Membrane Proteins Of Normal And Abnormal Red Cells

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 2R01HL038794-15 from National Heart, Lung, And Blood Institute, IRG: HEM

Abstract: The broad, long range goal of this project is to develop a detailed understanding of the molecular properties of spectrin and its role in red cell membrane deformability and stability. The specific aims are to 1) explore structural and functional implications of our recent, exciting discovery that lysines in the spectrin tetramer binding site are selectively, extensively carbamylated in vivo; 2) determine the submolecular basis of spectrin s unique flexibility/extensibility properties and rationalize these properties with existing high resolution structures of spectrin motifs; 3) define the structure of the red cell spectrin tetramer binding site and investigate the isoform specificity of this site; and 4) determine the spectrin isoform specificity of the dimerization initiation site. Three major hypotheses will be tested, which should result in a more accurate, detailed understanding of red cell membranes as well as membrane skeletons of other cell types. The first hypothesis is that physiological modification of specific spectrin lysines in the red cell affect its function and could play a critical role in red cell survival under both normal and pathological conditions possibly including renal failure, diabetes, and red cell aging. The second hypothesis is that red cell spectrin, which is more flexible than non-red cell isoforms, has a different conformation in solution than suggested by available high-resolution structures with long continuous helices connecting adjacent homologous motifs. The third hypothesis is that different spectrin isoforms share similar mechanisms of self-assembly but isoform-specific complementary recognition sites in the dimer initiation and tetramer binding regions control correct isoform assembly and determine association affinities. These hypotheses will be tested using isolated spectrin dimers and monomers, as well as recombinant domains of red cell and non-red cell spectrin isoforms. Functional properties including interactions between adjacent motifs, dimerization, and tetramer assembly will be studied using biochemical and biophysical techniques including isothermal titration calorimetry, analytical ultracentrifugation, and HPLC gel filtration. Structural properties will be analyzed using protein microchemistry methods that will include high-resolution 2D gels, in-gel protease digestion, mass spectrometry (MS), hydrogen-deuterium exchange/MS analyses, and related methods.

Keywords: congenital hemolytic anemia, erythrocyte, erythrocyte membrane, membrane protein, protein structure function, spectrin, binding site, biophysics, carbamate, conformation, dimer, glycosylation, intermolecular interaction, lysine, protein isoform, thermodynamics, high performance liquid chromatography, human tissue, mass spectrometry, microcalorimetry, monoclonal antibody, ultracentrifugation

Project start date: 1987-07-01

Project end date: 2007-03-31

2R01HL038794-15 (2002): $454038

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MEMBRANE PROTEINS OF NORMAL AND ABNORMAL HUMAN RED CELLS

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 2R01HL038794-06A1 from National Heart, Lung, And Blood Institute, IRG: HEM

Abstract: The broad, long range goal of this proposal is to elucidate the molecular basis of human red cell membrane destabilization resulting from hereditary hemolytic anemia related mutations. Ultimate achievement of this goal requires the development of a detailed understanding of the membrane skeleton s structural/functional/regulatory processes and its role in determining red cell stability in both normal and abnormal cells. The major immediate focus will be a detailed analysis of spectrin which is a major critical component of the red cell membrane. Normal spectrin and selected spectrin mutations will be studied in concert to attempt to better understand its role in stabilizing the red cell membrane. Three specific immediate goals are 1) to determine the mechanism of spectrin self-assembly to form heterodimers and tetramers, 2) to locate the submolecular sites involved in self-assembly, and 3) to investigate the molecular shape of spectrin under different ionic conditions. Normal spectrin, as well as naturally occurring polymorphisms and pathogenic mutations, will be studied using established and novel approaches. Spectrin domain peptides defined by mild tryptic cleavage at 0 degrees C will continue to be important tools for these submolecular functional and structural analyses. Modem protein chemical methods will be used, together with FPLC gel filtration based functional assays for spectrin assembly; completion of a 2D gel computerized database for spectrin domains; use of electron paramagnetic resonance (EPR) and fluorescence energy transfer analyses to measure intramolecular distances of spectrin in different environments; and expression of recombinant peptides to locate and characterize the minimum heterodimer nucleation site in the alpha subunit. These investigations should substantially contribute to at least five different conceptual aspects of spectrin that are currently poorly understood, but central to development of a comprehensive understanding of the red cell membrane. These critical questions are 1) how are conformational changes resulting from mutations or protein-protein assembly transduced over long molecular distances in spectrin; 2) what is the mechanism of spectrin assembly and the factors that regulate assembly; 3) where are the specific sites of protein-protein interaction and how are they regulated; 4) what is the actual molecular shape of spectrin in the intact cell, - is it an elastic, compact 60 nm or an extended 200 nm tetrameric actin crossbridge; 5) what is the conformation of spectrin?

Keywords: congenital hemolytic anemia, erythrocyte membrane, membrane protein, membrane structure, protein structure function, spectrin, chemical cleavage, conformation, dimer, gene mutation, genetic polymorphism, molecular pathology, peptide, antiserum, computer simulation, electron spin resonance spectroscopy, fluorescent dye /probe, high performance liquid chromatography, human subject, human tissue, human volunteer subject, laboratory rabbit, nucleic acid sequence, polymerase chain reaction, protein purification

Project start date: 1987-07-01

Project end date: 1997-03-31

2R01HL038794-06A1 (1993): $264981

MEMBRANE PROTEINS OF NORMAL/ABNORMAL RED CELLS

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 2R01HL038794-10 from National Heart, Lung, And Blood Institute, IRG: HEM

