional potential targets for drug action remains a matter of interest to define. To identify cellular targets, flavopiridol was immobilized. CDKs, particularly CDK 4, bound weakly to immobilized flavopiridol when ATP was absent but not in its presence. Two proteins with molecular weights of 40 kDa and 120 kDa had high affinities to the immobilized flavopiridol independent of the presence of ATP. They were present in all cell lines analyzed: cervical (HeLa), prostate and non‐small cell lung carcinoma (NSCLC) cell lines. A 60‐kDa protein, which was present only in NSCLC cells and bound similarly well to immobilized flavopiridol, was identified as cytosolic aldehyde dehydrogenase class 1 (ALDH‐1). The level of this protein correlated with the resistance of NSCLC cell lines to cytotoxicity caused by 500 nM flavopiridol but not higher flavopiridol concentrations. Despite binding to ALDH‐1, there was no inhibition of dehydrogenase activity by flavopiridol concentrations as high as 20 μM and flavopiridol was not metabolized by ALDH‐1. The results suggest that high cellular levels of ALDH‐1 may reduce cytotoxicity of flavopiridol and contribute to relative resistance to the drug. This is the first report that flavopiridol binds to proteins other than CDKs.

Inhibition of glycogen phosphorylase (GP) by CP-91,149 induces growth inhibition correlating with brain GP expression.

The role of glycogenolysis in normal and cancer cells was investigated by inhibiting glycogen phosphorylase (GP) with the synthetic inhibitor CP-91,149. A549 non-small cell lung carcinoma (NSCLC) cells express solely the brain isozyme of GP, which was inhibited by CP-91,149 with an IC(50) of 0.5 microM. When treated with CP-91,149, A549 cells accumulated glycogen with associated growth retardation. Treated normal skin fibroblasts also accumulated glycogen with G1-cell cycle arrest that was associated with inhibition of cyclin E-CDK2 activity. Overall, cells expressing high levels of brain GP were growth inhibited by CP-91,149 correlating with glycogen accumulation whereas cells expressing low levels of brain GP were not affected by the drug. Analyses of 59 tumor cell lines represented in the NCI drug screen identified that every cell line expressed brain GP but the profile was dominated by a few highly GP expressing cell lines with lower than mean GP-a enzymatic activities. The correlation program, COMPARE, identified that the brain GP protein measured in the NCI cell lines corresponded with brain GP mRNA expression, ADP-ribosyltransferase 3, and colony stimulating factor 2 receptor alpha in the 10,000 gene microarray database with similar correlation coefficients. These results suggest that brain GP is present in proliferating cells and that high protein levels correspond with the ability of CP-91,149 to inhibit cell growth.

The role of mammalian initiation factor eIF-4D and its hypusine modification in translation.

Initiation factor eIF-4D functions late in the initiation pathway, apparently during formation of the first peptide bond. The factor is post-translationally modified at a specific lysine residue by reaction with spermidine and subsequent hydroxylation to form hypusine. A precursor form lacking hypusine is inactive in the assay for methionyl-puromycin synthesis, but activity is restored following in vitro modification to deoxyhypusine, thereby suggesting that the modification is essential for function. Since formylated methionyl-tRNA is less dependent on eIF-4D in the puromycin assay, we postulate that eIF-4D and its hypusine modification may stabilize charged Met-tRNA binding to the peptidyl transferase center of the 60S ribosomal subunit. Analysis of eIF-4D genes in yeast indicate that eIF-4D and its hypusine modification are essential for cell growth.

Acute Severe Animal Model of Anti–Muscle-Specific Kinase Myasthenia: Combined Postsynaptic and Presynaptic Changes

OBJECTIVESnTo determine the pathogenesis of anti-muscle-specific kinase (MuSK) myasthenia, a newly described severe form of myasthenia gravis associated with MuSK antibodies characterized by focal muscle weakness and wasting and absence of acetylcholine receptor antibodies, and to determine whether antibodies to MuSK, a crucial protein in the formation of the neuromuscular junction (NMJ) during development, can induce disease in the mature NMJ. Design, Setting, andnnnPARTICIPANTSnLewis rats were immunized with a single injection of a newly discovered splicing variant of MuSK, MuSK 60, which has been demonstrated to be expressed primarily in the mature NMJ. Animals were assessed clinically, serologically, and by repetitive stimulation of the median nerve. Muscle tissue was examined immunohistochemically and by electron microscopy.nnnRESULTSnAnimals immunized with 100 μg of MuSK 60 developed severe progressive weakness starting at day 16, with 100% mortality by day 27. The weakness was associated with high MuSK antibody titers, weight loss, axial muscle wasting, and decrementing compound muscle action potentials. Light and electron microscopy demonstrated fragmented NMJs with varying degrees of postsynaptic muscle end plate destruction along with abnormal nerve terminals, lack of registration between end plates and nerve terminals, local axon sprouting, and extrajunctional dispersion of cholinesterase activity.nnnCONCLUSIONSnThese findings support the role of MuSK antibodies in the human disease, demonstrate the role of MuSK not only in the development of the NMJ but also in the maintenance of the mature synapse, and demonstrate involvement of this disease in both presynaptic and postsynaptic components of the NMJ.

