Mapping specific protein-protein interactions within the core component of the breast cell DNA synthesome

We have previously described the isolation and characterization of an intact multiprotein complex for DNA replication, designated the DNA synthesome, from human breast cancer cells and biopsied human breast tumor tissue. The purified DNA synthesome was observed to fully support DNA replication in vitro. We had also proposed a model for the breast cell DNA synthesome, in which DNA polymerases alpha, delta, and epsilon, DNA primase, and replication factor C (RF-C) represent members of the core component, or tightly associated, proteins of the complex. This model was based on the observed fractionation, chromatographic, and sedimentation profiles for these proteins. We report here that poly(ADP-ribose)polymerase (PARP) and DNA ligase 1 are also members of the breast cell DNA synthesome core component. More importantly, in this report we present the results of coimmunoprecipitation studies that were designed to map the protein-protein interactions between several members of the core component of the DNA synthesome. Consistent with our proposed model for the breast cell DNA synthesome, our data indicate that DNA polymerases alpha and delta, DNA primase, RF-C, as well as proliferating cell nuclear antigen (PCNA), tightly associate with each other in the complex, whereas DNA polymerase epsilon, PARP, and several other components were found to interact with the synthesome via a direct contact with only PCNA or DNA polymerase alpha. The association of PARP with the synthesome core suggests that this protein may serve a regulatory function in the complex. Also, the coimmunoprecipitation studies suggest that the three DNA polymerases alpha, delta, and epsilon all participate in the replication of breast cell DNA. To our knowledge this is the first report ever to describe the close physical association of polypeptides constituting the intact human breast cell DNA replication apparatus.     

Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry: potentials and limitations

Precursor ion scans have proven to be extremely useful for the characterization of unseparated peptide mixtures. In conjunction with the nanoelectrospray source, precursor ion scans provide a sensitive tool for the detection of posttranslationally modified peptides and have been used to determine phosphorylation sites of proteins digested in solution. In this report, we extend our previous work to the determination of protein phosphorylation sites of gel-isolated proteins. The in-gel digestion procedure developed in our laboratory for protein microsequencing was found to be suitable for phosphorylation mapping as well. The risk of losing hydrophilic peptides in the desalting step was decreased by using column packing material designed for the purification of oligonucleotides and by adjusting the pH conditions to the needs of phosphopeptide analysis. With this method, the tryptic phosphopeptides of beta-casein were detected after in-gel digestion at a sensitivity of 250 fmol of protein applied to the gel. The phosphorylation sites of two other proteins, Src-delta U and Op18, have similarly been mapped. Subpicomole to low-picomole amounts of protein starting material are needed in general, although we and others have reported attomole sensitivity for the detection of model phosphopeptides using precursor ion scans. This indicates that the success in determining phosphorylation sites depends crucially on the digestion, extraction, and detection efficiency for individual phosphopeptides.     

Determining protein-protein interactions by oxidative cross-linking of a glycine-glycine-histidine fusion protein

The Ni(II) complex of the tripeptide NH2-glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP). Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking. The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli. In the presence of Ni(OAc)2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGH-ecotin dimer with no observable protein degradation. The cross-linking reaction occurs between two ecotin proteins in a dimer complex. Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin. The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The target of the reaction is tyrosine, and the product is bityrosyl cross-links. The yield of the cross-linking is on the order of 15%. However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 A of a naturally occurring tyrosine. This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification.     

Ribosomal structure: RNA-protein and protein-protein interactions

Vitamin A and its derivatives have been postulated to play an important role in renal tubulogenesis and compensatory hypertrophy. This study examined the effects of two carboxylic derivatives of vitamin A on Lewis lung carcinoma-porcine kidney-1 (LLC-PK1) renal tubular epithelial cell mito- and motogenesis and cell size. It was found that all-trans and 13-cis retinoic acids exerted modest, dose-dependent effects to stimulate incorporation of 3H-thymidine into acid-precipitable material of LLC-PK1 cells. The effects of all-trans retinoic acid to promote 3H-thymidine uptake in LLC-PK1 cells modestly enhanced that seen with acidic fibroblastic growth factor. Similar findings of these two retinoic acid derivatives to promote 3H-thymidine uptake and to enhance 3H-thymidine uptake stimulated by another growth factor (platelet-derived growth factor BB) were also observed in cultured bovine aortic smooth muscle cells. Both retinoic acids promoted healing of denuded areas made within confluent monolayers of serum-starved LLC-PK1 cells. All-trans retinoic acid also stimulated recovery of mechanically denuded areas within bovine aortic smooth muscle monolayers. Neither all-trans nor 13-cis retinoic acids s affected cell size as assessed by forward light scatter with flow cytometry, suggesting lack of effect to induce hypertrophy. These results demonstrate that two carboxylic acid derivatives of vitamin A are capable of stimulation of basal and growth factor-induced incorporation of 3H-thymidine uptake into acid-precipitable material and healing of denuded areas in disparate cell types. These findings are compatible with a role for vitamin A and its analogues in the tissue repair process.     

Effects of protein-surface interactions on protein ion signals in MALDI mass spectrometry

The influence of polymer surface-protein binding affinity on protein ion signals in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is examined. The surfaces of poly(vinylidene fluoride) and poly(ethylene terephthalate) polymer substrates are modified by pulsed rf plasma deposition of allylamine. By varying the on/off duty cycle of the pulsed rf plasma, the polymer substrate surfaces are coated with thin films having varying densities of surface amine groups. The varying surface amine density is shown to lead to systematic changes in the surface binding affinity for the 125I-radiolabeled peptides angiotensin I and porcine insulin. Unlabeled angiotensin I and porcine insulin are then deposited on the pulsed rf plasma-modified substrates and analyzed by MALDI mass spectrometry. The experimental approach involves applying the peptide to the modified polymer surface in an aqueous phosphate-buffered saline solution and allowing the peptide solution to dry completely under ambient conditions. Subsequently, the MALDI matrix alpha-cyano-4-hydroxycinnamic acid in methanol and 10% trifluoroacetic acid in water are added to the peptide-coated modified polymer surfaces. The results of these studies demonstrate that, for the sample preparation method employed, increases in the surface peptide binding affinity lead to decreases in the peptide MALDI ion signal.     