Abstract: The broad, long range goal of this project is to eluciate the mechanisms of human red cell spectrin sef-assembly and the molecular bases for perturbing functional interactions by hereditary hemolytic anemia related mutations. In the current proposal, three working hypotheses will be explored. The first hypothesis is that phosphorylation of beta spectrin influences membrane stability by affecting the rate limiting step in tetramer formation. The second hypothesis is that alpha spectrin pathogenetic mutations located substantial molecular distances away from the tetramer binding site affect tetramer formation by affecting the closed yields open dimer equilibrium. A third hypothesis is that current spectrin models deduced from the crystal structure of a single motif where adjacent motifs are connected by a continuous helix are inaccurate; instead, we propose that spectrin s flexibility is maintained by in-register alignment of laterally paired alpha and beta motifs with flexible hinge regions connecting adjacent motifs. The specific goals of the current proposal are 1) to identify the beta spectrin phosphorylation sites and determine their function; 2) to determine whether the rate limiting closed yields open dimer equilibrium is affected by pathogenic mutations in the hairpin loop region; and 3)to determine how inter-motif and lateral interchain non- covalent interactions affect conformational stability. These specific goals will be pursuedusing molecular biology and protein chemical methods. Most experiments will utilize normal and mutagenized recombinant spectrin peptides, intact spectrin monomers, and in vivo phosphorylated spectrin dimers. Spectrin phosphorylation, assembly, and structure will be studied using HPLC peptide mapping, mass spectrometry, protein microsequencing, protein binding assays, microcalorimetry, and related methods. The four specific aims are 1) Structural and functional role phosphorylation in spectrin assembly; 2) Role of the closed hairpin loop in spectrin function; 3) Mechanism of membrane destabilization by pathogenic spectrin mutations locaed at lare molecular distances from the tetramerization site; 4) Analysis of lateral dimer interfaces and inter-motif interfaces and their effects on conformational stability of spectrin.

Keywords: congenital hemolytic anemia, erythrocyte, erythrocyte membrane, membrane protein, protein structure /function, spectrin, gene mutation, intermolecular interaction, oligonucleotide, phosphoprotein, protein sequence, recombinant protein, synthetic nucleic acid, high performance liquid chromatography, human subject, human tissue, mass spectrometry, microcalorimetry

Project start date: 1987-07-01

Project end date: 2002-03-31

2R01HL038794-10 (1997): $304165

Protein Modification With Oxidative Stress In ALI

David W Speicher, Professor, Director, Molecular And Cellu
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Grant 5P01HL079063-030003 from National Heart, Lung, And Blood Institute, IRG: HLBP

Abstract: We will employ state-of-the-art biochemical, biophysical, and proteomic approaches to explore the role of oxidative stress in acute lung injury (ALI). The overall goals are to 1) define the mechanism of peroxiredoxin 6 s (Prdx 6) antioxidant activity and its role in protection from oxidative stress; 2) gain novel insights into the molecular mechanism(s) of lung damage caused by oxidative stress by identifying critical lung proteins that are oxidatively modified under different experimental conditions in mouse models, and 3) identify human plasma biomarkers of lung injury and ALI development. A major focus (Aim 1) will be to investigate structure-function relationships of Prdx 6 in close collaboration. Specifically, we will use analytical ultracentrifugation, mass spectrometry, and related biochemical and biophysical approaches to characterize recombinant Prdx 6 self-assembly and interaction with wild type piGST and a piGST mutant. In parallel with these biophysical/structural studies, we will examine functional properties of Prdx 6, particularly substrate interactions and enzymology. Aim 1 will also evaluate oxidative modifications on Prdx 6 isolated from mouse lungs using the models of oxidative stress being studied. In related studies, Aim 2 will systematically identify other lung proteins that are oxidatively modified and evaluate changes in these modifications in mice under normoxic and hyperoxic conditions when Prdx 6 is expressed at normal levels, over-expressed, or underexpressed. The ability of nanocarrier anti-oxidant therapy to minimize oxidative damage of proteins after oxidative stress will also be assessed. In summary, Aims 1 and 2 will test the hypothesis that Prdx 6 plays a central role in protecting lung tissue from excessive oxidative damage under hyperoxic conditions. In addition, Aim 3 will test the related hypothesis that oxidative stress resulting from severe trauma in patients and the resulting lung tissue damage will induce changes in blood protein profiles that can form the basis for minimally invasive diagnostic tests of ALI. This aim will utilize a unique resource of plasma samples from patients in a trauma cohort study of ALI/ARDS. Two complementary novel proteomic methods will be used to identify novel biomarkers or biosignatures of ALI in these human plasma samples.

Keywords: acute disease /disorder, enzyme activity, lung injury, oxidative stress, peroxidase, protein structure function, proteomics, respiratory protein, adult respiratory distress syndrome, biomarker, blood protein, disease /disorder model, gene expression, glutathione transferase, hyperoxia, molecular assembly /self assembly, mutant, protein protein interaction, recombinant protein, respiratory disorder diagnosis, trauma, analytical ultracentrifugation, clinical research, genetically modified animal, laboratory mouse, mass spectrometry

CORE--PROTEIN MICROCHEMISTRY/MASS SPECTROMETRY FACILITY

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 3P30CA010815-34S29024 from National Cancer Institute, IRG:

Abstract: The Protein Microchemistry Laboratory was established in 1986 when Dr. David Speicher joined The Wistar Institute. Its services have evolved from simple amino acid analysis and N-terminal sequencing to more sophisticated analyses involving subpicomole protein and peptide sequencing, high throughput protein purification, and mass spectrometry. Currently, a full range of services for protein isolation and primary structural determination is available. New equipment in the facility, including updated high pressure liquid chromatography (HPLC) systems, mass spectrometer, and a protein sequencer, purchased since the last review cost about $625,000, of which The Wistar Institute provided $200,000 and the rest was from NIH equipment grants and industrial sources. The following additional equipment is available in the facility Speedvac, sonicator, 1D and 2D gel apparatus, electroblotting units, power supplies, HP 1090M HPLC with autoinjector for amino acid analysis, Beckman System Gold HPLC for routine microbore HPLC peptide mapping, Isco fraction collector with peak detector, and general laboratory equipment. The Protein Microchemistry/Mass Spectrometry facility is located in a 500 square foot area that was renovated in 1987 and is adjacent to Dr. Speicher s research laboratory. All Cancer Center investigators have access to the facility on a first-come, first-served basis, and there is an oversight committee composed of the five most frequent users. Twenty-one Cancer Center investigators have used the facility in the past five years, including some no longer at the Institute. The major use was by 15 research projects, but no data are provided about how many of these projects were funded by peer- reviewed grants. All of these users appear to be or have been Cancer Center members. In addition, 18 other investigators used the facility on a more limited basis, which included either routine analyses or consultations. Approximately 50 percent of the total facility effort is dedicated to consultation and/or collaboration, for which there is no charge. The reported percent use by peer-reviewed projects was 77 percent and 23 percent was by non-peer-reviewed projects. Dr. Speicher s own projects utilized 39.4 percent of the total usage of the facility.

