|TH E N I H C A T A L Y S T||JU L Y A U G U S T 2008|
Susan Buchanan received her Ph.D. from the Johann-Wolfgang-Goethe Universität in Frankfurt, Germany, in 1990. She completed postdoctoral fellowships at the MRC Laboratory of Molecular Biology, Cambridge, U.K., and at the University of Texas Southwestern Medical School Southwestern Medical Center, Dallas, before returning to the U.K. to establish a research group at Birkbeck College, London, in 1998. She joined NIDDK as an investigator in 2001 and is currently a senior investigator in the Laboratory of Molecular Biology, NIDDK.
My group uses X-ray crystallography to study the structures of integral membrane proteins. About 30 percent of the human genome codes for membrane proteins, and a similar distribution is found in lower organisms. Membrane proteins are difficult to work with, however, due to their low abundance in cells and to their preference to reside in a lipid bilayer. Although a large number of pharmaceutical targets are membrane proteins, they currently represent less than 1 percent of all solved protein structures.
We study transporters embedded in the outer membranes of Gram-negative bacteria, which are surface accessible and therefore have the potential to be good vaccine or drug targets against infectious diseases.
One area of particular interest is the transport of small molecules and large proteins across the outer membrane by a single family of membrane proteins. We focus on iron transporters from several bacterial pathogens. Iron is essential for bacterial proliferation: If iron uptake could be blocked, an infection could be eradicated.
So far, our structures have shown how iron transporters specifically recognize Fe3+ bound to small molecules such as enterobactin (a siderophore synthesized by Escherichia coli) and citrate. Each transporter has a unique binding pocket for its preferred small molecule. When the correct substrate binds, the transporter undergoes conformational changes that send a signal across the outer membrane and prepare the system for transport.
However, transport into the periplasm is complicated and involves another protein complex and energy in the form of protonmotive force. We are still working to understand the actual transport process.
Even without knowing exactly how they function, we believe that these iron transporters may make good vaccine or drug targets because they are surface exposed and often antigenic. We are currently testing this idea using an iron transporter from Yersinia pestis. Y. pestis causes plague, and deletion of the gene encoding an iron transporter abolishes virulence in a mouse model of bubonic plague. We recently solved the structure of the Y. pestis iron transporter in two states, alone and in complex with its cognate Fe3+-siderophore.
These structures allowed us to precisely define the binding pocket for the substrate. The next step is to use computational methods to screen for small molecules that effectively compete with the natural substrate for binding. This could lead to the design of novel antibiotics.
We also are collaborating with Joe Hinnebusch (NIAID), who is evaluating this protein and several others for a protective immune response in rat and mouse models of bubonic plague. We hope that this work will identify new vaccine targets.
Recently, we extended our work on small-molecule transporters to ask how proteins are ferried across the outer membrane. Some of the iron transporters that we study also facilitate the uptake of large protein toxins called colicins. Whether the transport mechanism is the same as found for small molecules or entirely different, we hope that our crystal structures will suggest answers.
We also have begun to study protein export through collaboration with Harris Bernstein (NIDDK). Together we have solved the structure of the transporter domain of an autotransporter from O157:H7 E. coli. This protein forms a transport channel similar to those found in iron transporters, but the secreted protein domain is very large. Exactly how it gets to the bacterial cell surface is still a mystery.
Our next major goal is to solve outer membrane protein structures from mitochondria. Most proteins residing in mitochondria are nuclear encoded and must be imported through a general protein-import channel. We aim to solve structures of this channel and others to see how similar these proteins are to bacterial outer membrane proteins, and how the passage of proteins across membranes varies between these systems.
Ramanujan S. Hegde received his M.D. and Ph.D. in 1999 from the University of California, San Francisco. After three years at the National Cancer Institute as an NCI Scholar, he joined the Cell Biology and Metabolism Program of NICHD in 2002, where he is currently a Senior Investigator heading the Protein Biogenesis Section.
