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Schnitzer Lab

Our research focuses on understanding the role of vascular endothelium and especially its surface proteins and transport vesicles in normal and pathological processes. Blood vessels are lined with a thin monolayer of specialized cells called vascular endothelium that forms a key biological interface and critical barrier controlling the exchange of circulating blood molecules, nutrients, cells and even drugs from the blood to the internal compartments and cells of the tissue. The molecular transport across this tissue-blood interface occurs predominantly within the smallest blood vessels called microvessels or capillaries and is critical for the normal growth, maintenance and survival of all tissues of the body. Abnormal transvascular exchange contributes to organ dysfunction, tissue cell death, and the pathogenesis of many cardiovascular diseases such as atherosclerosis and complications of diabetes. Furthermore, angiogenesis requires the migration and proliferation of endothelial cells and is mandatory for the growth and survival of most solid tumors.

This laboratory uses a combination of molecular and cellular biology, genomics, proteomics, and in vivo imaging techniques to analyze the role of the vascular endothelium in health and disease. We focus primarily on the luminal endothelial cell surface and its caveolae to better understand how they regulate vascular wall barrier function, vesicular trafficking, signaling and mechanotransduction, as well as how to exploit these functions to deliver antibodies, drugs, nanoparticles, and even gene vectors in a tissue- and disease specific manner. Our research can be divided into 5 broad, synergistic projects. Together, this research promises to advance basic understanding of vascular biology in vivo as well as produce powerful new therapies that can be used to prevent, diagnose, image, and treat single-organ diseases and cancer.

Proteomic Mapping: Vascular endothelial cells have proven difficult to characterize in vivo because they are only a tiny percentage of the entire tissue. Additionally, these cells are extremely sensitive to the local tissue microenvironment and rapidly dedifferentiate in vivo, making it essential to define their protein complement and explore function in vivo. We were the first to isolate endothelial plasma membranes and caveolae directly in normal and tumor tissues and now can reliably isolate high quality endothelial cell preparations in vivo from healthy and diseased organs in humans, mice, and rats.

This laboratory has developed, analyzed, and published a comprehensive map of the proteins found at the endothelial cell surface of pulmonary vasculature. Because the endothelium forms a vital interface between the blood and lung tissue, this proteome will undoubtedly provide new insight into cell function in vivo as well as yield new targets for tissue- and disease-specific delivery. Our data show that each organ presents a unique constellation of proteins. For several proteins and proteins family, localization at the cell membrane is a novel finding. Though rodents and humans are quite similar, species differences can be prevalent. Therefore, we also plan to extend characterization to human organs. Ultimately, this research will produce a whole-body proteomic map and allow us to target each organ individually.

Caveolae Function in Health and Disease: A distinctive feature of continuous endothelium is the prevalence of caveolae which are plasmalemmal invaginations at the cells surface. We have developed a novel process for isolating highly purified preparations of luminal endothelial cell plasma membranes directly from tissue and then subfractionating these membranes to further purify their caveolae. Using this technology, we have discovered that, similar to other vesicular carriers, caveolae contain the vSNARE apparatus for specific docking and fusion with target membranes. We have also shown that caveolae can bud directly from the cell surface to form free transport vesicles via a process requiring dynamin and GTP hydrolysis. Thus, caveolae do indeed function in vesicular trafficking, including receptor-mediated endocytosis of blood-borne molecules into and even across the endothelium.

The organization of signaling molecules in distinct subcompartments or microdomains at the cell surface may be very important for signal transduction and even mechanotransduction (signal stimulation in cells by mechanical stressors such as fluid shear). We and others find that caveolae play an important role in the efficient propagation of cell surface signaling cascade into the cell by compartmentalizing specific signaling molecules on the cell surface. We have discovered that mechanotransduction occurs very rapidly in vivo at the luminal endothelial cell surface in tissue and that caveolae appear to function as the "acute flow or mechanical sensor" on the endothelial cell surface. Caveolae contain key regulatory molecules including eNOS, select G proteins, Src-like kinase, receptor tyrosine kinases and various other signaling molecules. We are continuing to investigate the molecular mechanisms regulating function of caveolae with a primary emphasis now on how caveolae integrate signaling with vesicular transport.

Perhaps most importantly, we have shown that caveolae at the surface of endothelial cells can act as active pumps to transport molecules out of the blood and into the tissue, even against a concentration gradient. Antibodies targeted against caveolar proteins are also pumped across the endothelium and into the underlying tissue. This process occurs in both healthy and tumor tissue. If antibodies are radiolabeled, this can lead to the complete focal destruction of the tumors. Thus, caveolae provide a novel pathway to deliver imaging agents, nanoparticles, biologics, drugs, and even gene vectors to specific tissues in vivo.