Keywords: biomedical facility, mass spectrometry, neoplasm /cancer, protein purification, protein structure, protein structure function, recombinant protein, electrospray ionization mass spectrometry, gas chromatography mass spectrometry, analytical ultracentrifugation, biosensor, high performance liquid chromatography, protein sequence

Development Of Methods For Cancer Biomarker Detection

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 5R33CA094360-03 from National Cancer Institute, IRG: ZCA1

Abstract: The overall goal of this three-year R33 proposal is to develop a novel proteomic strategy for quantitative comparisons of serum protein profiles capable of detecting proteins down to the low ng/ml level. This is about 100- to 1000-fold more sensitive than direct analysis of serum on either broad range or narrow range 2-D gels, and it represents the lower end of the concentration range for most known tumor serological markers. This high sensitivity proteome strategy will be developed and tested using chimeric mouse serum that contains human proteins secreted by tumors grown subcutaneously from injected human melanoma or pancreatic carcinoma cell lines. Systematic analyses of secreted human proteins using the new methods developed in this project should identify multiple new potential diagnostic targets for cancer screening. This proteome strategy will incorporate several novel supporting methods in addition to further optimization of a very promising proteome fractionation device and method recently invented in our laboratory. The fractionation method, "microscale solution isoelectrofocusing" (microsol-IEF) uses a variable number of tandem small volume chambers separated by semipermeable partitions containing immobilines at specific pH?s. Proof-of-principle experiments have been completed on several types of samples including mouse serum and demonstrate that this method can cleanly fractionate at least several mg of crude extracts into a small number of fractions. The resulting well-resolved fractions can be separated on a series of slightly overlapping narrow range 2-D gels using much higher protein loads than when unfractionated samples are used. Although the feasibility of this prefractionation method has been demonstrated, more reliable devices must be developed and separation parameters must be optimized to maximize resolution and reliability before this method can become a reliable robust proteomics method. Other novel features of the overall proteome analysis strategy include use of custom-made slightly overlapping narrow pH range gradients to ensure proteins are not lost at gel boundaries and use of dual sensitivity stains to increase dynamic range of detected spots. Proteins that appear to be specific to tumor-bearing mice will be identified and their species of origin (mouse = host or human = tumor) will be determined using LC-MS/MS methods. The Specific Aims are 1) Develop an optimized microsol-IEF device and method, 2) Develop high sample capacity microsol-IEF devices, 3) Develop high sensitivity post microsol-IEF analysis methods, and 4) Systematically apply integrated proteome analysis strategy to serum samples containing human melanoma or pancreatic carcinoma tumors.

Keywords: biomarker, diagnosis design /evaluation, method development, neoplasm /cancer diagnosis, proteomics, blood protein, early diagnosis, pancreas neoplasm, secretion, blood fractionation, cell line, clinical research, electrofocusing, gel electrophoresis, human tissue, laboratory mouse

Project start date: 2002-08-01

Project end date: 2006-06-30

5R33CA094360-03 (2004): $416082

Discovery And Validation Of Novel Serological Biomarkers Of Colon Cancer

David W Speicher, Professor, Director, Molecular & Cellula
Wistar Institute

Grant 5R01CA120393-03 from National Cancer Institute, IRG: CBSS

Abstract: The overall goal of this proposal is to identify novel serological biomarkers of human colon cancer and evaluate their capacities to predict disease occurrence or clinical outcome. To achieve this goal, we will use a powerful new 4-D protein profiling method recently developed in our lab that can identify many low abundance serum proteins. This method utilizes three tandem orthogonal protein separations consisting of major protein depletion, solution IEF and 1-D SDS gels. Each gel lane is cut into uniform slices, and digested with trypsin. Each tryptic digest, which still contains many proteins, is then analyzed by LC-MS/MS using a nanocapillary reverse phase column coupled to a high performance linear ion trap mass spectrometer. Candidate human biomarkers in SCID mice bearing human tumors will be identified by distinguishing human and mouse proteins based on species sequence differences. Validation of candidate biomarkers will be conducted in two stages. Initially, candidate human biomarkers will be tested using medium throughput quantitative mass spectrometer assays and Western blots to evaluate sera from a small group of colon cancer patients and matched controls. Higher throughput sandwich ELISA assays will then be developed for the most promising biomarkers, and these assays will be used to systematically test sera from a larger number of early and late stage cancer patients and matched controls. Levels of individual biomarkers as well as groups of biomarkers will be evaluated for their capacity to predict disease occurrence and clinical outcome. This ambitious but feasible five year project involves the following Specific Aims 1) Identify candidate human colon cancer biomarkers in a SCID mouse xenograph model system using a novel multi-dimensional protein profiling method; 2) Validate candidate serum biomarkers in patient and control sera using medium throughput assays; 3) Develop high throughput quantitative immunoassays for the most promising biomarkers from initial patient screens; and 4) Compare patient and control serum using high throughput immunoassays. Relevance to public health. Discovery of novel protein biomarkers of colon cancer that can be measured using a simple blood test has great potential to decrease the suffering and loss of life associated with this disease. Recently developed, powerful mass spectrometry-based methods provide a unique opportunity to systematically discovery groups of colon cancer biomarkers. It is most likely that groups of biomarkers (biomarker signatures) will have greater power to monitor cancer than individual biomarkers

Keywords: biomarker, colon neoplasm Carnivora, SCID mouse, antibody, base, blood, blood protein, blood test, carcinoma, cell, cell line, colon, death, early diagnosis, endoscopy, gastrointestinal imaging /visualization, gel, health care, human, implant, ion, lead, low socioeconomic status, mass spectrometry, model, monoclonal antibody, neoplasm /cancer, neoplasm /cancer classification /staging, neoplasm /cancer diagnosis, neoplastic cell, peptide, performance, physician, plasma, protein, proteomics, public health, serum, solution, therapy, tissue, trypsin, western blotting, xenotransplantation clinical research