Cells have many thousands to millions of individual proteins that must be folded, processed, assembled and localized correctly to maintain normal organismal physiology. Conversely, defective protein maturation and trafficking cause a wide range of diseases ranging from cystic fibrosis to neurodegenerative disorders. Our laboratory is interested in the basic problem of how protein maturation normally occurs, how these events are regulated, and how misregulation leads to cellular dysfunction and disease. We are focused on four intertwined aspects of secretory and membrane protein metabolism.
Translocational regulation. A decisive step in the maturation of secretory and membrane proteins is their entry, or translocation, into the endoplasmic reticulum (ER). We have discovered that protein translocation is under regulatory control for at least two important reasons. In some cases, regulating translocation allows a single protein to have multiple locations — for example, the ER and cytosol-where it can serve independent functions. In other cases, translocation is regulated to limit the entry of certain proteins into the ER, for example, during ER stress when maturation capacity is limited. We are currently studying both mechanistic and physiologic aspects of this newly emerged field of translocational regulation.
Quality control. Our discovery of translocational regulation has raised a previously unappreciated question: What happens to secretory and membrane proteins that fail to be segregated into the ER? Because these proteins are often quite hydrophobic, they represent a high risk for aggregation and inappropriate interactions. Indeed, we have discovered that they can contribute to the propagation of cytosolic protein aggregates that typify various neurodegenerative diseases. Thus, the pathway for the selective recognition and degradation of non-translocated secretory and membrane proteins is critical for normal cellular homeostasis. We are now identifying the machinery for selective recognition and degradation of these non-translocated proteins, and anticipate their important role in diseases of protein misfolding and aggregation.
Membrane protein insertion. Insertion of proteins into biological membranes is vital to all organisms. Although most membrane proteins utilize a highly conserved insertion pathway discovered over 25 years ago, others are inserted by yet unknown pathways. We have recently identified a novel ATPase that functions in insertion of "tail-anchored" membrane proteins. Tail-anchored proteins are found on essentially all cellular membranes in every organism and have diverse functional roles ranging from intracellular trafficking to regulation of cell death. Thus, this novel membrane insertion pathway is of broad biological importance for the cell. We are now taking various approaches to identify additional factors in this pathway and to define their mechanistic and physiologic roles in membrane protein insertion.
Neurodegeneration mechanisms. Neurodegeneration is the most common pathologic consequence of aberrant protein folding and metabolism. However, the pathways leading from misregulated protein metabolism to neuronal dysfunction are very poorly understood. We are applying insights from our mechanistic studies on secretory and membrane protein biogenesis to neurodegenerative diseases caused by the prion protein. We discovered that some of these diseases are a direct consequence of altered prion protein translocation into the ER. We are finding that exposure of what is normally a cell surface protein to the cytosol may cause disease via inappropriate interactions with cytosolic proteins involved in regulating lysosomal trafficking. In parallel studies, we showed that other disease-causing mutations in the prion protein may cause lysosomal dysfunction directly by its inappropriate intracellular trafficking. We are now identifying the specific pathways of mutant prion protein trafficking, and the cellular consequences of its mislocalization.
We anticipate that our laboratory's efforts will help elucidate the machinery and mechanisms for several basic cellular pathways of secretory and membrane protein metabolism. These insights will have direct implications for our understanding of various protein misfolding diseases, eventually leading to new therapeutic strategies.
Paolo Lusso received his medical degree from the University of Turin, Italy, and his Ph.D. from the Ministry of Scientific and Technologic Research, Rome. He came to NIH for the first time in 1986 to work at the NCI Laboratory of Tumor Cell Biology. He returned to Italy in 1994, where he became Chief of the Laboratory of Human Virology at the San Raffaele Scientific Institute in Milan and Associate Professor of Infectious Diseases at the University of Cagliari. In 2004, he was elected Member of the European Molecular Biology Organization (EMBO). In March 2008, he was appointed Senior Investigator at the NIAID Laboratory of Immunoregulation, where he heads the Unit of Viral Pathogenesis.
