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Leonid Chernomordik received his Ph.D. from the Frumkin Institute of Electrochemistry of the Russian Academy of Sciences in Moscow in 1979. He did postdoctoral work and then led a research group there before joining the Laboratory of Theoretical and Physical Biology (now the Laboratory of Cellular and Molecular Biophysics) of NICHD in 1991. He is now a senior investigator in this laboratory and heads the Unit on Lipid Intermediates in Fusion.
Exocytosis, protein trafficking, and viral infection
have in common the process of membrane fusion. Whereas the majority of scientists
working on fusion are concentrating on identification and characterization
of the proteins involved in diverse fusion reactions, my research goal is
to characterize the fusion pathway and to uncover the physical forces that
drive the rearrangement of two membrane lipid bilayers into one.
To understand how lipid bilayers fuse, my colleagues and I in the Frumkin
Institute studied fusion of protein-free lipid bilayers. The specific
dependence of different fusion stages on the lipid composition of bilayers
led us to develop the "stalk-pore" model of membrane fusion.
This model is based on the fact that two pairs of membrane monolayers,
contacting and distal, must each bend during fusion. We hypothesized that
first the contacting monolayers of membranes bend toward each other to
form a stalk - a local connection between membranes. Then distal membrane
monolayers come together and bend in the direction opposite to the stalk,
which opens a fusion pore whose expansion concludes fusion. The monolayer's
propensity to bend depends on the molecular shape of the lipids forming
the monolayer. By adding lipids of different molecular shapes to different
monolayers, we can manipulate different fusion stages. For instance, inverted-cone-shaped
lysophosphatidylcholine, which cannot bend into the curvature of a stalk,
inhibits stalk formation when added to contacting membrane monolayers.
The same lipid promotes pore formation when present in the distal membrane
monolayers. The effects of cone-shaped lipids such as phosphatidylethanolamine
and arachidonic acid are opposite to those of lysophosphatidylcholine.
To understand how lipid bilayers fuse, my colleagues and I in the Frumkin Institute studied fusion of protein-free lipid bilayers. The specific dependence of different fusion stages on the lipid composition of bilayers led us to develop the "stalk-pore" model of membrane fusion. This model is based on the fact that two pairs of membrane monolayers, contacting and distal, must each bend during fusion. We hypothesized that first the contacting monolayers of membranes bend toward each other to form a stalk - a local connection between membranes. Then distal membrane monolayers come together and bend in the direction opposite to the stalk, which opens a fusion pore whose expansion concludes fusion. The monolayer's propensity to bend depends on the molecular shape of the lipids forming the monolayer. By adding lipids of different molecular shapes to different monolayers, we can manipulate different fusion stages. For instance, inverted-cone-shaped lysophosphatidylcholine, which cannot bend into the curvature of a stalk, inhibits stalk formation when added to contacting membrane monolayers. The same lipid promotes pore formation when present in the distal membrane monolayers. The effects of cone-shaped lipids such as phosphatidylethanolamine and arachidonic acid are opposite to those of lysophosphatidylcholine.
To test the relevance of this model to protein-mediated fusion in biological membranes, our group at NICHD has focused on fusion of the influenza virus and baculovirus envelope membranes with the endosome membranes of their host cells at the early stages of the viral infection. If the proteins that mediate these reactions - influenza hemagglutinin and baculovirus gp64 - bend the fusing membranes to form stalk intermediates, we can make it easier or more difficult for these proteins to act by altering the lipid composition of membranes. We have now identified an early fusion stage that is dependent on the composition of contacting membrane monolayers. As suggested by our hypothesis, lysophosphatidylcholine inhibits, and arachidonic acid promotes, fusion at a stage after the refolding of viral glycoproteins into a fusion-competent conformation but before lipid mixing and fusion - pore opening.
In addition to the gp64 - and hemagglutinin-mediated fusion reactions triggered by acidification of the endosomal contents, the same lipid, lysophosphatidylcholine, inhibits Ca++ -triggered exocytosis in sea urchin eggs, GTP-gamma-S-triggered mast-cell degranulation, and GTP-dependent microsomemicrosome fusion. Thus, while these different fusion processes use different triggers, they apparently share a common trigger-independent step involving membrane merger. At this lipid-sensitive stage, fusion proteins may promote fusion by decreasing the elastic energy of stalk-like fusion intermediates.
Altering the lipid composition of membranes to
be nonpermissive for fusion allowed us to isolate the "activated"
fusion state, in which the fusion proteins remain frozen for hours in
a fusion-competent conformation. We plan to characterize this conformation,
find out the number of viral fusion proteins required for a functional
fusion machine, and identify fusion intermediates downstream of stalk
formation. I hope these studies will help us to better understand how
Ezekiel Emanuel received his M.D. from Harvard Medical School in 1988 and his Ph.D. in political philosophy from Harvard in 1989. He completed a fellowship in ethics at Harvard's Kennedy School of Government, an internal medicine residency at Boston's Beth Israel Hospital, and an oncology fellowship at the Dana-Farber Cancer Institute. He has served as a senior consultant in bioethics to the NIH Clinical Center and is the director-designate of the Clinical Center Department of Clinical Bioethics, as well as an associate professor of medical ethics at Harvard.
For more than a decade, my research has focused on care for patients at the end of life. During the first phase of this, which concentrated on living wills and advance-care directives, my wife, Linda Emanuel, and I designed and clinically evaluated an advance-care directive, called The Medical Directive, which enables an individual to stipulate medical-care choices in the event of mental incompetency.
