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The Giovanni Armenise-Harvard Foundation Session 5: Cell Cycle, Senescence, Programmed Cell Death Overview Nothing goes on forever, including the capacity of a normal, well-nourished cell to keep dividing. When a cell becomes senescent, it stops dividing but remains metabolically active for a time, so that it gradually fades away. Programmed cell death is a dramatically different scenario, in which healthy cells act decisively to commit suicide. When senescence and programmed cell death occur at the right time and place, they are an integral part of the life and death of a normal organism. When they occur inappropriately, however, developmental abnormalities or disease can result. Senescence and programmed cell death are complex phenomena that require a veritable symphony of intracellular signals and processes. Some of the individual contributors were examined by presentations in this session. The first paper described how temperature-sensitive small molecules can be used to selectively block the transport of materials from one organelle to the next. The second concerned programmed death in plant cells, which turns out to bear a surprising resemblance to apoptosis in animal cells. The genetic control of premature senescence in acute promyelocytic leukemia was the focus of the third, and the session ended with an update on the dynamics of LDL-receptor binding.
Dr. Kirchhausen's laboratory specializes in clathrin, a protein involved in the formation of vesicles that sort and transport materials from the membrane to intracellular organelles. Several years ago, he and his colleagues used X-ray crystallography to solve the structure of clathrin. But they've had less success using yeast knock-out models to analyze its interactions with certain proteins. Chemical genetics offered them a new way to identify substances that can selectively block clathrin's biosynthetic pathway. Chemical genetics is a novel method for identifying small molecules that disrupt gene or protein function. A fluorescent microscope is used to detect activity in a sample tray with nearly 400 wells, each containing whole cells to which candidate molecules and markers have been added. Dr. Kirchhausen used a temperature-sensitive glycoprotein whose migration from ER to golgi, then from golgi to membrane, can be controlled by temperature manipulation. This enabled the researchers to visualize the activity of different chemicals at selected points in biosynthesis. Dr. Kirchhausen's team screened approximately 10,000 chemicals, and identified more than two dozen that disrupt the clathrin pathway. They found 2 chemicals that block the exit of newly-synthesized proteins from the ER, and 6 more that block exits from the golgi to the membrane. Eight others altered the structure of the golgi in various ways. The investigators were surprised to find 10 agents that could induce the formation of vacuoles in human cells, and to see that this process could be reversed without damaging the cells.
When the p53 gene functions normally as a tumor suppressor, it induces cellular senescence in response to oncogenic signals. Although the activity of the P53 protein is modulated by protein stability and post-translational modification, including phosphorylation and acetylation, exactly how the p53 gene is activated by oncogenes in the first place has been a mystery. Now Dr. Pearson and his colleagues report that a tumor suppressor gene called PML, first identified in a mouse model for acute promylocytic leukemia, acts in concert with p53 to induce senescence. This gene appears to regulate p53's response to oncogenic signals from Ras. Expression of this oncogene causes p53 to accumulate and PML expression to increase, PML over-expression acetylates p53 at lysine-382, and this makes p53 biologically active. The outcome is senescence. The researchers have also shown that Ras stimulation causes p53 and the acetyltransferase CBP to form a trimeric p53-PML-CBP complex within the nuclear bodies, a site where PML occurs even in normal cells. Further evidence for PML's role comes from knock-out experiments, which showed that PML-/- fibroblasts lose Ras-induced p53 acetylation, p53-CBP complex stability, and senescence. These data establish a link between PML and p53 and indicate that unless PML is on hand, signals from Ras will go unheard by the cell.
The hypersensitive response is a classic defense strategy of plants, in which resistance genes trigger programmed cell death (PCD) at the site of a pathogen invasion. In order to study signal transduction in this form of cell suicide, Dr. Stone and her colleagues developed a pathogen-free system they could use to trigger focal cell death in Arabidopsis. When they treated Arabidopsis with fumonisin B1 (FB1), a fungal toxin, the resulting lesions had the hallmarks of the hypersensitive response, including accumulation of phenolics, callose, and camalexin, production of reactive oxygen intermediates, and induction of pathogenesis-related gene expression. Although this model yielded cleaner data than they could have gotten using a whole pathogen, Dr. Stone's team thought they could do better still if they used an even simpler model. They switched to using protoplasts, which are plant cells stripped of their walls, grown in culture. When these cells were challenged with FB1, the resulting cell death was consistent with PCD in Arabidopsis: it was dependent on de novo transcription, translation, and protein phosphorylation. Dr. Stone also observed that salicylate-, jasmonate- and ethylene-dependent pathways contribute to FB1-induced PCD, as indicated by FB1 susceptibility of mutants. To identify other factors contributing to FB1-induced PCD, they selected FB1-resistant (fbr) mutants by sowing seeds on FB1-containing agar media. In this hostile environment, two resistant mutants, fbr1 and fbr2, were able to grow. When these mutants were challenged with a different bacterial pathogen, a type of Pseudomonas syringae pv. Maculicola that expresses the avirulence gene avrRpt2, they exhibited no resistance. However, fbr1 and fbr2 displayed enhanced resistance to an isogenic strain that did not express avrRpt2. These results indicate that this protoplast system can be used to reveal mutants with pathogen phenotypes, and suggest that triggering host PCD is a common feature of compatible plant-pathogen interactions.
The LDL receptor (LDLR) is the primary mechanism that animal cells use to take up particles of low-density lipoproteins from the blood. Healthy cells make LDL receptors and insert them into the plasma membrane when they need cholesterol for membrane synthesis. This normal process is disrupted in people with familial hypercholesterolemia (FH), who inherit defective genes for the LDLR. Because these mutated receptors cannot bind circulating cholesterol, abnormally high levels of lipoproteins accumulate in the blood and these individuals are at high risk for coronary artery disease. Many researchers have studied the amino-terminal domain of the receptor, which is responsible for binding LDL and consists of seven tandemly repeated LDL-A modules. Each LDL-A module is ~40 residues long, and contains six cysteine residues engaged in three disulfide bonds. The fifth of these modules is regarded as being most critical for LDL binding, but the specific contact points between LDL and its receptor were unknown. Dr. Blacklow's lab used nuclear magnetic resonance (NMR) imaging to take a closer look at the five-six pair (LR5-LR6) of the LDL-A module. Comparison of proton and multidimensional heteronuclear NMR spectra of individual modules to those of the module pair indicates that most of the significant spectroscopic changes lie within the linker region of the molecules, and that the cores of modules 5 and 6 have scant interaction with one another. The four-residue linker that separates the two modules is highly flexible, and its shape may be mediated by pH-dependent calcium binding. This, in turn, may involve mutations in the epidermal growth factor receptor. What this work-in-progress shows, Dr. Blacklow said, is that normal LDL binding can probably be disrupted by mutations in any of several transmembrane receptors.
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Chemical
genetics of membrane traffic