My research focuses on understanding the mechanisms of viral pathogenesis, with the aim of developing novel strategies for the control and prevention of viral infections. Specifically, my interest has been concentrated on herpesviruses, in particular human herpesvirus 6 (HHV-6) and HIV.
In 1995, my research group was the first to establish a connection between the fields of HIV and the chemokine system with the discovery that three chemokines of the CC family (RANTES, MIP-1α and MIP-1β) act as specific endogenous inhibitors of HIV-1. This discovery, along with the subsequent identification of specific chemokine receptors as critical components of the HIV receptor complex, has led to the elucidation of several aspects of AIDS pathogenesis and opened new perspectives for the development of effective therapies and vaccines.
The identification of host factors that control HIV infection in vivo and thereby influence the natural course of HIV infection remains one of the major goals of my research. In fact, although RANTES, RANTES, MIP-1α and MIP-1β represent major components of the anti-HIV activity produced by different cells of the immune system, several lines of evidence point to the existence of additional, still unrecognized, suppressive factors, which may play an important role in the in vivo control of HIV replication, particularly in subjects with long-term non-progressive infection.
I am currently using an integrated approach, combining classic protein purification methods with state-of-the-art transcriptomics and proteomics analyses, to identify the nature of novel HIV-suppressive factors produced ex vivo by stimulated immune cells.
A further step following the identification of new suppressive factors will be the characterization of their mechanism of action and their potential clinical relevance in HIV transmission and disease progression. This will involve extensive analysis of clinical samples to establish a correlation between the in vivo levels of the factors and the pace of disease progression, as well as the search for genetic polymorphisms linked to the variable clinical course of the disease in different individuals.
Endogenous suppressive factors such as CCR5-binding chemokines are believed to play a role also in determining the in vivo evolution of HIV-1, with the so-called "phenotypic switch" from the prevalent CCR5-tropic viral variants to the less frequent CXCR4-using variants, which typically emerge during the late stages of disease and are insensitive to inhibition by RANTES. I plan to continue investigating the mechanisms that restrict the early in vivo emergence of CXCR4-using HIV-1 strains, as well as those that eventually promote or allow their emergence, albeit belated.
Another major goal of my research is to develop novel strategies for the therapy and prevention of HIV infection. Over the past decade, with my collaborators in Italy, I have attempted to design specific HIV-1 inhibitors targeting CCR5, the chemokine receptor used for entry by the vast majority of wild-type HIV-1 isolates. Based on our previous identification of the primary determinants of CCR5 recognition and HIV-1 blockade in RANTES, we have rationally designed short synthetic peptides that block HIV-1 entry at low nanomolar concentrations while exerting no agonistic effects on CCR5.
I plan to continue investigation of the structure-function relationships in RANTES and related chemokines, with the aim of defining more precisely the receptor-ligand interface. While further molecular refinement of our biologically active peptides is in progress, we are currently exploring potential strategies for their in vivo delivery both as systemic therapeutics and as topical microbicides.
Maria I. Morasso received her Ph.D. from the Instituto Venezolano de Investigaciones Cientificas (IVIC) in Caracas, Venezuela, in 1991. She was a postdoctoral fellow in the NICHD Laboratory of Molecular Genetics, led by Thomas Sargent, where she developed an interest in developmental skin biology. In 2000, she joined NIAMS as a tenure-track investigator. She is currently a senior investigator and head of the NIAMS Developmental Skin Biology Section.
My lab explores how epidermal cells differentiate and ectodermal appendages (hair, teeth) form during embryonic development. My research has focused in characterizing the regulation and function of the Dlx homeobox transcription factor, a member of the murine Dlx family, with essential roles in epidermal, osteogenic and placental development.
The importance of Dlx3 in the patterning and development of ectodermal structures derived from epithelial-mesenchymal interactions during embryogenesis (i.e. tooth, hair) is corroborated by the effects of DLX3 mutations in patients with the autosomal dominant Tricho-Dento-Osseous (TDO) syndrome.