Project start date: 2006-09-29

Project end date: 2011-07-31

5R01CA120393-02 (2007): $320521

1R01CA120393-01A1 (2006): $403974

Novel Comprehensive Proteome Analyses Of Metastasis

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 5R33CA092725-04 from National Cancer Institute, IRG: ZCA1

Abstract: The overall goal of this proposal is to apply several novel techniques to comprehensive quantitative comparisons of human breast cancer protein profiles focusing on changes associated with invasive/ metastatic potential. Our global proteome strategy should result in reliable quantitative comparisons of up to 10,000 proteins instead of the 1,500 to 2,500 protein spots typically detected on high resolution 2D gels. The most novel and key part of this strategy is a new method that we recently developed for prefractionating whole cell extracts prior to 2D PAGE. This method, termed microscale solution IEF (mu sol-IEF), uses small chambers separated by large pore acrylamide discs with immobilines at specific pH s to separate cell extracts into well resolved pools based upon pI s. A second novel feature of our global proteome strategy is use of slightly overlapping (about +/-0.1 pH) custom-made narrow pH range IPG gels tailored to match mu sol-IEF pools, rather than existing commercial 1 pH unit IPG gels with either 0 or 0.5 pH unit overlaps. A third novel feature is use of high-resolution 1D gels coupled with LC- MS-MS to analyze the insoluble proteins and large soluble proteins (100-500+ kDa). These two protein groups represent >25% of total cell protein mass, but are usually ignored in 2D protein profile comparisons. Finally, we recently optimized femtomole in-gel trypsin digestion to improve sensitivity and reliability of MALDI MS and LC-MS/MS identifications of targeted proteins. Our preliminary results show each of these techniques is very promising, but the feasibility of reliably quantitating 7,500 to 10,000+ proteins in human tumor cells remains to be demonstrated. The one year R21 phase will further optimize and integrate these methods to demonstrate proof-of-principle of our integrated global proteome analysis strategy using human breast cancer cells. The three year R33 phase will then apply the optimized global strategy to comparisons of multiple human breast cancer cell lines and tumors to identify protein changes associated with increased invasion and metastatic potential.

Keywords: breast neoplasm, metastasis, neoplasm /cancer invasiveness, oncoprotein, proteomics, cell migration, MCF7 cell, SCID mouse, gel electrophoresis, liquid chromatography mass spectrometry

Project start date: 2001-07-01

Project end date: 2006-08-31

5R33CA092725-04 (2004): $602469

1R21CA092725-01 (2001): $165904

STRUCTURE/FUNCTION OF THE CO17-1A/GA733 ANTIGEN

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 5P01CA074294-050001 from National Cancer Institute, IRG:

Abstract: The overall goal of this project is to study the molecular basis of GA733 Ag function in colorectal tumor and normal epithelial cells by investigating structural and functional properties of this protein. The Co17-1A/GA733 antigen (GA733 Ag) is expressed at high levels on most human colorectal carcinomas and on some normal epithelial cells. It is intriguing the monoclonal antibody CO17-1A has shown clinical efficacy without inducing toxicity related to binding of the antibody to normal tissues. This lack of toxicity is probably not due simply to differences in antigen density on tumor and normal cell surfaces; instead it is more likely that there are important structural and/or functional differences of GA733 Ag expressed by tumor versus normal tissues, which will be studied here. The detailed elucidation of structural and functional properties of this putative homotypic cell adhesion protein as described in this project should provide insights into mechanisms for potential anti-tumor effects and potential toxicities in clinical therapy. Molecular biology and protein chemical approaches will be used to identify important structural features of the GA733 Ag and to determine their effects on cell adhesion. One working hypothesis is that cell-cell adhesion mediated by GA733 Ag is signaled across the membrane by a mechanism. involving tyrosine phosphorylation and/or change in cis oligomer state of the cytoplasmic domain, which results in altered interactions with the cytoskeleton. The role of cis dimer site and to engineer secretable, adhesion-active dimers. The cell-cell adhesion binding site will also be mapped using mutagenesis. Standard protein chemical methods, including mass spectroscopy, N-terminal sequencing, HPLC peptide mapping, and chemical crosslinking will be used to identify post- translational modifications, oligomeric state, and other structural features that may affect function. Finally, cell transfetions will be used to evaluate the role of GA733 Ag and its mouse homolog (mEGP) on cell growth, morphology, invasion, and tumorigenicity.

Keywords: colorectal neoplasm, protein structure function, tumor antigen, active site, cell adhesion, cytoplasm, cytoskeleton, gene expression, phosphorylation, posttranslational modification, protein sequence, mass spectrometry, monoclonal antibody, recombinant protein, site directed mutagenesis, tissue /cell culture, transfection

MEMBRANE PROTEINS OF NORMAL AND ABNORMAL HUMAN RED CELLS

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 5R01HL038794-05 from National Heart, Lung, And Blood Institute, IRG: HEM

Abstract: The long range goal of this proposal is to identify the molecular bases for certain human congenital hemolytic anemias and to develop a detailed concept of the membrane skeleton s role in maintenance of red cell stability and shape. Two major immediate aims will be to identify precise mutations responsible for red cell membrane instability in certain hemolytic anemias and to elucidate mechanisms contributing to this destabilization using established and novel approaches. General clinical classifications of hereditary elliptocytosis (HE), hereditary spherocytosis (HS), and hereditary pyropoikilocytosis (HPP) actually correspond to a large number of unrelated biochemical disorders. These disorders are poorly characterized biochemically and very little is known about the mechanisms involved in membrane destabilization. Spectrin, a complex, major protein with a central role in the red cell membrane skeleton, will be studied first and in the greatest detail. Red cell spectrin has recently been directly implicated in several unrelated hemolytic disorders and these initial observations will be extended. Effects of specific mutations on structural and functional properties, and their contributions to membrane destabilization will be investigated. Synthetic peptides and mutant peptides will be used in known functions. In conjunction with functional characterizations, mechanisms responsible for long range transducing effects in the spectrin molecule will be investigated. Current evidence indicates that mutations can be located substantial distances from the destabilized functional site. The basis for this remarkable long range effect is not known, although it is probably directly related to spectrin conformation. Further analysis of the 106 amino acid repeat unit and spectrin conformation will use new computer prediction methods coupled with synthetic peptide experiments to develop and test new models. Characterization of the precise mutations in selected hemolytic anemias coupled with more detailed analyses of structural and functional properties of proteins in the membrane skeleton will make vital contributions to a refined understanding of the role of the membrane skeleton in normal and diseased human red cells.

