Short-lived alpha-helical intermediates in the folding of beta-sheet proteins.
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Several lines of evidence point strongly toward the importance of highly alpha-helical intermediates in the folding of all globular proteins, regardless of their native structure. However, experimental refolding studies demonstrate no observable alpha-helical intermediate during refolding of some beta-sheet proteins and have dampened enthusiasm for this model of protein folding. In this study, beta-sheet proteins were hypothesized to have potential to form amphiphilic helices at a period of <3.6 residues/turn that matches or exceeds the potential at 3.6 residues/turn. Hypothetically, such potential is the basis for an effective and unidirectional mechanism by which highly alpha-helical intermediates might be rapidly disassembled during folding and potentially accounts for the difficulty in detecting highly alpha-helical intermediates during the folding of some proteins. The presence of this potential was confirmed, indicating that a model entailing ubiquitous formation of alpha-helical intermediates during the folding of globular proteins predicts previously unrecognized features of primary structure. Further, the folding of fatty acid binding protein, a predominantly beta-sheet protein that exhibits no apparent highly alpha-helical intermediate during folding, was dramatically accelerated by 2,2,2-trifluoroethanol, a solvent that stabilizes alpha-helical structure. This observation suggests that formation of an alpha-helix can be a rate-limiting step during folding of a predominantly beta-sheet protein and further supports the role of highly alpha-helical intermediates in the folding of all globular proteins.
Published Version (Please cite this version)
Chen, E, ML Everett, ZE Holzknecht, RA Holzknecht, SS Lin, DE Bowles and W Parker (2010). Short-lived alpha-helical intermediates in the folding of beta-sheet proteins. Biochemistry, 49(26). pp. 5609–5619. 10.1021/bi100288q Retrieved from https://hdl.handle.net/10161/4008.
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The increasing number and the improvement in the success of heart and lung transplantation in recent years make now the most exciting time to be involved in research related to the scientific issues surrounding this field. Among those issues, two problems are currently recognized to be the major impediment to the optimal application of transplantation in patients with end-stage cardiopulmonary disease. First, there is a lack of consistent long-term graft survival, which is constrained by the current immunosuppressive regimen and its side effects; compared to other solid organ transplants, lung and heart-lung allografts are particularly susceptible to this problem. Second, there is a dire shortage of donor organs; although this problem is especially pronounced in lung and heart-lung transplantation, it is also seen in transplantation of other organs. The Duke Cardiopulmonary Transplantation Laboratory addresses both of these problems and examines the factors associated with chronic allograft failure as well as the approaches aimed at circumventing the donor organ shortage.
Clinical lung transplantation has only recently begun to consistently enjoy some short-term success. Compared to other solid organ transplants, however, the long-term survival is still limited. For example, the 5-year patient survival rate for primary transplants, according to the October 2004 Organ Procurement and Transplantation Network data, is only 43.9% for pulmonary allografts and 70.6%, 70.3%, and 67.2% for cardiac, cadaveric renal, and hepatic allografts, respectively. Chronic rejection, specifically in the form of bronchiolitis obliterans, appears to be the predominant factor contributing to the poor long-term survival of lung transplant recipients.
Despite the less than optimal long-term outcome today, there has undoubtedly been a gradual improvement in the overall success of lung transplantation in recent years; and with this success comes more demand for organ donors. For example, the waiting list for heart transplantation in October of 2004 is just under 3400 long while that for lung transplantation is over 3900. Furthermore, the median waiting time for lung transplantation during 2001-2002 ranges from 636 to 834 days, depending on the recipient ABO blood type.
Focus of Scientific Research
In searching for solutions to these problems, our laboratory now focuses on three main areas of scientific investigation within the field of cardiopulmonary transplantation:
(1)Mechanisms underlying the chronic rejection, especially that of lung and heart-lung allografts,
(2)Induction of immunologic tolerance to reduce the morbidity and improve the long-term survival of heart and lung transplantation, and
(3)Xenotransplantation, with the ultimate goal of alleviating the problem of donor organ shortage but the more immediate goal of gaining general knowledge about transplantation immunobiology using this exciting experimental model.
Our current work in these three areas is described briefly below:
(1) Chronic Rejection of Pulmonary and Cardiopulmonary Allografts
Chronic rejection can occur months to years after a transplant when a graft escapes hyperacute and acute rejection, which are typically mediated by humoral and cellular components, respectively. For pulmonary allografts, chronic rejection is typically manifested histologically in the form of bronchiolitis obliterans, a process in which the small airways are severely narrowed from subepithelial fibrosis.