Tissue- and Disease-Specific Markers: Tissue-specific targeting has long been a goal of molecular medicine. However, most current antibody targeted strategies have focused on identifying specific targets expressed by cells deep within tissue, and therefore generally fail in vivo because <0.01% of the intravenously-injected dose reaches the target tissues. Small drugs present an alternative to relatively large antibodies because they can much more readily enter nearly all tissues. As a result, these agents are rapidly diluted, cleared from the blood, and excreted. For both large and small molecules, it appears that only a small proportion of the injected dose actually reaches the inside of tissue where they can be effective. Thus, higher doses must be administered to reach effective levels within the diseased tissue, increasing the chance of severe systemic side effects.

Comprehensively identifying the proteins present at the endothelial cell surface has identified key tissue- and disease-specific proteins that facilitate in vivo targeting of antibodies and attached cargos. Some of these proteins are in direct contact with the blood, and thus inherently accessible. Solid tumors appear to act as a unique type of tissue with its own distinctive molecular signature. Targeting lung-specific caveolar proteins provides a portal into the underlying tissue. This rapid, tissue-specific vascular targeting and transcytosis has numerous therapeutic implications. Therapeutic and imaging agents can be rapidly delivered to a specific tissue, decreasing harmful side effects through interaction with other tissue. Drugs are effectively concentrated such that lower doses might be needed. Active pumping of antibodies across the endothelial cell layer and into tissue may also prevent degradation and increase the therapeutic potential.

We are currently investigating the functional role that tissue and tumor-specific markers play in health and disease. This will allow us to identify new therapeutic targets as well as better understand the processes underlying tumor progression. We have extensively shown that we can target the lungs and solid tumors in animal models. We must now apply these findings to humans. We know these proteins targets exist in humans, that they’re found in caveolae, and that they show the same specificity as in rodents. If caveolae themselves function in humans as they do in rodents, then the research we have done can be rapidly applied to human to provide powerful new treatments. The health impact of this is almost unlimited. By targeting healthy tissue, we can prevent disease, provide protection against assault, image function in vivo, and image pathological changes. By targeting diseased tissue, we can treat disease specifically with far less chance of systemic side effects. We can also image disease progression in vivo without the need for invasive procedures. To better understand the strength and limitations of the antibody-based delivery of therapeutics, we are developing and testing a number of novel cancer therapeutics. We have also begun a number of pilot projects to investigate utility in different pulmonary disease models. Ultimately, we hope to use these antibodies to target treatment to human infectious, genetic, and acquired lung and heart diseases.

Nanoparticle Development: Nanoparticles are tiny engineered particles that can serve as molecular complexes to deliver cell-penetrating agents for the study of disease pathways, for the imaging of tissue mass and disease progression, and for tissue-specific drug delivery. Because these particles can be engineered and optimized independently from any single drug, they can be applied to a wide range of therapies. In spite of the clear theoretical power of nanoparticles, several major roadblocks have thus far prevented broad application of nanoparticles to both clinical medicine and basic research. Roadblocks include nonspecific effects, rapid uptake by the reticulo-endothelial system, and a lack of fundamental biological awareness of how these particles interact with cells and tissues in vivo. To be most effective with minimal side effects, nanoparticles need a portal into cells. Without this access, only a fraction of the potential of nanoparticles will ever be realized because the reticulo-endothelial system traps most of the injected dose so that little nanoparticle ever reaches the target.

Targeting caveolar proteins is a novel strategy that can deliver nanoparticles to specific tissues or disease states and also provide a route across the endothelium and into tissue. We have developed a plethora of multifunctional nanoparticles to help define caveolae-based transport, including dendrimers, quantum dots, liposomes, and gold-, silver-, and silica-based nanoparticles. To be able to rapidly test novel antibodies as well as novel nanoparticles in vivo, we have recently developed a method to self-assemble biotinylated antibodies into nanoparticles we call nanostreptabodies. The first step in this process is creating enzymatically biotinylated antibodies. Though chemical biotinylation is common practice and simple, one has no control over the degree or location of biotinylation. We have overcome this problem by incorporating a genetically encoded biotinylation site in antibodies or antibody fragments. Using a nucleus of avidin, these antibodies can self-assemble with other biotinylated compounds. This nanostreptabody system can be readily expanded to attach targeting antibodies to biotinylated nanoparticles. This allows different antibodies and different nanoparticles to be rapidly tested in vivo. This linkage system forms the basis for all of our targeted nanoparticles, as well as the targeted gene vectors we’re developing.