Keywords: chemical structure function, congenital nonspherocytic hemolytic anemia, erythrocyte membrane, gene mutation, hereditary elliptocytosis, hereditary spherocytosis, membrane protein, membrane structure, protein engineering, spectrin, chemical binding, conformation, crosslink, model design /development, molecular pathology, mutant, nucleic acid sequence, peptide, protein sequence, structural model, antiserum, computer simulation, endonuclease, gel electrophoresis, high performance liquid chromatography, human clinical subject, human tissue from nonrelated source, human volunteer subject, laboratory rabbit, monoclonal antibody, nucleic acid probe, synthetic peptide

Project start date: 1987-07-01

Project end date: 1993-03-31

DOMAIN STRUCTURE OF HUMAN SMOOTH MUSCLE FILAMIN

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute
3601 Spruce Street
philadelphia, Pa 191044265

Grant 5R01AR039158-05 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases, IRG: CTY

Abstract: The long range goal of this proposal is to characterize the functional roles of filamin, an important major component of smooth muscle and most other tissues. It is now clear that this protein is a major organizer of three dimensional actin filament arrays and it is likely to play a critical role in normal and diseased smooth muscle. Our long range goal will be approached through investigation of structural and functional properties of both the intact molecule and domain peptides. Functional sites on the filamin molecule will be more precisely located and characterized in greater detail. A major emphasis will involve the study of the regulation of filamin functions (self-association and actin binding) and factors that are likely to regulate these interactions. Possible changes in filamin organization, quantity and isoform distribution in several pathological states involving smooth muscle will also be investigated. For most studies, mild proteolysis will be used to divide this large multifunctional protein into smaller peptide units or domains. To facilitate the characterization of functional sites, a preliminary domain map of the molecule will be completed using conventional and HPLC peptide mapping. Functional properties of filamin will be investigated using established and novel methods for measuring protein - protein interactions in vitro. Pairwise protein associations and more complex multiprotein complexes will be analyzed. Attempts will be made to more precisely locate binding sites on the filamin molecule using several methods including chemical crosslinking, site directed antibodies, and synthetic peptides. Biochemical and protein chemical methods will be used to search for factors that control and modulate known functional interactions. Possible regulatory factors that will be investigated include phosphorylation and other posttranslational modifications, proteins or other factors whose interactions with filamin have not yet been identified, calpain cleavage, and ionic environment (especially Ca2+ levels). Also, biochemical methods and immunological probes developed in the above studies will be used to characterize filamin content, isoform expression and subcellular localization in normal and diseased smooth muscle

Keywords: hypertension, laminin, leiomyoma, smooth muscle, uterus neoplasm, vascular smooth muscle actin, binding protein, crosslink, myofibril, phosphorylation, platelet, protein isoform, protein sequence antibody, high performance liquid chromatography, human tissue, laboratory rabbit, laboratory rat, proteolysis

Project start date: 1988-12-01

Project end date: 1993-11-30

5R01AR039158-05 (1993): $161856

5R01AR039158-04 (1992): $159356

MEMBRANE PROTEINS OF NORMAL AND ABNORMAL HUMAN RED CELLS

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 5R01HL038794-08 from National Heart, Lung, And Blood Institute, IRG: HEM

Abstract: The broad, long range goal of this proposal is to elucidate the molecular basis of human red cell membrane destabilization resulting from hereditary hemolytic anemia related mutations. Ultimate achievement of this goal requires the development of a detailed understanding of the membrane skeleton s structural/functional/regulatory processes and its role in determining red cell stability in both normal and abnormal cells. The major immediate focus will be a detailed analysis of spectrin which is a major critical component of the red cell membrane. Normal spectrin and selected spectrin mutations will be studied in concert to attempt to better understand its role in stabilizing the red cell membrane. Three specific immediate goals are 1) to determine the mechanism of spectrin self-assembly to form heterodimers and tetramers, 2) to locate the submolecular sites involved in self-assembly, and 3) to investigate the molecular shape of spectrin under different ionic conditions. Normal spectrin, as well as naturally occurring polymorphisms and pathogenic mutations, will be studied using established and novel approaches. Spectrin domain peptides defined by mild tryptic cleavage at 0 degrees C will continue to be important tools for these submolecular functional and structural analyses. Modem protein chemical methods will be used, together with FPLC gel filtration based functional assays for spectrin assembly; completion of a 2D gel computerized database for spectrin domains; use of electron paramagnetic resonance (EPR) and fluorescence energy transfer analyses to measure intramolecular distances of spectrin in different environments; and expression of recombinant peptides to locate and characterize the minimum heterodimer nucleation site in the alpha subunit. These investigations should substantially contribute to at least five different conceptual aspects of spectrin that are currently poorly understood, but central to development of a comprehensive understanding of the red cell membrane. These critical questions are 1) how are conformational changes resulting from mutations or protein-protein assembly transduced over long molecular distances in spectrin; 2) what is the mechanism of spectrin assembly and the factors that regulate assembly; 3) where are the specific sites of protein-protein interaction and how are they regulated; 4) what is the actual molecular shape of spectrin in the intact cell, - is it an elastic, compact 60 nm or an extended 200 nm tetrameric actin crossbridge; 5) what is the conformation of spectrin?