Cyclin D1 Downregulation is Important for Permanent Cell Cycle Exit and Initiation of Differentiation Induced by Anchorage-Deprivation in Human Keratinocytes

To understand the relationship between permanent cell cycle exit and differentiation the immortalized keratinocyte cell line, SIK and the squamous cell carcinoma, SCC9 were compared during differentiation induced by anchorage‐deprivation. The SIK cells when placed in suspension culture promptly lost almost all ability to reinitiate growth by 2 days concomitantly expressing the differentiation specific proteins, transglutaminase (TGK) and involucrin. These cells rapidly underwent G1 cell cycle arrest with complete disappearance of phosphorylated RB. In contrast SCC9 cells neither showed TGK expression nor increase in involucrin. They decreased their colony‐forming ability much more slowly, which coordinated well with a gradual decrease in phosphorylated RB, demonstrating the significant resistance to loss of colony‐forming ability and cell cycle exit. In accordance, cyclin D1, a positive regulator of cyclin‐dependent kinase (CDK) 4/6 which phosphorylates RB decreased drastically in anchorage deprived SIK but not in SCC9 cells. Endogenous cyclin D1 knockdown in SCC9 cells by siRNA enhanced loss of the colony‐forming ability during anchorage‐deprivation. Conversely enforced expression of cyclin D1 in SIK cells and in another immortalized keratinocyte cell line, HaCaT, partly prevented loss of their colony‐forming abilities. Cyclin D1 overexpression antagonized Keratin 10 expression in suspended HaCaT cells. The result demonstrates the importance of cyclin D1 down regulation for proper initiation of keratinocyte differentiation. J. Cell. Biochem. 106: 63–72, 2009.

Glycogen synthesis correlates with androgen-dependent growth arrest in prostate cancer

BackgroundAndrogen withdrawal in normal prostate or androgen-dependent prostate cancer is associated with the downregulation of several glycolytic enzymes and with reduced glucose uptake. Although glycogen metabolism is known to regulate the intracellular glucose level its involvement in androgen response has not been studied.MethodsWe investigated the effects of androgen on glycogen phosphorylase (GP), glycogen synthase (GS) and on glycogen accumulation in the androgen-receptor (AR) reconstituted PC3 cell line containing either an empty vector (PC3-AR-V) or vector with HPV-E7 (PC3-AR-E7) and the LNCaP cell line.ResultsAndrogen addition in PC3 cells expressing the AR mimics androgen ablation in androgen-dependent prostate cells. Incubation of PC3-AR-V or PC3-AR-E7 cells with the androgen R1881 induced G1 cell cycle arrest within 24 hours and resulted in a gradual cell number reduction over 5 days thereafter, which was accompanied by a 2 to 5 fold increase in glycogen content. 24 hours after androgen-treatment the level of Glucose-6-P (G-6-P) had increased threefold and after 48 hours the GS and GP activities increased twofold. Under this condition inhibition of glycogenolysis with the selective GP inhibitor CP-91149 enhanced the increase in glycogen content and further reduced the cell number. The androgen-dependent LNCaP cells that endogenously express AR responded to androgen withdrawal with growth arrest and increased glycogen content. CP-91149 further increased glycogen content and caused a reduction of cell number.ConclusionIncreased glycogenesis is part of the androgen receptor-mediated cellular response and blockage of glycogenolysis by the GP inhibitor CP-91149 further increased glycogenesis. The combined use of a GP inhibitor with hormone therapy may increase the efficacy of hormone treatment by decreasing the survival of prostate cancer cells and thereby reducing the chance of cancer recurrence.