Gene therapy has great promise to image, treat, and cure a wide variety of diseases, including cystic fibrosis, pulmonary fibrosis, pulmonary hypertension, and acute respiratory distress syndrome. Ideally for systemic therapies, the viral vector would be injected intravenously and then go immediately and completely only to the desired site of action, usually a cell deep within a single tissue. Unfortunately, the current reality is that rapid scavenging by the reticulo-endothelial system, broad tropism, and host immunogenicity have, so far, prevented safe and effective viral-mediated gene therapy. If caveolae can pump targeted virus out of the blood like they do antibodies alone, small doses of virus could have robust, specific effects on the lungs, while sparing other organs, especially the liver. Recent preliminary experiments show that, when linked to antibodies that target lung caveolae, viruses are rapidly retargeted to the lung with minimal liver uptake. Understanding how viruses interact with endothelial cells, whether they are endocytosed and/or transcytosed, how they interact with the underlying tissue, and perhaps most importantly, which cells can be readily transduced to express the virally delivered transgene will provide the critical and fundamental knowledge to design viral vectors for pulmonary imaging and treatment of a wide variety of pulmonary diseases.

Tumor Models: Animal tumor models are necessary for preclinical efficacy studies, but most therapies that are effective in animal models fail in humans. We have created and are in the process of validating a new tumor model. This model uses native tissue implanted in a dorsal skinfold window chamber to provide an orthotopic tissue environment while maintaining the ease and power of IVM imaging. This model allows us to image tumor targeting, penetration, and therapeutic response in the same animal overtime. This powerful model will allow us to follow tumor growth dynamically in vivo, carefully parse out the effects of tumor vs. microenvironmental factors, and evaluate new targeted therapies in vivo in a rigorous system that appears to closely resemble human tumors, and determine the strengths and limitations of caveolae-based transport. 

