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The Giovanni Armenise-Harvard Foundation Session 5: Proteolysis/Apoptosis/Cell Cycle Overview The topics covered in this session-protein degradation, cell cycle regulation, and apoptosis-may hold the keys to the development of better cancer therapeutics, Dr. Giulio Draetta said in his opening remarks. Unlike treatments for infectious diseases, which can be directed against features of the pathogen that aren't found in the human host, most cancer treatments must aim at molecules that are normally present. And this, of course, explains why so many anti-cancer treatments are so toxic to patients. In the future, molecular oncologists hope to have treatments that selectively kill cancer cells while leaving normal ones alone. Someday it may be possible for physicians to obtain a molecular fingerprint of the patient's cancer, then to select inhibitors that will moderate responses of the individual patient and the specific tumor. The presentations in session focus on signaling cascades that could ultimately prove relevant to the ultimate goal of finding less toxic treatments for cancer, Dr. Draetta said. He predicted that the next step will be looking for cross-talk among signaling pathways, in addition to exploring individual pathways, and that this will lead to even more ideas for high-specificity, low-toxicity treatments.
In his presentation on the first day of the symposium, Harvard's Dr. Alfred Goldberg described how cells use the ubiquitin-proteasome pathway to degrade proteins that would cause trouble if they were allowed to accumulate. Dr. Finley's work sheds more light on this crucial pathway. He used a yeast model to understand the opening and closing of the doorway through which ubiquitin-protein conjugates enter the lumen of the gigantic proteasome's 28-subunit core particle (CP). Once inside this chamber, tagged proteins are reduced to confetti. Although it would be easy to view the proteasome as a monolith, it is actually formed by the association of the CP with the 19S regulatory particle (RP), which sits over the CP channel and selects ubiquitin-conjugates for degradation. In addition, Dr. Finley's research indicates that the RP is a complex structure that fully unfolds substrates so they will fit through the 13-wide opening that leads to the CP lumen. The yeast RP contains 17 subunits, 6 of them ATPases, and when viewed with an electron microscope this structure looks like a set of jaws that open and close to admit selected proteins. The lid is an 8-subunit subcomplex which can be dissociated from yeast proteasomes in vitro. The base is also an 8-subunit complex but it cannot be separated from the CP. The base contains all 6 proteasomal ATPases, which may both join the RP to the CP and propel some of the proteasome's targets into the CP for destruction, Dr. Finley said. By experimenting with various RP mutations, he and his colleagues have determined that rpt2, one of the ATPases found in the base, is needed to open the gated channel into the CP. The base is sufficient to activate the CP for degradation of peptides, perhaps indicating that it is competent to open the channel into the CP. However, the proteasome needs the lid to recognize ubiquitin-conjugates. To determine whether the base of this assembly unfolded proteins in addition to opening the door, the researchers used citrate synthase (CS) as a model substrate. As they predicted, base ATPases acted as molecular chaperones for CS. Only after it was unfolded could CS be bound by the base, Dr. Finley said, and this reaction was independent of ubiquitin tagging. These data suggest that ubiquitin-protein conjugates are initially tethered to the proteasome via specific recognition of their ubiquitin chains, followed by a nonspecific interaction between the base and the target protein, which is coupled to unfolding and translocation of the target protein into the CP.