Keywords: congenital hemolytic anemia, erythrocyte membrane, membrane protein, membrane structure, protein structure function, spectrin, chemical cleavage, conformation, dimer, gene mutation, genetic polymorphism, molecular pathology, peptide, antiserum, computer simulation, electron spin resonance spectroscopy, fluorescent dye /probe, high performance liquid chromatography, human subject, human tissue, laboratory rabbit, nucleic acid sequence, polymerase chain reaction, protein purification

Project start date: 1987-07-01

Project end date: 1997-03-31

5R01HL038794-08 (1995): $300346

5R01HL038794-07 (1994): $278440

MEMBRANE PROTEINS OF NORMAL/ABNORMAL RED CELLS

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 5R01HL038794-14 from National Heart, Lung, And Blood Institute, IRG: HEM

Abstract: The broad, long range goal of this project is to eluciate the mechanisms of human red cell spectrin sef-assembly and the molecular bases for perturbing functional interactions by hereditary hemolytic anemia related mutations. In the current proposal, three working hypotheses will be explored. The first hypothesis is that phosphorylation of beta spectrin influences membrane stability by affecting the rate limiting step in tetramer formation. The second hypothesis is that alpha spectrin pathogenetic mutations located substantial molecular distances away from the tetramer binding site affect tetramer formation by affecting the closed yields open dimer equilibrium. A third hypothesis is that current spectrin models deduced from the crystal structure of a single motif where adjacent motifs are connected by a continuous helix are inaccurate; instead, we propose that spectrin s flexibility is maintained by in-register alignment of laterally paired alpha and beta motifs with flexible hinge regions connecting adjacent motifs. The specific goals of the current proposal are 1) to identify the beta spectrin phosphorylation sites and determine their function; 2) to determine whether the rate limiting closed yields open dimer equilibrium is affected by pathogenic mutations in the hairpin loop region; and 3)to determine how inter-motif and lateral interchain non- covalent interactions affect conformational stability. These specific goals will be pursuedusing molecular biology and protein chemical methods. Most experiments will utilize normal and mutagenized recombinant spectrin peptides, intact spectrin monomers, and in vivo phosphorylated spectrin dimers. Spectrin phosphorylation, assembly, and structure will be studied using HPLC peptide mapping, mass spectrometry, protein microsequencing, protein binding assays, microcalorimetry, and related methods. The four specific aims are 1) Structural and functional role phosphorylation in spectrin assembly; 2) Role of the closed hairpin loop in spectrin function; 3) Mechanism of membrane destabilization by pathogenic spectrin mutations locaed at lare molecular distances from the tetramerization site; 4) Analysis of lateral dimer interfaces and inter-motif interfaces and their effects on conformational stability of spectrin.

Keywords: congenital hemolytic anemia, erythrocyte, erythrocyte membrane, membrane protein, protein structure function, spectrin, gene mutation, intermolecular interaction, oligonucleotide, phosphoprotein, protein sequence, recombinant protein, synthetic nucleic acid, high performance liquid chromatography, human subject, human tissue, mass spectrometry, microcalorimetry

Project start date: 1987-07-01

Project end date: 2002-03-31

5R01HL038794-14 (2001): $342342

5R01HL038794-13 (2000): $332371

5R01HL038794-12 (1999): $322689

5R01HL038794-11 (1998): $313289

Membrane Proteins Of Normal & Abnormal Red Cells

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 5R01HL038794-19 from National Heart, Lung, And Blood Institute, IRG: HEM

Abstract: The broad, long range goal of this project is to develop a detailed understanding of the molecular properties of spectrin and its role in red cell membrane deformability and stability. The specific aims are to 1) explore structural and functional implications of our recent, exciting discovery that lysines in the spectrin tetramer binding site are selectively, extensively carbamylated in vivo; 2) determine the submolecular basis of spectrin s unique flexibility/extensibility properties and rationalize these properties with existing high resolution structures of spectrin motifs; 3) define the structure of the red cell spectrin tetramer binding site and investigate the isoform specificity of this site; and 4) determine the spectrin isoform specificity of the dimerization initiation site. Three major hypotheses will be tested, which should result in a more accurate, detailed understanding of red cell membranes as well as membrane skeletons of other cell types. The first hypothesis is that physiological modification of specific spectrin lysines in the red cell affect its function and could play a critical role in red cell survival under both normal and pathological conditions possibly including renal failure, diabetes, and red cell aging. The second hypothesis is that red cell spectrin, which is more flexible than non-red cell isoforms, has a different conformation in solution than suggested by available high-resolution structures with long continuous helices connecting adjacent homologous motifs. The third hypothesis is that different spectrin isoforms share similar mechanisms of self-assembly but isoform-specific complementary recognition sites in the dimer initiation and tetramer binding regions control correct isoform assembly and determine association affinities. These hypotheses will be tested using isolated spectrin dimers and monomers, as well as recombinant domains of red cell and non-red cell spectrin isoforms. Functional properties including interactions between adjacent motifs, dimerization, and tetramer assembly will be studied using biochemical and biophysical techniques including isothermal titration calorimetry, analytical ultracentrifugation, and HPLC gel filtration. Structural properties will be analyzed using protein microchemistry methods that will include high-resolution 2D gels, in-gel protease digestion, mass spectrometry (MS), hydrogen-deuterium exchange/MS analyses, and related methods.

Keywords: congenital hemolytic anemia, erythrocyte, erythrocyte membrane, membrane protein, protein structure function, spectrin, binding site, biophysics, carbamate, conformation, dimer, glycosylation, intermolecular interaction, lysine, protein isoform, thermodynamics, high performance liquid chromatography, human tissue, mass spectrometry, microcalorimetry, monoclonal antibody, ultracentrifugation

Project start date: 1987-07-01

Project end date: 2008-03-31

5R01HL038794-19 (2006): $425931

5R01HL038794-18 (2005): $423475

5R01HL038794-17 (2004): $411141

5R01HL038794-16 (2003): $399165

CORE--BIOCHEMISTRY CORE

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 2P01CA025874-20A19004 from National Cancer Institute, IRG:

Abstract: The Biochemistry was established in the previous grant period to address a growing need by the individual projects for protein isolation and structural characterization. As our methods for isolation and analysis of the small amounts of proteins that are available from melanoma and melanocyte cell lines have improved, they have developed to the point where most required procedures are now relatively routine, although they remain highly sophisticated and technically demanding. In addition, use of proteome analysis methods based on 2D gel separations of tumor cell proteins to compare changes in protein patterns under different experimental conditions is emerging as an important complement to nucleic acid based microarray method. This core will be used by all investigators within the program project. Overall, this Core will continue to provide sophisticated, high sensitivity biochemical and protein chemical technologies to the participating laboratories.

