Glyceraldehyde-3-phosphate dehydrogenase and other enzymatic activity in normal mouse lung and in lung tumors

The histogenesis of chemically induced mouse lung adenomas is currently being debated. Tumors induced by a variety of chemicals and in a number of different strains exhibit growth patterns having a solid/alveolar appearance, a papillary appearance, or a mixture of both. Ultrastructural observations suggest that solid tumors are derived from the alveolar type II pneumocyte and that papillary tumors arise from the bronchiolar Clara cell. However, recent immunocytochemical investigations have concluded that most mouse lung tumors are derived solely from the alveolar type II cell. Enzyme histochemical methods have previously been utilized to identify Clara cells in pulmonary cell isolates and also to characterize mouse lung tumors. This report demonstrates a difference in glyceraldehyde-3-phosphate dehydrogenase (G3PD) activity in type II pneumocytes and Clara cells. Solid tumors and type II cells appear to have a similar G3PD activity, and this activity is different from that observed in papillary tumors and bronchiolar cells. These findings support morphological evidence that suggests mouse lung tumors are phenotypically different and may arise from at least two different cells of origin.     

Centrosome positioning and directionality of cell movements

In several cell types, an intriguing correlation exists between the position of the centrosome and the direction of cell movement: the centrosome is located behind the leading edge, suggesting that it serves as a steering device for directional movement. A logical extension of this suggestion is that a change in the direction of cell movement is preceded by a reorientation, or shift, of the centrosome in the intended direction of movement. We have used a fusion protein of green fluorescent protein (GFP) and γ-tubulin to label the centrosome in migrating amoebae of Dictyostelium discoideum, allowing us to determine the relationship of centrosome positioning and the direction of cell movement with high spatial and temporal resolution in living cells. We find that the extension of a new pseudopod in a migrating cell precedes centrosome repositioning. An average of 12 sec elapses between the initiation of pseudopod extension and reorientation of the centrosome. If no reorientation occurs within approximately 30 sec, the pseudopod is retracted. Thus the centrosome does not direct a cell’s migration. However, its repositioning stabilizes a chosen direction of movement, most probably by means of the microtubule system.     

Bcl-xL protects adult septal cholinergic neurons from axotomized cell death

Bcl-xL suppresses apoptotic cell death induced by diverse stimuli in cell lines in vitro. To examine the mechanism by which axotomized cholinergic neurons die in vivo, lentiviral vectors expressing Bcl-xL, human nerve growth factor (hNGF), or green fluorescent protein were injected into the septum 3 weeks before transection of the fimbria fornix. Three weeks after transection, Bcl-xL- and hNGF-injected animals showed significantly higher numbers of spared cholinergic neurons compared with control (green fluorescent protein) injected animals. These results provide evidence that adult axotomized cholinergic neurons die of apoptotic death that can be prevented by local delivery of hNGF or intracellular delivery of Bcl-xL.     

Glyceraldehyde-3-phosphate dehydrogenase is required for the transport of nitric oxide in platelets.

Nitric oxide (NO) or NO-generating compounds like sodium nitroprusside (SNP) increase cellular levels of cGMP and produce S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase [GAPDH; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12]. In search of a reagent that could discriminate between these two effects, we used the sesquiterpene antibiotic koningic acid, which binds to GAPDH at the Cys-149 of the active site. Koningic acid inhibited basal and sodium nitroprusside-stimulated NAD-dependent covalent modification of purified rabbit muscle GAPDH in a dose-dependent manner. Furthermore, we tested the effect of koningic acid on human platelets. Approximately 90% of GAPDH is present in the cytosol of human platelets, and the exposure of platelet cytosol to koningic acid inhibited GAPDH activity, while the soluble guanylyl cyclase (basal and sodium nitroprusside-stimulated) activity remained unaltered. Pretreatment of intact platelets with koningic acid slowed the rate of aggregation induced by a submaximal concentration of thrombin. In addition, the antibiotic also inhibited the cGMP increases triggered by SNP, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosyndomidine (SIN-1) but failed to prevent an increase in cGMP caused by nitrosylated albumin. Under the same conditions, koningic acid also inhibited basal and SNP- SNAP-, and SIN-1-stimulated NAD-dependent modification of GAPDH and its enzymatic activity. These results suggest that the mechanism of delivery of NO from SNP, SNAP, and SIN-1 to platelets may require the active form of GAPDH. When NO is delivered by nitrosylated albumin, active GAPDH was not necessary.     