Selected References

  1. Schnitzer, J.E., Oh, P., Pinney, E. & Allard, J. NEM inhibits transcytosis, endocytosis and capillary permeability: implication of caveolae fusion in endothelia. Am. J. Physiol., 37, H48-55, 1995
  2. Schnitzer, J.E., Oh, P., Jacobson, B.S. & Dvorak, A.M. Caveolae from luminal plasmalemma of rat lung endothelium: Microdomains enriched in caveolin, Ca++-ATPase and IP3 receptor. Proc. Nat. Acad.Sci. (USA), 92, 1759-1763, 1995
  3. Schnitzer, J.E., Lui, J. & Oh, P. Endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexins and GTPases. J. Biol. Chem., 270, 14399-14404, 1995
  4. Schnitzer, J.E., McIntosh, D.P., Dvorak, A.M., J. Liu & Oh, P. Separation of caveolae from associated microdomains of GPI-anchored proteins. Science, 269, 1435-1439, 1995
  5. Jacobson, B.S., Stolz, D.B. & Schnitzer, J.E. Identification of endothelial cell surface proteins as potential targets for diagnosis and treatment of disease. Nature Med., 2, 482-484, 1996
  6. Schnitzer, J. E. McIntosh, D.P. & Oh, P. Role of GTP hydrolysis in fission of caveolae directly from plasma membranes, Science, 274, 239-242, 1996 [printer's erratum-Science, 274,p.1069, 1996]
  7. Lui, J., Oh, P., Horner, T., Rogers, R.A. & Schnitzer, J. Organized cell surface signal transduction in caveolae distinct from GPI-anchored protein microdomains, J. Biol. Chem., 272, 7211-7222, 1997
  8. Oh, P., McIntosh, D.P. & Schnitzer, J.E. Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium, J. Cell Biol., 141, 101-114, 1998
  9. Rizzo, V., Sung, A., Oh, P. & Schnitzer, J.E. Rapid mechanotransduction in situ at the luminal cell surface of vascular endothelium and its caveolae, J. Biol. Chem., 273, 26323-26329, 1998
  10. Rizzo, V., McIntosh, D.P., Oh, P., & Schnitzer, J. E. Flow activates eNOS in caveolae at the luminal cell surface of endothelium in situ with rapid caveolin dissociation and calmodulin association J. Biol. Chem., 273, 34724-34729, 1998
  11. Schnitzer, J.E. Clinical Implications of Basic Research - "Vascular targeting as a strategy for cancer therapy" New Eng. J. Med., 339: 472-4, 1998
  12. Oh, P. & Schnitzer, J.E. Immuno-isolation of caveolae with high affinity antibodies binding the caveolin cage: Towards understanding the basis of purification, J. Biol. Chem., 274, 23144-23154, 1999
  13. McIntosh, D.P., Oh, P. & Schnitzer, J.E. Caveolae require intact VAMP-2 for targeted transport in vascular endothelium, Am. J. Physiol., 277: H2222-H2232, 1999
  14. Oh, P. & Schnitzer, J.E. (2001) Segregation of heterotrimeric G proteins in cell surface microdomains. G(q) binds caveolin to concentrate in caveolae, whereas G(i) and G(s) target lipid rafts by default. Mol. Biol. Cell 12:685-698.
  15. Schnitzer, J., Caveolae: from basic trafficking mechanisms to targeting transcytosis for tissue-specific drug and gene delivery in vivo, Adv. Drug Delivery Rev.., 1, 1-16, 2001.
  16. McIntosh, D.P., Tan, X.Y., Oh, P., & Schnitzer, J.E. (2002) Targeting endothelium and its dynamic caveolae in vivo for tissue-specific delivery and transcytosis in vivo: A pathway to overcome cell barriers to drug and gene delivery. Proc. Natl. Acad. Sci. USA, 99, 1996-2001.
  17. Zabel, U., Oh, P., Kleinschnitz, C., Nedvedsky, P., Smolenski, A., Kugler, P., Walter, U., Schnitzer, J.E , & Schmidt, H.H.H.W. (2002) Calcium-dependent membrane association sensitizes soluble guanylyl cyclase to NO. Nature Cell Biol., 4: 307-311.
  18. Czarny, M., Liu, J. & Schnitzer, J.E. Transient mechanoactivation of neutral sphingomyelinase in caveolae to generate ceramide. J. Biol Chem 278, 4424-30 (2003).
  19. Carver, L. and Schnitzer, J.E. Caveolae: Mining little caves for new cancer targets; Nature Rev Cancer 3:571-581 (2003).
  20. Rizzo, V., Morton, C., DePaola N., Schnitzer J.E., Davies P.F.(2003) Recruitment of endothelial caveolae into mechanotransduction pathways by flow-conditioning in vitro. Am J Physiol 285(4):H1720-9.
  21. Carver LA, Schnitzer JE, Anderson RG, Mohla A. (2003) Role of caveolae and lipid rafts in cancer: Workshop summary and future needs. Cancer Res, (20):6571-4.
  22. Oh, P., Li, Y., Yu, J., Durr, E., Krasinska, K. Carver, L., Testa, J.E., and Schnitzer, J.E. (2004) Subtractive proteomic mapping of the endothelial surface in lung and solid tumors for tissue-specific therapy, Nature, 429:629-35.
  23. Durr E., Yu J., Krasinska K.M., Carver L.A., Yates J.R. III, Testa J.E., Oh P., Schnitzer J.E. (2004) Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture. Nature Biotechnol. 22(8):985-92.
  24. Czarny M., Schnitzer J.E. (2004) Neutral sphingomyelinase inhibitor scyphostatin prevents and ceramide mimics mechanotransduction in vascular endothelium. Am J Physiol. 287(3):H1344-52.
  25. Valadon P, Garnett JD, Testa JE, Bauerle M, Oh P, Schnitzer JE. (2006) Screening phage display libraries for organ-specific vascular immunotargeting in vivo. Proc Natl Acad Sci U S A. 103(2):407-412.
  26. Koziol JA, Feng AC, Schnitzer JE.(2006) Application of Capture—Recapture Models to Estimation of Protein Count in MudPIT Experiments. Analytical Chemistry 78,3203-3207.
  27. Oh P., Borgström P., Witkiewicz H., Li Y., Borgström B.J., Chrastina A., Iwata K., Zinn K.R, Baldwin R., Testa J.E., & Schnitzer J.E. (2007) Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung. Nature Biotechnol 25(3) 327-337.
  28. Simonson, A.B. and Schnitzer, J.E., (2007) Vascular proteomic mapping in vivo. J Thombosis and Haemostasis 5 (Suppl 1): 183-187.
  29. Koziol, JA, Feng AC, Yu J, Griffin NM, Schnitzer JE (2008) Range Charts for Agreement in Measurement Comparison Studies, with Application to Replicate Mass Spectrometry Experiments. J. Proteomics Bioinformatics 1(6):287-292.
  30. Griffin NM, and Schnitzer JE. (2008) Proteomic mapping of the vascular endothelium in vivo for vascular targeting. Methods Enzymol. 445:177-208.
  31. Testa JE, Chrastina A, Li Y, Oh P, and Schnitzer JE. (2009) Ubiquitous yet distinct expression of podocalyxin on vascular surfaces in normal and tumor tissues in rat. J Vasc Res. 46(4):311-324.
  32. Li Y, Yu J, Wang Y, Griffin NM, Long F, Shore S, Oh P, Schnitzer JE. (2009) Enhancing identifications of lipid-embedded proteins by mass spectrometry for improved mapping of endothelial plasma membranes in vivo. Mol. Cell. Proteomics 8: 1219-1235.
  33. Testa J. E., Chrastina, A., Oh, P., Li, Y., Witkiewicz, H., Czarny, M., Buss, T. and Schnitzer, J.E.. Immunotargeting and cloning of two CD34 variants exhibiting restricted expression in adult rat endothelia in vivo. A. J. Physiol. Lung 297(2): p. L251-L262
  34. Massey K. A. and Schnitzer J.E., (2009) Targeting and Imaging Signature Caveolar Molecules in Lungs. Proc. Am. Thor. Soc., 6: 419-430
  35. Yi M. and Schnitzer JE. Impaired tumor growth, metastasis, angiogenesis and wound healing in annexin A1-null mice. Proc Natl Acad Sci U S A. 106:17889-91.
  36. Valadon P, Darsow B, Buss TN, Czarny M, Griffin NM, Nguyen HN, Oh P, Chrastina A, Borgstrom P, Schnitzer JE. (2009) Designed auto-assembly of nanostreptabodies for rapid tissue-specific targeting in vivo. J Biol Chem. Epub ahead of print.
  37. Griffin NM, Yu J, Long F, Oh P, Shore S, Li Y, Koziol JA, Schnitzer JE, (2009) Label-free, normalized quantification of complex mass spectrometry data for proteomics analysis Nat Biotechnol (in press).