The incidence of bronchiolitis obliterans in lung and heart-lung transplant patients has remained relatively constant despite the development of newer therapeutic agents that successfully prevent alloimmunity in other organ transplants. This observation suggests that factors unique to the lung and perhaps not strictly immune-mediated may adversely affect the survival of pulmonary allografts.
One important characteristic of lung transplantation is that the pulmonary allograft might be constantly exposed to inhaled or aspirated exogenous materials. There is extensive evidence in the literature suggesting that lung injury can be caused by repetitive aspiration episodes as a result of gastroesophageal reflux. It can be hypothesized that such an injury could result in the stimulation and augmentation of the recipient's immune response against the pulmonary allograft. Consistent with this idea, clinical studies from our institution have demonstrated that gastroesophageal reflux disease is a contributing factor to pulmonary allograft dysfunction and suggested that treatment of gastroesophageal reflux disease with fundoplication could abrogate bronchiolitis obliterans.
Despite the clinical evidence linking gastroesophageal reflux disease to chronic rejection in lung transplantation, scientific studies in this area are lacking. One of the projects ongoing in our laboratory is to use an experimental animal model to investigate the histologic, immunologic, and physiologic effects of chronic aspiration, as it might occur in gastroesophageal reflux disease, on pulmonary allografts and to study the pathogenesis of reflux-induced pulmonary allograft dysfunction.
(2) Immunologic Tolerance
Clinical transplantation is currently feasible because of the medications that intentionally lead to immunosuppression of the recipient. Although new immunosuppressive drugs have been produced and are continuing to surface over the past few decades, there are still significant problems with these new drugs, particularly related to infections and toxicity from suppression of the overall immune system. To a transplant clinician, the ideal scenario would be for the recipient to achieve a state of immunologic tolerance, a condition in which the recipient’s immune system, after only a brief period of treatment around the time of transplantation, remains completely competent to all immunologic stimuli except for those from the allograft. Thus, immunologic tolerance is a specific response, and, by definition, there is little risk in weakening the recipient’s immune system to the point where routine infectious agents cannot be overcome.
There are two reasons why we believe that the potential application of immunologic tolerance needs to be critically investigated for lung transplantation today. First, for patients suffering from end-stage pulmonary disease, there is no other comparable alternative therapy, such as ventricular assist device in heart failure or dialysis in renal failure; induction of tolerance would minimize the morbidity associated with the conventional immunosuppression currently being used in clinical transplantation. Second, immunologic tolerance, by definition, could minimize the problems with chronic rejection and reduce the need for more donor organs for repeat transplants, thus alleviating the shortage of donor organs. Third, living-related transplantation has become a real option for lungs in recent years, and induction of tolerance in this subset of transplant recipients, because of their genetic similarities to the donors, is theoretically more likely to be achieved.
One of the most daunting problems with clinical transplantation today is the shortage of donor organs. In terms of the numbers, the most definitive solution would be to bring forth the clinical application of xenotransplantation, or transplantation of an organ from one species to another. The use of organs from the pig would especially be suitable, not only because of the abundance in the number of this species, but also because of its appropriate size match with typical human adults as well as our ability to genetically manipulate this animal with the biotechnology available today. However, the degree of biological incompatibility still prohibits us from successfully using pig organs for clinical xenotransplantation. Some of the factors leading to this biological incompatibility relate to immunologic differences, and others appear to be more non-immunologic, especially in pulmonary xenotransplantation.
Using a pig-to-non-human primate pulmonary transplantation model, our laboratory has been actively investigating these immunologic and non-immunologic reasons that are contributing to the demise of these lung xenografts. In particular, we have been focusing on complement-mediated effects as well as issues of incompatibility in coagulation factors that appear to be more pronounced in pulmonary xenotransplantation. As in the projects dealing with immunologic tolerance in the allotransplant model, the potential use of tolerance induction in the setting of xenotransplantation is also being examined in our laboratory. There is no question that the ultimate and ideal goal of research in xenotransplantation is to apply this concept in the clinical arena; however, even without achieving that goal, successful laboratory research in this area would certainly provide new knowledge and advances, not just in transplantation and endothelial cell biology but also in biotechnology in general.
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