Anomalies in epithelial-mesenchymal-derived organs are characteristics of a group of human heritable pathological disorders defined as ectodermal dysplasias (EDs). DLX3 is among the few genes for which mutations have been linked directly with EDs.
We have developed inducible and knockin mouse models to pursue studies on the effects of the mutant protein in hair, bone (intramembraneous and endochondral) and tooth development. We are also performing analysis of conditional knockout lines to elucidate the signaling and regulatory pathways requiring normal morphogenesis of these tissues during embryogenesis.
David M. Wilson, III, received his Ph.D. from Loyola University of Chicago in 1993 and performed his postdoctoral training at the Harvard School of Public Health. In 1997, he became a Senior Biomedical Scientist at Lawrence Livermore National Laboratory in the Biology and Biotechnology Research Program. He started at NIA as a tenure-track investigator in the Laboratory of Molecular Gerontology (LMG) in 2002.
My laboratory focuses on elucidating the molecular mechanisms of the base excision DNA repair (BER) pathway and delineating the contribution of core and auxiliary BER proteins to disease manifestation, therapeutic agent responsiveness and aging.
The free radical theory of aging proposes that the gradual accumulation of macromolecular oxidative damage over the lifespan of an organism leads to a gradual decline in cellular function and eventual death. It is our hypothesis that deficits or a decrease in the repair of oxidative DNA damage will translate into premature aging phenotypes and age-related disease. Evidence supporting the idea that genome surveillance systems are a major factor in adetermining longevity and cell functionality comes from studies of model organisms and human segmental progerias (e.g. Werner and Cockayne syndrome). In addition, defects in DNA damage responses have been causally linked to the age-associated diseases, cancer and neurodegeneration.
Many types of lesions are formed via attack of reactive oxygen species of DNA, with the most prominent being base modifications (e.g. 8-oxoguanine), abasic sites and single-strand breaks harboring non-conventional 3' or 5' termini. If unrepaired, these damages can promote cellular dysfunction or genetic instability. BER is the primary pathway for coping with spontaneous, oxidative and alkylative DNA damage. Using basic molecular and biochemical approaches, we have determined how specific human, core BER proteins recognize and process target lesions and/or coordinate with other components of the pathway. This research has centered largely on apurinic/apyrimidinic endonuclease 1 (APE1), the major mammalian repair protein for abasic sites in DNA, and x-ray cross-complementing 1 (XRCC1), a key non-enzymatic scaffold protein for the efficient operation of single-strand break processing.
Current efforts on APE1 revolve around (i) determining which of its many identified biochemical activities are biologically important using strategic knockdown and complementation strategies, (ii) evaluating the role of APE1 (and BER more broadly) in clinical DNA-damaging agent resistance, and (iii) assessing the potential relationship of reduced BER capacity to disease development using established and in-development biochemical repair assays and defined population sets.
Studies centered on XRCC1 involve (i) elucidating the role of XRCC1 (and more broadly single-strand break repair) in oxidative stress resistance in non-dividing, neuronal cells, given the recent linkage of defects in single-strand break processing to inherited spinocerebellar ataxias, (ii) evaluating the involvement of XRCC1 deficiency on age-related pathologies using a heterozygous mouse model, and (iii) determining the contribution of XRCC1 to DNA damage responses, genome stability and telomere maintenance.
Finally, Cockayne syndrome (CS) is a rare, autosomal recessive disorder characterized by growth failure, impaired development of the nervous system, cutaneous photosensitivity and premature aging. Recent studies indicate that the pathophysiology of CS might arise, at least in part, due to a defect in the repair of endogenous DNA damage. My group, in collaboration with Dr. Vilhelm Bohr in LMG, recently identified a novel interaction with the CS complementation group B (CSB) protein and APE1. Efforts are now underway to delineate the biochemical roles of CSB and its precise molecular involvement in the BER response as an auxiliary factor.