 

Latterich Lab

Our main focus is on understanding disease, such as cancer and genetically caused aging and degenerative disorders, at the systems level. The ultimate goal of this research is to define biomarkers to create better molecular diagnoses and companion diagnostic assays, as well as to understand the pathways and networks affected by disease in an effort to define future drug targets. We use genomic sequencing, epigenomic profiling, RNA profiling, proteomics, and traditional biochemistry and molecular biology to capture the dynamic changes taking place during disease development, followed by genetic and biochemical validation methods in well-defined experimental model systems. Our current interest is in tumorigenesis, the regulation of protein degradation in health and disease, and aging at the molecular level.

Systems Analysis of Tumorigenesis: One of the hallmarks of cancer is a significantly altered gene and protein expression pattern in comparison to normal tissue. Recent studies examining gene expression patterns in tumors have shown a correlation between gene expression patterns, disease prognosis and therapeutic responses. Ultimately, this may allow for more personalized therapy targeted to an individual’s specific tumor type. However, this genetic approach has the inherent challenge that gene expression patterns are often transient and do not always correlate to protein expression levels and protein modifications. This simplistic picture is further complicated by the fact that most solid tumors are not homogeneous, but include cell populations derived from tumor, stroma and bone marrow. The focus of our lab is to apply systems approaches to study tumor models developed in the Schnitzer lab, which allow the isolation of cell populations of tumor, stroma and bone marrow derived cells. Measuring and assembling a map of genomic, epigenomic, gene expression and proteomic profiles over time will lead to a fine resolution map of the events responsible for solid tumor formation. This information will ultimately aid the design of new therapies designed to interfere in the tumorigenesis process, as well as aid in the design of tumor-stage diagnostic assays.

Regulation of Protein Degradation: The AAA-protein VCP is key in many protein degradation and protein remodeling pathways, yet its precise function is not clear, even after over a decade of intense research. There is mounting evidence that VCP functions in the ubiquitin pathway, the ER-associated degradation pathway and the N-end rule degradation pathway (Figure 1). Select mutations in VCP’s N-terminus cause an autosomal dominant syndrome upon onset of middle age: hereditary inclusion body myopathy with Paget disease of bone and frontotemporal dementia (hIBMPFD).

We are currently studying the detailed sequence of events of a novel mechanism by which muscles and bones degenerate in hIBMPFD syndrome. A more detailed understanding of this process will have eventual diagnostic and therapeutic implications for patients and their families affected by hIBMPFD as well as similar degenerative diseases, such as muscular dystrophy and Paget disease of bone. Other byproducts of this research will ultimately lead to a better mechanistic understanding in areas as diverse as DNA damage repair, pathological and normal aging, cancer, myocardial infarction, muscle wasting, degenerative dementias and premature senescence.

Molecular Aging: Aging at the molecular level is a complex series of pathways that work together to repair cellular and genomic damage, while maintaining replicative potential without allowing for uncontrolled growth. While the genetic basis of the premature aging disease Werner syndrome is understood, it is not entirely clear how the Werner syndrome protein WRNp integrates aging and DNA-damage related signals prior to its function in DNA damage repair and replication. Fred Indig, a former postdoctoral fellow in my lab, established that WRNp is sequestered in a novel VCP-containing protein complex, which is disrupted in the presence of DNA damaging agents and participates in an important aspect of DNA damage control. The AAA-protein VCP functions as a decision switch in protein ubiquitination and deubiquitination reactions, but the interaction between VCP and WRNp is independent of ubiquitin. Thus we predict that VCP plays an entirely novel role related to sequestration and pre-assembly of a protein complex that serves to maintain WRNp as an inactive component with a WRNp protein modification machinery preassembled. This complex can appropriately modify and release WRNp upon receiving a DNA-damage signal. We plan a number of entirely independent experimental strategies that aim to create a model of how VCP and WRNp are modulated by DNA damage signals, how the complex is modulated at the molecular level, and what role the plethora of post-translational modification play in controlling complex disassembly, ultimately leading to activation of WRNp in DNA repair. We are confident that providing a more detailed model of WRNp’s role in the diseased aging process will lead to a better understanding of Werner syndrome, which in turn will greatly benefit our understanding of the normal aging process. Experimental or drug-induced modulation of this pathway may eventually allow us to modulate DNA-damage response and ultimately augment the replicative potential of cells and tissues.