Congenital myasthenic syndrome caused by two non-N88K rapsyn mutations

To the Editor: Mutations in the human rapsyn gene (RAPSN) are associated with a congenital myasthenic syndrome (CMS) characterized by deficient clustering of acetylcholine receptors (AChRs) (1–5). With rare exceptions, reported patients with CMS caused by RAPSN mutations are homozygotes for the N88K mutation or carry N88K combined with another mutation (6, 7). We describe here a patient with a CMS caused by two heteroallelic non-N88K RAPSN mutations. The patient is a 16-month-old girl from a nonconsanguineous couple. At birth, she was hypotonic and required mechanical ventilation and gastric tube feeding. The examination revealed normal cognition, bilateral ptosis, normal ocular movements and mild facial and proximal limb weakness. Electromyogram with repetitive nerve stimulation showed 25% decrement. In vitro electrophysiologic studies performed in an anconeus muscle biopsy demonstrated marked reduction of amplitudes of miniature endplate potentials (0.43 0.09 mV, n 1⁄4 14 vs 1.26 0.36 nA, n 1⁄4 14 in a control, p , 0.001) and a moderate decrease of the endplate potential quantal content (3.55 1.76, n 1⁄4 6 vs 7.74 3.0, n 1⁄4 6 in a control, p , 0.05). Endplate acetylcholinesterase reaction was normal. Electron microscopy of neuromuscular junctions demonstrated a marked simplification of the post-synaptic membrane with no abnormality of subsynaptic sarcoplasm or the nerve terminal. Direct sequencing of the AChR a-, b-, d-, and esubunit genes using genomic DNA revealed no mutations. However, analysis of RAPSN revealed a heterozygous G/A change in exon 1 at nucleotide 133 and in exon 2 at nucleotide 284 predicting V45M and E162K (Fig. 1a and b). V45M is located in the first tetratricopeptide region (TPR), while E162K is located in the linker between the fourth and fifth TPRs. V45 and E162 are conserved (Fig. 1c). Restriction analysis revealed that the patient and her father were heterozygotes for V45M, while the patient and her mother were heterozygotes for E162K (Fig. 1d). To evaluate the effect of V45M and E162K on the protein function, we introduced these mutations in a wild-type RAPSN–GFP construct and transfected HEK cells with either wild type or mutants as described elsewhere (1). Data extracted from three to four experiments revealed that cells transfected with the wild-type RAPSN–GFP showed green fluorescence arranged in clusters in 49.3 2.8% of the cells (n 1⁄4 269), while the rest displayed green fluorescence homogeneously. Cells transfected with RAPSN V45M-GFP or RAPSN E162K-GFP displayed green fluorescence arranged in clusters in 50.6 5.2% (n1⁄4 290) and 51.5 2.5% (n1⁄4 210), respectively. Cotransfection of HEK cells with cDNA of the four AChR-subunits with wild-type RAPSN– GFP revealed colocalization of green fluorescence granules with red fluorescence granules representing rhodamine-alpha-bungarotoxin bound to AChRs in 68.4 3.1% of the cells (n 1⁄4 68) (Fig. 2a–c and j). In contrast, cotransfection of the AChR-subunit cDNAs with RAPSN V45MGFP or RAPSN E162K-GFP revealed coclustering of greenand red-fluorescence granules in only 26.6 3.0% (n 1⁄4 76) and 24.3 2.8% (n 1⁄4 67) of the cells, respectively (Fig. 2d–j) (p , 0.001). Hence, although neither V45M nor E162K mutants hinder rapsyn self-association, both mutants markedly decrease the ability of rapsyn to cluster with AChRs. Interestingly, it was recently reported that two rapsyn mutations not found associated with N88K also diminish coclustering of AChRs with rapsyn without impairing rapsyn self-association (7). V45M and E162K are located in the TPR domain of rapsyn, which controls rapsyn selfassociation; however, because neither truncates the TPR domain, it is not surprising that they do not hinder rapsyn self-association. However,

Animal models of antimuscle-specific kinase myasthenia

Antimuscle‐specific kinase (anti‐MuSK) myasthenia (AMM) differs from antiacetylcholine receptor myasthenia gravis in exhibiting more focal muscle involvement (neck, shoulder, facial, and bulbar muscles) with wasting of the involved, primarily axial, muscles. AMM is not associated with thymic hyperplasia and responds poorly to anticholinesterase treatment. Animal models of AMM have been induced in rabbits, mice, and rats by immunization with purified xenogeneic MuSK ectodomain, and by passive transfer of large quantities of purified serum IgG from AMM patients into mice. The models have confirmed the pathogenic role of the MuSK antibodies in AMM and have demonstrated the involvement of both the presynaptic and postsynaptic components of the neuromuscular junction. The observations in this human disease and its animal models demonstrate the role of MuSK not only in the formation of this synapse but also in its maintenance.