Keywords: biochemistry, biomedical facility, melanoma, molecular oncology, oncoprotein, protein purification, high performance liquid chromatography, mass spectrometry

CORE--PROTEOMICS FACILITY

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 2P30CA010815-359032 from National Cancer Institute, IRG: NCI

Abstract: Revised ) Advances in mass spectrometry (MS) capacities and availability of complete genomes for multiple organisms have recently dramatically changed how proteins are identified and how primary sequence modifications are analyzed. Most significant, it is now possible to identify proteins from 1-D or 2-D gels or in solution mixtures at unprecedented speeds and sensitivities. Hence, during the past grant period the Proteomics Facility has been extensively expanded and modified to better meet the needs of Cancer Center members who are taking targeted proteomic approaches to cancer research or require analysis of individual proteins at low femtomole sensitivities. To better reflect the current scope and major activities of this expanded facility, the name has been changed from "Protein Microchemistry" to "Proteomics". The facility now primarily focuses on high sensitivity identifications of proteins using MS methods and also provides essential complementary analyses of protein primary structure and post-translational modifications. The following services are provided MALDI mass spectrometry; Microbore HPLC peptide mapping; Femtomole level in-gel protease digestion; Nano-capillary LC-MS/MS based protein identification; MALDI MS peptide fingerprint analysis; Edman sequencing; ESI mass analysis of proteins. Nearly all the present equipment in the facility has been acquired during the past grant period and staff has been expanded to 4.8 FTE s. The major reason for this recent growth has been the rapidly escalating demand by Cancer Center members for nano-capillary LC-MS/MS protein identifications at sub-picomole levels from 1-D or 2-D gels of samples isolated from human cells or other organisms with complete or nearly complete genomes. Cancer Center demand for state-of-the-art MS-based analysis methods is expected to continue to grow for the foreseeable future because current Cancer Center members are increasingly shifting at least part of their research efforts to systems biology approaches using targeted or global proteomics strategies. In addition, many of the faculty who will be recruited during the next grant period are expected to have substantial needs for MS-based methods provided by the facility.

Keywords: biomedical facility, neoplasm /cancer, proteomics, protein sequence, mass spectrometry

Project start date: 2003-09-01

Project end date: 2008-02-28

Technologies For Identifying Proteins Secreted By Tumors

David W Speicher, Professor, Director, Molecular And Cellula
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 5R01CA077048-08 from National Cancer Institute, IRG: ZRG1

Abstract: Reliable serum markers for cancer diagnosis should lead to reduced mortality and morbidity through earlier initial detection of cancer and recurring tumors. Unfortunately, most of the handful of known serum diagnostic targets do not show adequate specificity. However, these serum markers have usually been identified one-at-a-time and based on indirect clues rather than through systematic analyses of serum proteins. A systematic proteomics search for tumor-specific serum proteins is much more likely to identify better serum markers and/or identify groups of proteins secreted by the tumors that will be more reliable than any one or two individual serological markers. Effective serum proteomics-based analyses will require development of new methods capable of detecting proteins at the low ng/ml level or less, because cancer biomarkers are typically low abundance proteins in the presence of very abundant serum proteins, The central working hypothesis of this proposal is that systematic analysis of biological fluids with new proteome methods will produce either several novel serological markers or alternatively will define biosignatures that can be used to diagnose diseases including cancer. The two major goals of this proposal are1) to develop multi-stage serum prefractionation strategies coupled with improved high sensitivity 1-D gel and nanocapillary LC-MS/MS methods for systematic identifications of tumor-specific proteins, and 2) to use these new methods to identify biomarkers secreted by human ovarian tumors into SCID mouse serum. The feasibility of using serum from SCID mice containing human tumors to identify secreted tumor proteins has recently been demonstrated in this laboratory. Ovarian cancer will be studied because early tumors are asymptomatic, the organ is relatively inaccessible to examination, and there are no reliable early detection methods. As a result, ovarian cancer has a very poor survival rate and is the leading cause of death from gynecological malignancies and the fourth most common cause of cancer deaths in women. The four Specific Aims are 1) Develop a robust nanoscale in-gel digestion method for higher sensitivity protein identification; 2) Develop and test an automated high throughput nanoscale in-gel digestion method; 3) Compare the 1-D gel pixelation approach with MudPIT analyses using prefractionated serum; and 4) Produce and systematically evaluate serum samples from SCID mice with ovarian tumors

Keywords: biomarker, blood protein, gel electrophoresis, mass spectrometry, method development, neoplasm /cancer, secretion, diagnosis design /evaluation, early diagnosis, electrofocusing, high throughput technology, liquid chromatography mass spectrometry, nanotechnology, ovary neoplasm, physical separation, proteomics, robotics, serology /serodiagnosis, SCID mouse, cell line, clinical research, human genetic material tag, neoplastic cell

Project start date: 1997-09-30

Project end date: 2007-06-30

5R01CA077048-08 (2005): $325610

5R01CA077048-07 (2004): $313085

5R01CA077048-06 (2003): $375222

Purchase Of An Ion Trap Mass Spectrometer

David W Speicher, Professor, Director, Molecular & Cellula
Wistar Institute
3601 Spruce Street
philadelphia, Pa 191044265

Grant 1S10RR022456-01A1 from National Center For Research Resources, IRG: ZRG1

Abstract: Funds are requested to purchase a ThermoFinnigan LTQ Orbitrap mass spectrometer that will be housed in the Wistar Institute Proteomics Core Facility. This instrument will provide critically needed increased capacity to identify proteins and posttranslational modifications with high confidence in complex samples. A major user group of 8 investigators will utilize 75% of instrument time and 8 minor users will account for an additional 21% of instrument use. Most investigators in both the major and minor user groups have multiple NIH funded research projects that will utilize this instrument. Hence, a linear ion trap mass spectrometer that is fully available to the major user group is critically needed and will greatly advance NIH funded research at the Wistar Institute. The purchase of an LTQ Orbitrap mass spectrometer is strongly preferred rather than a stand- alone LTQ instrument because the LTQ Orbitrap will also provide high resolution and high accuracy measurements of precursor and fragment ion masses. This will greatly enhance the identification of low abundance components in complex mixtures with improved confidence, and it should also improve the confidence of identifications of posttranslational modifications. Even with the addition of the requested LTQ Orbitrap, conservative projections of anticipated workload for 2007 indicate that both this instrument and an existing LTQ will need to be operated near full capacity, 24 hours per day, 7 days a week to keep up with demand. The requested instrument will be incorporated into a Proteomics Core Facility that has provided LC- MS/MS analysis services for the past 6 years to many academic investigators. In 2005, a total of 30 different laboratories used these services and similar numbers of users are anticipated for the future. The Wistar Institute is committed to housing, maintaining and staffing the requested mass spectrometer for its useful lifetime as verified by an accompanying letter from the Institute President and CEO