Selected Publications

  1. Patel, S.K., Indig, F.E., Olivieri, N., Levine, N., and Latterich, M. Organelle membrane fusion: A novel function for the syntaxin homolog Ufe1p in ER membrane fusion. Cell.  1998;92:611-620.
  2. Lin, A., Patel, S., and Latterich, M. Regulation of organelle membrane fusion by Pkc1p. Traffic 2, 2001;698-704.
  3. Kuwana, T., Mackey, M.R., Perkins, G., Ellisman, M., Latterich, M., Schneiter, R., Green, D.R., and Newmeyer, D.D. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 2002;111, 331–342.
  4. Partridge, J.J., Lopreiato, J.O. Jr, Latterich, M., and Indig, F.E. DNA damage modulates nucleolar interaction of the Werner protein with the AAA ATPase p97/VCP. Mol. Biol. Cell 2003;14, 4221-4229.
  5. Indig, F.E., Partridge, J.J., von Kobbe, C., Aladjem, M.I., Latterich, M. and Bohr, V.A. Werner Syndrome Protein Directly Binds to the AAA ATPase p97/VCP in ATP-dependent Fashion. J. Struct. Biol. 2004;146, 251-259.
  6. Barker, D.L., Hansen, M.S.T., Faruqui, A.F., Giannola, D., Irsula, O.R., Lasken, R.S., Latterich, M., Makarov, V., Oliphant, A., Pinter, J.H., Shen, R., Sleptsova, I., Ziehler, W. and Lai, E. Two methods of whole genome amplification enable accurate genotyping across a 2,320-SNP linkage panel. Genome Res. 2004;14, 901-907.
  7. Latterich, M. Molecular Systems Biology at the Crossroads: To know less about more, or to know more about less. Proteome Science 2005;3, 8.
  8. Halawani, D. and Latterich, M. p97: The Cell’s Molecular Purgatory? Molecular Cell 2006;22, 713-717.
  9. Latterich, M. p97 adaptor choice regulates organelle biogenesis. Dev. Cell 2006;11, 755-757.
  10. Binette, J., Dubé, M., Mercier, J., Halawani, D., Latterich, M., Cohen E.A. Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum. Retrovirology 2007;4, 75.
  11. Tewfik, M.A., Latterich, M., Di Falco, M.D., Samaha, M. Proteomics of Nasal Mucus in Chronic Rhinosinusitis. J. Am. Rhinology 2007;21, 680-685.
  12. Caruso, M.E., Jenna, S., Bouchecareilh, M., Baillie, D.L., Boismenu, D., Halawani, D., Latterich, M., and Chevet, E. GTPase-mediated regulation of the unfolded protein response in Caenorhabditis elegans is dependent on the AAA+ ATPase CDC-48.  Mol Cell Biol. 2008;28, 4261- 4274.
  13. Latterich, M., and Corbeil, J. Label-free detection of biomolecular interactions in real time with a nano-porous silicon-based detection method. Proteome Science 2008; 4;6:31.
  14. Latterich, M., Abramovitz, M., Leyland-Jones, B. Proteomics: New technologies and clinical applications. Eur. J. Cancer 2008; 44, 2737-2741.
  15. Halawani, D., LeBlanc, A., Rouiller, I., Michnick, S., Servant, M. and Latterich, M. Hereditary inclusion body myopathy-linked p97/VCP mutations in the NH2-domain and the D1 ring modulate p97/VCP ATPase activity and D2 AAA+ ring conformation. Mol Cell Biol. 2009; 29, 4484-4494.

Testa Lab

Dr. Testa’s research has focused on producing and characterizing monoclonal antibodies for diagnostic and therapeutic uses in human disease. Prior to joining PRISM, she helped develop several metastasis-inhibiting mAbs produced by a technique called subtractive immunization (7, 8, 9). While at PRISM, Dr. Testa has used both classical and subtractive immunization to produce a panel of monoclonal antibodies to a variety of proteins expressed on the luminal surface of endothelial cells in normal and tumor tissues. These antibodies, which bind to pan-endothelial markers and organ-specific proteins as well as caveolar and extra-caveolar proteins, are being used to further our understanding of the distribution and function of proteins on the luminal surface of vascular endothelial cells.