Differential ability of T cell subsets to undergo activation-induced cell death

Human T cell clones were analyzed for their susceptibility to activation-induced cell death (AICD) in response to CD3/T cell receptor ligation. AICD was observed only in Th1 clones and was Fas-mediated, whereas Th2 clones resisted AICD. Analysis of a panel of Th0 clones, characterized by their ability to secrete both Th1 and Th2 cytokines, revealed that this subset included both AICD-sensitive (type A) and -resistant (type B) clones. Resistance to AICD by Th2 and Th0-type B clones was not due to lack of expression of either Fas receptor or its ligand. Paradoxically, the AICD-resistant clones were susceptible to apoptosis when Fas receptor was directly ligated by anti-Fas antibodies. However, prior activation of the resistant clones by monoclonal antibodies to CD3/TCR complex induced resistance against Fas-mediated apoptosis. Thus, the Fas–FasL pathway is critical for the induction of AICD in T cells, and moreover this pathway can be negatively regulated in the AICD-resistant clones by signals that are generated from ligation of the CD3/TCR complex.     

Glyceraldehyde-3-phosphate dehydrogenase antisense oligodeoxynucleotides protect against cytosine arabinonucleoside-induced apoptosis in cultured cerebellar neurons.

Cytosine arabinonucleoside (AraC) is a pyrimidine antimetabolite that kills proliferating cells by inhibiting DNA synthesis and, importantly, is also an inducer of apoptosis. We recently reported that age-induced apoptotic cell death of cultured cerebellar neurons is directly associated with an over-expression of a particulate 38-kDa protein, identified by us as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). We now show that the AraC-induced neuronal death of immature cerebellar granule cells in culture is effectively delayed by actinomycin-D, cycloheximide, or aurintricarboxylic acid (a DNase inhibitor). Furthermore, two GAPDH antisense, but not their corresponding sense, oligodeoxyribonucleotides markedly arrested AraC-induced apoptosis. This protection was more effective than that induced by the above-mentioned classical inhibitors of apoptosis. Prior to AraC-induced neuronal death, GAPDH mRNA levels increased by approximately 2.5-fold, and this mRNA accumulation was blocked by actinomycin-D and the GAPDH antisense (but not sense) oligonucleotide. Like actinomycin-D, a GAPDH antisense oligonucleotide also suppressed the AraC-induced over-expression of the 38-kDa particulate protein (i.e., GAPDH), while the corresponding sense oligonucleotide was totally ineffective. Thus, the present results show that GAPDH over-expression is involved in AraC-induced apoptosis of cultured cerebellar granule cells.     

Transglutaminase-catalyzed inactivation of glyceraldehyde 3-phosphate dehydrogenase and α-ketoglutarate dehydrogenase complex by polyglutamine domains of pathological length

Several adult-onset neurodegenerative diseases are caused by genes with expanded CAG triplet repeats within their coding regions and extended polyglutamine (Q_n) domains within the expressed proteins. Generally, in clinically affected individuals n ≥ 40. Glyceraldehyde 3-phosphate dehydrogenase binds tightly to four Q_n disease proteins, but the significance of this interaction is unknown. We now report that purified glyceraldehyde 3-phosphate dehydrogenase is inactivated by tissue transglutaminase in the presence of glutathione S-transferase constructs containing a Q_n domain of pathological length (n = 62 or 81). The dehydrogenase is less strongly inhibited by tissue transglutaminase in the presence of constructs containing shorter Q_n domains (n = 0 or 10). Purified α-ketoglutarate dehydrogenase complex also is inactivated by tissue transglutaminase plus glutathione S-transferase constructs containing pathological-length Q_n domains (n = 62 or 81). The results suggest that tissue transglutaminase-catalyzed covalent linkages involving the larger poly-Q domains may disrupt cerebral energy metabolism in CAG/Q_n expansion diseases.     