Keywords: housing, ion, posttranslational modification, proteomics measurement, protein

Project start date: 2007-06-01

Project end date: 2009-05-31

1S10RR022456-01A1 (2007): $500000

TECHNOLOGIES FOR IDENTIFYING PROTEINS SECRETED BY TUMORS

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 5R01CA077048-04 from National Cancer Institute, IRG: ZCA1

Abstract: Applicant s Description) The overall goal of this proposal is to develop and test protein-based technologies for characterizing the protein profiles of complex tumor-related samples, particularly proteins and protein fragments secreted by human tumor tissues. The proposed studies will focus primarily on developing improved technologies in several critical areas. One working hypothesis is that effective analyses of human proteomes will require improved protein separation methods capable of resolving most of the proteins in the proteome, which are estimated to contain more than 10,000 protein components. A second working hypothesis is that existing in situ proteolysis methods are very inefficient when using femtomole protein amounts, which limits overall sensitivity of mass spectrometry (MS)-based protein identifications. A major emphasis will be to develop reproducible, robust 2D or 3D protein methods capable of separating >10,000 protein components. These efforts will include improve conventional immobiline-based 2D gel, increase 2D gel resolution by using overlapping pH gradients, and develop 3D separation methods using pre-isofocusing ion exchange chromatography to produce several discrete reproducible groups of proteins. A second major emphasis will be to systematically evaluate and improve femtomole level proteolysis of gel spots for subsequent MS analyses, and to improve the confidence level of protein assignments using MS and MS-MS data. One objective will be to ensure extensive coverage of the entire protein sequence to help distinguish between closely related protein isoforms, alternatively spliced forms of proteins, and highly homologous protein modules shared by different proteins in complex human proteomes. This proposal involves four Specific Aims 1) develop high resolution, higher capacity 2D or 3D proteome separation methods; 2) develop reliable microscale protease digestion of gel spots for MS and MS-MS analyses, 3) determine parameters that contribute to definitive protein identification using MS and MS-MS data; and 4) develop high throughput proteome identification methods by automating appropriate stages of the process.

Keywords: mass spectrometry, method development, oncoprotein, endopeptidase, physical separation, protein sequence

Project start date: 1997-09-30

Project end date: 2002-06-30

5R01CA077048-04 (2000): $309280

1R01CA077048-01 (1997): $297575

5R01CA077048-03 (1999): $313139

STRUCTURE FUNCTION OF MELCAM, A CELL ADHESION PROTEIN

David W Speicher, Professor, Director, Molecular And Cellu
Wistar Institute 3601 Spruce Street Philadelphia, Pa 191044265

Grant 5R01CA066671-05 from National Cancer Institute, IRG: BIO

Abstract: Adapted from  s ) The long-term objective of this project is to understand the role of MelCAM and its complementary ligand in cell-cell adhesion. MelCAM is a recently discovered member of the immunoglobulin superfamily of CAMs, which shows dramatically elevated expression in many melanoma and breast cancer cell lines. Structure-function analysis of MelCAM and its 95 kDa ligand will use protein chemical and molecular biology methods to study both natural proteins from melanoma cells and recombinants expressed in insect cells, E. coli, and stably-transfected human cells. Methods will include HPLC peptide mapping, N-terminal sequencing, mass spectrometry, microcalorimetry, circular dichroism, immunological methods, and cell binding assays. The functionally-important interactions that will be studied in this project are (1) lateral association of MelCAM within a single plasma membrane (cis homodimers); (2) heterotypic intercellular binding to a novel 95 kDa ligand, and (3) association with the cytoskeleton. The overall hypothesis is that heterotypic intercellular association with MelCAM with its 95 kDa ligand forms important cell-cell adhesions that play important, but poorly defined, roles in primary tumor growth as well as the invasive stages of metastasis. A specific hypothesis that will be directly tested is that these moderate affinity intercellular interactions are dependent upon the oligomeric state of MelCAM within the cell membrane, i.e., a cis MelCAM homodimer is essential for adhesion. In addition, association of MelCAM with the cytoskeleton does occur and is likely to be involved in intercellular adhesion and/or transmembrane signaling. Signal transduction and/or interaction with the cytoskeleton may involve phosphorylation of the MelCAM cytoplasmic domain. The specific aims of this proposal are (1) clone and characterize the heterotypic 95 kDa ligand; (2) map and characterize MelCAM binding sites; and (3) identify cytoskeletal proteins associated with MelCAM and define their role. MelCAM expression correlates closely with the transformed phenotype; only trace expression occurs on normal melanocytes in vivo, while very high MelCAM expression is observed in aggressively growing tumors and in most melanoma cell lines. The MelCAM-ligand adhesion system is both essential and sufficient for cell-cell aggregation of melanoma cells. This project will produce a detailed understanding of the structural and functional properties of MelCAM-mediated cell-cell adhesion, which should provide critical insights concerning its importance during tumor progression.

Keywords: cell adhesion, cell adhesion molecule, ligand, protein structure /function, chemical binding, cytoskeletal protein, dimer, membrane protein, phosphorylation, posttranslational modification, recombinant protein, structural biology, circular dichroism, high performance liquid chromatography, mass spectrometry, microcalorimetry, molecular cloning, protein purification, protein sequence, site directed mutagenesis, transfection

Project start date: 1996-02-12

Project end date: 2001-11-30

5R01CA066671-05 (2000): $318690

5R01CA066671-04 (1999): $306433