Of particular importance to PRISM’s research endeavor are those monoclonal antibodies which bind to accessible caveolar targets. Caveolae, which function as active pumps to rapidly transport targeting molecules out of the blood and into the tissue, represent a novel strategy with which to access extravascular tissue compartments. High affinity monoclonal antibodies developed in the laboratory provide the targeting moiety for numerous classes of cargo, including other antibodies, drugs, gene vectors, and nanoparticles. Radiolabeled monoclonal antibodies can also serve as agents of radioimmunotherapy. Currently in development are several caveolae targeting mAbs to distinct lung-specific proteins for use in treating a variety of chronic, genetic or infectious diseases. Also in development is an antibody to a unique target expressed in the caveolae of tumor blood vessels which has been used in laboratory experiments to image and treat tumors in vivo. The antigen recognized by this antibody is expressed not only on endothelial cells in rodent tumors but in a variety of human tumor types as well. PRISM plans on beginning clinical trials with this antibody within the next one to two years.

The examples mentioned above are part of an ever-growing panel of monoclonal antibodies that target the endothelial cell surface and/or caveolae which will allow us to fine-tune delivery to the vasculature surface, to the interior of endothelial cells, or to underlying tissue. Fully understanding how targeted antibodies interact with endothelial cells in vivo is necessary to rationally design therapies to treat cancer and other human diseases.

Selected Publications

  1. Testa JE, Chrastina A, Oh P, Li Y, Witkiewicz H, Czarny M, Schnitzer JE.  2009. Immunotargeting and cloning of two CD34 variants exhibiting restricted expression in adult rat endothelia in vivo. Am J Physiol Lung Cell Mol Physiol. 297(2):L251-62
  2. Testa JE, Chrastina A, Li Y, Oh P, Schnitzer JE. 2009. Ubiquitous yet Distinct Expression of Podocalyxin on Vascular Surfaces in Normal and Tumor Tissues in the Rat.  J Vasc Res. 10;46(4):311-324.
  3. Oh P., Borgstrom P., Witkiewicz H., Li Y., Borgstrom B.J., Chrastina A., Iwata K., Zinn K.R., Baldwin R., Testa J.E., and Schnitzer J.E.  2007.   Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung.  Nat Biotechnol.   25(3):327-37.
  4. Valadon, P., Garnett, J.D., Testa, J.E., Bauerle, M., Oh, P., and Schnitzer J.E.  Screening phage display libraries for organ-specific vascular immunotargeting in vivo. 2006.  Proc Natl Acad Sci U S A. 103(2):407-12.
  5. Oh, P., Li, Y., Yu, J., Durr, E., Krasinska, K. Carver, L., Testa, J.E., and Schnitzer, J.E. (2004) Subtractive proteomic mapping of the endothelial surface in lung and solid tumors for tissue-specific therapy, Nature, 2004 429:629-35.
  6. Durr E., Yu J., Krasinska K.M., Carver L.A., Yates J.R. III, Testa J.E., Oh P., Schnitzer J.E. (2004) Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture. Nat Biotechnol.22(8):985-92.
  7. Zijlstra, A., J.E. Testa, and J.P. Quigley.  2003.  Targeting the proteome/epitome, implementation of subtractive immunization.  Biochem Biophys Res Commun. 303:733-744.
  8. Hooper, J.D., A. Zijlstra, R.T. Aimes, H. Liang, G.F. Claassen, D. Tarin, J.E. Testa, J.P. Quigley.  2003.  Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen. Oncogene. 22:1783-94.
  9. Testa, Jacqueline E., Peter C Brooks. Jian-Min Lin, and James P. Quigley. 1999. Eukaryotic expression cloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis. Cancer Research. 59:3812-3820.
  10. Nielsen-Preiss, Sheila, James P. Quigley, and Jacqueline E. Testa. 1999. Co-inoculation of human and murine carcinoma cells induces reciprocal suppression of metastasis by both cell lines. Clin. Exp. Metastasis. 17:489-496.
  11. Noiri, Eisei, Eugene Lee, Jacqueline Testa, James Quigley, David Colflesh, Charles R. Keese, Ivar Giaever, and Michael S. Goligorski. 1998.  Podokinesis in endothelial cell migration: role of nitric oxide.  Am. J. Physiol.  274 (Cell Physiol. 43):C236-C244.
  12. Sipley, John D., Daniela S. Alexander, Jacqueline E. Testa, and James P. Quigley.  1997.  Introduction of an RRHR motif into chicken urokinase-type plasminogen activator (ch-uPA) confers sensitivity to plasminogen activator inhibitor-1 (PAI-1) and PAI-2 and allows ch-uPA-mediated extracellular matrix degradation to be controlled by PAI-1.  Proc. Natl. Acad. Sci., USA  94:2933-2938.
  13. Testa, Jacqueline E, Steingrimur Stefansson, Tracy Sioussat, and James P. Quigley.  1995  Avian urokinase-type plasminogen activator (uPA) lacks the putative binding site for plasminogen activator inhibitor (PAI) and is resistant to inhibition by human PAI-1 and PAI-2. Fibrinolysis 9:93-99.
  14. Coleman, James L., Timothy J. Sellati, Jacqueline E. Testa, Richard R. Kew, Martha B. Furie, and Jorge L. Benach. 1995.  Borrelia burgdorferi binds plasmin(ogen) resulting in enhanced penetration of endothelial monolayers. Infection and Immunity  63:2478-2484.
  15. Testa, Jacqueline E. 1992. Loss of the metastatic phenotype by a human epidermoid carcinoma cell line, HEp-3, is accompanied by increased expression of tissue inhibitor of metalloproteinases-2 (TIMP-2).  Cancer Res. 52:5597-5603.