Yersinia signals macrophages to undergo apoptosis and YopJ is necessary for this cell death

Pathogenic Yersinia spp. carry a large common plasmid that encodes a number of essential virulence determinants. Included in these factors are the Yersinia-secreted proteins called Yops. We analyzed the consequences of wild-type and mutant strains of Yersinia pseudotuberculosis interactions with the macrophage cell line RAW264.7 and murine bone marrow-derived macrophages. Wild-type Y. pseudotuberculosis kills ≈70% of infected RAW264.7 macrophages and marrow-derived macrophages after an 8-h infection. We show that the cell death mediated by Y. pseudotuberculosis is apoptosis. Mutant Y. pseudotuberculosis that do not make any Yop proteins no longer cause host cell death. Attachment to host cells via invasin or YadA is necessary for the cell death phenotype. Several Yop mutant strains that fail to express one or more Yop proteins were engineered and then characterized for their ability to cause host cell death. A mutant with a polar insertion in YpkA Ser/Thr kinase that does not express YpkA or YopJ is no longer able to cause apoptosis. In contrast, a mutant no longer making YopE or YopH (a tyrosine phosphatase) induces apoptosis in macrophages similar to wild type. When yopJ is added in trans to the ypkAyopJ mutant, the ability of this strain to signal programmed cell death in macrophages is restored. Thus, YopJ is necessary for inducing apoptosis. The ability of Y. pseudotuberculosis to promote apoptosis of macrophages in cell culture suggests that this process is important for the establishment of infection in the host and for evasion of the host immune response.     

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) induces cancer cell senescence by interacting with telomerase RNA component

Oxidative stress regulates telomere homeostasis and cellular aging by unclear mechanisms. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key mediator of many oxidative stress responses, involving GAPDH nuclear translocation and induction of cell death. We report here that GAPDH interacts with the telomerase RNA component (TERC), inhibits telomerase activity, and induces telomere shortening and breast cancer cell senescence. The Rossmann fold containing NAD(+) binding region on GAPDH is responsible for the interaction with TERC, whereas a lysine residue in the GAPDH catalytic domain is required for inhibiting telomerase activity and disrupting telomere maintenance. Furthermore, the GAPDH substrate glyceraldehyde-3-phosphate (G3P) and the nitric oxide donor S-nitrosoglutathione (GSNO) both negatively regulate GAPDH inhibition of telomerase activity. Thus, we demonstrate that GAPDH is regulated to target the telomerase complex, resulting in an arrest of telomere maintenance and cancer cell proliferation.     

Gravity and the orientation of cell division

A novel culture system for mammalian cells was used to investigate division orientations in populations of Chinese hamster ovary cells and the influence of gravity on the positioning of division axes. The cells were tethered to adhesive sites, smaller in diameter than a newborn cell, distributed over a nonadhesive substrate positioned vertically. The cells grew and divided while attached to the sites, and the angles and directions of elongation during anaphase, projected in the vertical plane, were found to be random with respect to gravity. However, consecutive divisions of individual cells were generally along the same axis or at 90° to the previous division, with equal probability. Thus, successive divisions were restricted to orthogonal planes, but the choice of plane appeared to be random, unlike the ordered sequence of cleavage orientations seen during early embryo development.     

Glyceraldehyde-3-phosphate dehydrogenase on the surface of group A streptococci is also an ADP-ribosylating enzyme.

We recently identified an enzymatically active glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; GAPDH) as a major protein on the surface of group A streptococci (SDH), which exhibits multiple binding activity to various mammalian proteins. We now report that the SDH molecule also functions as an ADP-ribosylating enzyme, which, in the presence of NAD, is auto-ADP-ribosylated. In a crude cell wall extract of group A streptococci, SDH is the only protein that is ADP-ribosylated. SDH found in the streptococcal cytoplasmic fraction could not be ADP-ribosylated in the presence of NAD. Treatment of ADP-ribosylated SDH with the cytoplasmic fraction removed the ADP-ribose from SDH, suggesting the presence of an ADP-ribosyl hydrolase in the cytoplasmic compartment. The covalent linkage of ADP-ribose to SDH was stable to neutral hydroxylamine, sensitive to HgCl2, and inhibitable by free cysteine, indicating that the modification was at a cysteine residue of SDH. In addition to its auto-ADP-ribosylation activity, purified SDH or streptococcal cell wall extracts were able to transfer the ADP-ribose moiety of NAD specifically to free cysteine, resulting in a true thioglycosidic linkage. Treatment of purified SDH or the crude cell wall extract with sodium nitroprusside, which spontaneously generates nitric oxide, was found to stimulate the ADP-ribosylation of SDH in a time-dependent manner. ADP-ribosylation and nitric oxide treatment inhibited the GAPDH activity of SDH. Since ADP-ribosylation and nitric oxide are involved in signal transduction events, the ADP-ribosylating activity of SDH may enable communication between host and parasite during infection by group A streptococci.     