My research then focused on requests for euthanasia and physician-assisted suicide. Surprisingly, we found that cancer patients experiencing pain were not likely to be interested in either of these interventions; indeed, they tended to find them unethical. Cancer patients with depression and psychological distress, on the other hand, were much more inclined to consider these measures.
I am now interviewing about 1,000 terminally ill patients and their family caregivers to see what factors make the dying experience easier for patients and their families and what factors make it worse. Thus far, it appears that high caregiving demands on the family - the need to provide transportation, nursing care, homemaking services, and the like - are associated with poorer outcomes, including increased depression of the patients' caregivers. Home-health-care services seem to improve the situation, especially reducing rates of caregiver depression.
On a more theoretical plane, I've been defining and elaborating important characteristics of the physician-patient relationship, such as choice, compassion, and continuity. This work has proceeded in two directions: first, I've defined different ideal types, or models, of the relationship based on different ways of relating to the patient; second, I'm considering how changes in the health- care system, especially increased managed care, are likely to affect this relationship.
This last-mentioned topic complements the most recent focus of my research: organizational and institutional ethics. With the shift of medicine from solo practitioners to organized managed-care systems, the major medical-ethical issues revolve not so much around an individual physician's problematic cases as the need to develop overarching institutional policies and procedures.
Therefore, I have been working on different types of accountability that might exist and how to enhance ethical performance of managed - care organizations. Some areas I am now working on relate to the just allocation of health-care resources within managed-care organizations and to identifying ethical criteria for physicians' financial incentives.
With respect to my work at the Clinical Center,
the Department of Clinical Bioethics is launching several clinical research
projects that will explore advance directives for research (for example,
indicating beforehand a willingness to participate in research in the
event of mental incompetence), motivations for people who participate
in Phase I studies, and incorporating respect for communities into considerations
of the ethics of research. Another major endeavor is to better define
the ethical values at stake in medical privacy and confidentiality. We
will also be looking for ethical issues affecting patient care and research
that arise in the daily work of clinicians and researchers at NIH, with
a view toward entering into collaborative theoretical and empirical research
focusing on these issues.
Allan Weissman received his M.D. degree from the Albert Einstein College of Medicine in 1981 and trained in internal medicine at Barnes Hospital in St. Louis before coming to the Cell Biology and Metabolism Branch of NICHD in 1984 as a medical staff fellow. He joined the Experimental Immunology Branch of NCI in 1989 and is now a senior investigator in the Laboratory of Immune Cell Biology, NCI.
The ability of a cell to maintain homeostasis and respond to external stimuli requires acute regulation of protein levels. Although the importance of regulated protein synthesis is taken for granted, the significance of regulated and spe cific protein degradation is just now becoming fully appreciated. The conjugation of proteins with ubiquitin and their degradation in the multicatalytic 26S proteasome plays a central role in this process. A major focus of our laboratory is the ubiquitin-conjugating system and the consequences of this modification, particularly on membrane-bound proteins.
Ubiquitination involves a cascade of enzymes known as E1 (ubiquitin-activating enzyme), E2s (ubiquitin-conjugating enzymes), and E3s (ubiquitin-protein ligases). E1 activates ubiquitin in an ATP-dependent manner. Substrate specificity for ubiquitination is conferred primarily by E3s and, to a lesser extent, by E2s.
We became interested in ubiquitination when we discovered that T-cell-antigen receptor (TCR) components are ubiquitinated in response to receptor ligation. At that time (1992), few naturally occurring ubiquitination substrates had been identified. Until our studies on the TCR, it was generally accepted that only one specific lysine within a protein is modified with ubiquitin. We demonstrated that ubiquitination occurs on multiple lysines within TCR subunits, and we recently established that this TCR modification is tyrosine kinase dependent. However, we now know that lysosomal rather than proteasomal degradation is the major route by which assembled cell-surface TCRs are degraded in response to ligand; thus, the role of ligand-induced ubiquitination is not obvious. Findings in yeast suggest that ubiquitination of cell-surface receptors serves as a targeting signal for internalization and lysosomal degradation, a possibility we are actively investigating for the TCR.
Although proteasomes do not play a substantive role in the degradation of cell-surface TCRs, ubiquitination and proteasomal degradation play an important function in determining the fate of unassembled TCR components. It has been known for some time that TCR components that are not assembled into complete receptors are degraded by an ill-defined process that has been referred to as "ER degradation," which ensures that only fully assembled receptors reach the cell surface. We recently determined that the TCR CD3-d subunit, which is largely disposed of by ER degradation, actually undergoes ubiquitination and is extracted and degraded from the ER by a coupled process that requires the catalytic activity of proteasomes. Interestingly, and unexpectedly, ER degradation of CD3-d is dependent on trimming of N-linked sugars in the ER. Our findings should provide new insights into the molecular bases by which quality control in protein folding and assembly is regulated within the central secretory system.
We have cloned and characterized a family of closely related E2 enzymes that function in the human papilloma virus' E6-mediated ubiquitination of p53, and we have characterized an E3 enzyme, Nedd-4. Also, using the yeast two-hybrid system, we have begun to identify novel binding partners for E2s and E3s in lymphocytes. Among the binding partners being characterized are a protein previously identified as a tumor suppressor and a novel E3 enzyme unrelated to any other known E3. Finally, with Michael Kuehn, NCI, we are studying the ubiquitin-conjugating system during embryogenesis.