Valadon Lab

To protect from invaders, our body has developed one of the most fascinating tools in protein engineering, namely antibodies. Over the past 30 years, the machinery behind antibody generation has been dissected and varied techniques have been developed to allow the in vitro generation of antibodies with desirable and tunable properties. Developing novel antibodies that can target the vasculature in vivo and deliver proper payloads offers challenges in both antibody selection and design. Overcoming these challenges is the main focus of Dr. Valadon’s current research.

Vascular targeting: Vascular targeting depends on the specific and unique expression of target proteins on the luminal surface of blood vessels in different tissues and disease states, but their lack of accessibility renders the process of panning (i.e. the process of selecting binders among a large library of candidate antibodies) very inefficient. Alternative methods based on phage display are actively developed.

Engineering payloads: We have created a multifunctional antibody targeting system that we call nanostreptabodies. In this system, a 15-amino acid biotin-acceptor sequence is incorporated into full length antibodies or Fab’ fragments, allowing for efficient and specific enzymatic biotinylation in vivo to generate bisbiotinylated antibodies and monobiotinylated Fab’ fragments. To assemble nanostreptabodies, streptavidin is first mixed with biotinylated antibodies. Because streptavidin can bind four biotin residues, a diverse array of complexes can be formed by attaching other moieties to the streptavidin. Adding a second type of antibody produces a bivalent antibody; adding an imaging and/or drug components produces multifunctional complexes. When injected intravenously, these auto-assembled complexes could target the lung in vivo and were rapidly transcytosed across the EC barrier. Targeting antibody and biotinylated NPs, probes, imaging agents, viruses and even biotinylated siRNA’s can be readily combined to provide a flexible, rapid, and efficient way to assemble antibody-based targeting platforms.

Selected Publications:

  1. Valadon P, Nussbaum G, Boyd LF, Margulies DH, Scharff MD. (1996).  Peptide libraries define the fine specificity of anti-polysaccharide antibodies to Cryptococcus neoformans. J Mol Biol. 1996;261(1):11-22.
  2. Nussbaum G, Cleare W, Casadevall A, Scharff MD, Valadon P. (1997).  Epitope location in the Cryptococcus neoformans capsule is a determinant of antibody efficacy. J Exp Med. 1997;185(4):685-94.
  3. Young AC, Valadon P, Casadevall A, Scharff MD, Sacchettini JC. (1997).  The three-dimensional structures of a polysaccharide binding antibody to Cryptococcus neoformans and its complex with a peptide from a phage display library: implications for the identification of peptide mimotopes. J Mol Biol. 1997; 274(4):622-34.
  4. Valadon P, Nussbaum G, Oh J, Scharff MD. (1998).  Aspects of antigen mimicry revealed by immunization with a peptide mimetic of Cryptococcus neoformans polysaccharide. J Immunol. 1998;161(4):1829-36.
  5. Beenhouwer DO, May RJ, Valadon P, Scharff MD. (2002).  High affinity mimotope of the polysaccharide capsule of Cryptococcus neoformans identified from an evolutionary phage peptide library. J Immunol. 2002;169(12):6992-9.
  6. Valadon P, Garnett JD, Testa JE, Bauerle M, Oh P, Schnitzer JE. (2006).  Screening phage display libraries for organ-specific vascular immunotargeting in vivo. Proc Natl Acad Sci U S A. 2006;103(2):407-12.
  7. Valadon P, Darsow B, Buss TN, Czarny M, Griffin NM, Nguyen HN, Oh P, Borgstrom P, Chrastina A, Schnitzer JE. (2009). Designed auto-assembly of nanostreptabodies for rapid tissue-specific targeting in vivo. J Biol Chem. 2009 Oct 22. [Epub ahead of print]