Mitochondria are often referred to as the "power plants" of the cell because they specialize in synthesizing ATP. The most striking feature of these organelles is that they have a double membrane, which divides them into two compartments: the intermembrane space and the matrix space in the center of the structure. Mitochondria make few proteins in-house, and for the most part they import proteins from the cytosol which form complexes with mitochondria-made proteins. Since the early 1990s, mitochondria have been under close scrutiny as regulators of apoptosis, and as potential targets for therapeutic interventions directed at accidental or programmed cell death. Some researchers suggest that the permeability transition pore (PTP) is a major player in mitochondrial apoptotic signalling. They postulate that when this high-conductance inner membrane channel expands to admit solutes, leading to tremendous swelling of the mitochondria, this may trigger release of intermembrane apoptosis-inducing factor and possibly of cytochrome c. In mechanistic terms, however, it is difficult to understand how this pore might be linked to the release of death factors by the organelle's inner membrane, Dr. Bernardi said. One barrier to understanding this process is that in vitro studies of cell-free mitochondria may not correspond well with in vivo events. Working with populations of mitochondria in suspension, Dr. Bernardi and his colleagues manipulated a variety of factors to try and mimic the in vivo opening and closing of the PTP. They found that depolarization always leads to opening of the pore, but that the reverse is not true. Further, they determined that a closed pore did not necessarily mean that the pump that drives ATP synthesis was also out of commission. More recently, the researchers devised a novel method for studying mitochondria in intact cells, which involves chemically blotting out background activity in the cell so that mitochondrial events stand out. So far it appears that depolarization can result not only from opening of the PTP, but also from increased ATP demand or calcium influx. It also appears that GD3 ganglioside opens the pore and increases the likelihood of apoptosis, Dr. Bernardi said, and this is being studied further.
In order for a cell to die or commit suicide, its mitochondrial "power plants" must be shut down. But which of the many proteins involved in intracellular apoptosis pathways delivers the fatal blow to these organelles? The answer appears to be BID, a new protein that Dr. Wagner and his colleagues have identified in the complicated Fas signal transduction pathway. BID appears to link intracellular death signals to the mitochondria, where it sets in motion a chain of events that culminates with the activation of fatal caspace enzymes. When the researchers used NMR spectroscopy to determine the structure of BID, they were surprised to find that this pro-apoptotic protein looks much like Bcl-xL, a protein known to inhibit apoptosis. Models of BID and Bcl-xL binding indicate that the two join easily in the presence of caspace-8, a death enzyme that loosens Bcl-xL's ordinarily tight structure. The complex of BID and Bcl-xL may interfere with the anti-apoptotic effects of APAF-1, which ordinarily binds with Bcl-xL. This sets the stage for lethal caspace activation that knocks out the mitochondria.
In mammalian cells, the retinoblastoma proteins (pRB) are key regulators of the cell cycle, serving as one of the main brakes on progress around the cell-division cycle. These proteins are essential for fundamental decisions about whether a cell should proliferate, differentiate, or undergo apoptosis. Of the numerous cellular proteins that interact with members of the pRB family, the best characterized are the E2F transcription factors. It is widely believed that the ability of the pRB family proteins to restrict cell proliferation depends on their ability to inhibit E2F transcriptional activity. E2Fs are important for normal cell function, and dysregulation of these proteins has many consequences. Dr. Helin and his colleagues have generated cell lines expressing E2F-1, E2F-2, and E2F-3, each fused to the estrogen receptor ligand binding domain (ER), an innovation that makes it possible to manipulate ERE2F levels with hydroxy tamoxifen. Using this system, the researchers have found that activation of all three E2Fs can relieve the mitogen requirement for entry into S phase, and activation of the E2Fs leads to a shortening of the G0-G1 phase of the cell cycle by 6-7 hours. E2F can also induce apoptosis even in cells fed growth factors that would ordinarily sustain them, Dr. Helin reported. The researchers have also demonstrated that several genes containing E2F DNA binding sites are efficiently induced by the E2Fs in the absence of protein synthesis. More recently, Dr. Helin's laboratory has identified two novel targets for E2F transcription factors, both cell-division-cycle genes. One, cdc25A, is a tyrosine phosphatase essential for the activation of certain cyclin-dependent kinases and S-phase initiation; it is also overexpressed in many tumors. The second, cdc6, is not only needed for the cell cycle to advance but also is a very sensitive marker for cell proliferation; in some cancers it may be a marker for the aggressiveness of tumor cells. In the future, E2Fs can be used to scan the human genome for additional genes important for apoptosis, cell proliferation, or DNA replication, Dr. Helin said.
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The regulatory particle of the proteasome