Neurotrophin regulation of ionic currents and cell size depends on cell context

Trk receptor activation by neurotrophins is often considered to have a defined set of actions on target neurons, including supporting neuronal survival, inducing morphological differentiation, and regulating a host of target genes that specify neuronal phenotype. It is not known if all such regulatory effects are obligatory, or if some may vary depending on the cell context in which the receptors are expressed. We have examined this issue by comparing neurotrophin effects on the regulation of electrical excitability and morphological differentiation in two strains of PC12 cells. We found that while neurotrophins induced neurite extension and increased calcium currents in both PC12 cell types, sodium current levels were regulated in only one of these strains. Moreover, we found little correlation between calcium current levels and the extent of morphological differentiation when compared in individual cells of a single strain. Thus, the regulatory effects of neurotrophins on cell phenotype are not fully determined by the Trk receptors that they activate; rather, they can vary with differences in cell context that arise not only between different cell lineages, but also between individual cells of clonal relation.     

MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death

Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems. We have isolated a gene, termed MRIT, that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclX_L and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.     

Human deoxycytidine kinase is located in the cell nucleus

Human deoxyribonucleoside kinases are required for the pharmacological activity of several clinically important anticancer and antiviral nucleoside analogs. Human deoxycytidine kinase and thymidine kinase 1 are described as cytosolic enzymes in the literature, whereas human deoxyguanosine kinase and thymidine kinase 2 are believed to be located in the mitochondria. We expressed the four human deoxyribonucleoside kinases as fusion proteins with the green fluorescent protein to study their intracellular locations in vivo. Our data showed that the human deoxycytidine kinase is located in the cell nucleus and the human deoxyguanosine kinase is located in the mitochondria. The fusion proteins between green fluorescent protein and thymidine kinases 1 and 2 were both predominantly located in the cytosol. Site-directed mutagenesis of a putative nuclear targeting signal, identified in the primary structure of deoxycytidine kinase, completely abolished nuclear import of the protein. Reconstitution of a deoxycytidine kinase-deficient cell line with the wild-type nuclear or the mutant cytosolic enzymes both restored sensitivity toward anticancer nucleoside analogs. This paper reports that a deoxyribonucleoside kinase is located in the cell nucleus and we discuss the implications for deoxyribonucleotide synthesis and phosphorylation of nucleoside analogs.     

Telomere lengthening and telomerase activation during human B cell differentiation

The function of the immune system is highly dependent on cellular differentiation and clonal expansion of antigen-specific lymphocytes. However, little is known about mechanisms that may have evolved to protect replicative potential in actively dividing lymphocytes during immune differentiation and response. Here we report an analysis of telomere length and telomerase expression, factors implicated in the regulation of cellular replicative lifespan, in human B cell subsets. In contrast to previous observations, in which telomere shortening and concomitant loss of replicative potential occur in the process of somatic cell differentiation and cell division, it was found that germinal center (GC) B cells, a compartment characterized by extensive clonal expansion and selection, had significantly longer telomeric restriction fragments than those of precursor naive B cells. Furthermore, it was found that telomerase, a telomere-synthesizing enzyme, is expressed at high levels in GC B cells (at least 128-fold higher than those of naive and memory B cells), correlating with the long telomeres in this subset of B cells. Finally, both naive and memory B cells were capable of up-regulating telomerase activity in vitro in response to activation signals through the B cell antigen receptor in the presence of CD40 engagement and/or interleukin 4. These observations suggest that a novel process of telomere lengthening, possibly mediated by telomerase, functions in actively dividing GC B lymphocytes and may play a critical role in humoral immune response by maintaining the replicative potential of GC and descendant memory B cells.