Peptide modulators of protein-protein interactions in intracellular signaling

Signal transduction cascades involve multiple enzymes and are orchestrated by selective protein-protein interactions that are essential for the progression of intracellular signaling events. Modulators of these protein-protein interactions have been used to dissect the role of individual components of each signaling cascade. We describe several methods that have been developed for the identification of peptides that inhibit the interaction between signaling proteins and hence selectively modulate their functions. Such peptide modulators provide important tools for basic research and have great potential as leads for the development of new classes of therapeutic drugs.     

Distinguishing small molecular mass differences of proteins by mass spectrometry

This study was undertaken to determine if acidic or basic fibroblast growth factor (FGF1 or FGF2) or vascular endothelial growth factor (VEGF) alters the radiation response of small bowel after total-body irradiation (TBI). Female C3H mice were treated with various doses of angiogenic growth factor administered intravenously 24 h before or 1 h after TBI. Radiation doses ranged from 7 to 18 Gy. End points measured were the number of crypts in three portions of the small bowel, the frequency of apoptosis of crypt cells at various times after TBI, and the LD50/30 (bone marrow syndrome) and LD50/6 (GI syndrome). Fibroblast growth factors alone, without TBI, decreased the number of crypts per circumference significantly. Among the factors tested, FGF2 caused the greatest decline in baseline crypt number. Despite this decrease in the baseline crypt number, after irradiation the number of surviving crypts was greater in animals treated with growth factor. The greatest radioprotection occurred at intermediate doses of growth factor (6 to 18 pg/mouse). Mice treated with FGF1 and FGF2 had crypt survival curves with a slope that was more shallow than that for saline-treated animals, indicating radiation resistance of crypt stem cells in FGF-treated mice. The LD50/6 was increased by approximately 10% for all treatments with angiogenic growth factors, whether given before or after TBI. Apoptosis of crypt cells was maximum at 4 to 8 h after TBI. The cumulative apoptosis was decreased significantly in animals treated with angiogenic growth factors, and the greatest protection against apoptosis was seen in animals treated with FGF2 prior to TBI. All three angiogenic growth factors tested were radioprotective in small bowel whether given 24 h before or 1 h after irradiation. The mechanism of protection is unlikely to involve proliferation of crypt stem cells, but probably does involve prevention of radiation-induced apoptosis or enhanced repair of DNA damage of crypt cells.     

DNA sequence analysis by MALDI mass spectrometry

Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products. Gel casting and electrophoresis are the time limiting steps, and the gel separation is occasionally imperfect due to aberrant mobility of certain fragments, leading to erroneous sequence determination. Furthermore, illegitimately terminated products frequently cannot be distinguished from correctly terminated ones, a phenomenon that also obscures data interpretation. In the present work the use of MALDI mass spectrometry for sequencing of DNA amplified from clinical samples is implemented. The unambiguous and fast identification of deletions and substitutions in DNA amplified from heterozygous carriers realistically suggest MALDI mass spectrometry as a future alternative to conventional sequencing procedures for high throughput screening for mutations. Unique features of the method are demonstrated by sequencing a DNA fragment that could not be sequenced conventionally because of gel electrophoretic band compression and the presence of multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the sequence was deduced, and the nature of the non-specific termination could be determined. The method described here increases the fidelity in DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation.     

Thermodynamics of iron-nickel alloys by mass spectrometry

A Knudsen cell-mass spectrometer combination was used to determine the activities of iron and nickel in solid and liquid iron-nickel alloys in the temperature range 1500 to 1900 K. This has provided thermodynamic data which are consistent in both the solid and liquid regions. The δH^M@#@ values obtained are in fair agreement with calorimetric data. A subregular model gives a good representation of the thermodynamic properties of this system.     

Thermodynamics of iron-nickel alloys by mass spectrometry

A Knudsen cell-mass spectrometer combination was used to determine the activities of iron and nickel in solid and liquid iron-nickel alloys in the temperature range 1500 to 1900 K. This has provided thermodynamic data which are consistent in both the solid and liquid regions. The δH^M@#@ values obtained are in fair agreement with calorimetric data. A subregular model gives a good representation of the thermodynamic properties of this system.     

Investigation of protein folding by mass spectrometry

Bone repair by regeneration as we know it continues to undergo changes, with advances approaching that may change our treatment of patients with craniofacial deformities and skeletal defects. Perhaps by the turn of the century, patients born with asymmetric deformities due to lack of growth will be treated early in life by skeletal stretching, and then later in life by skeletal distraction that is followed by use of accelerating factors to assist the healing processes. All of these available modalities are part of the regeneration of new bone formation. The future of such changes is very interesting, and our ability to help our patients will be maximized. We may even look back 25 years from now at bone grafting and find it to be obsolete and crude. It is hoped that with the new modalities being developed, we will not deviate from the use of a bone grafting procedure, which is the workhorse of the craniofacial surgeon. Bone grafting is used by all surgeons working on the craniofacial skeleton despite the problems of unpredictability of healing and an inability to calculate what percentage of the original graft will survive. The transplantation issue will be solved. The problems with donor site morbidity will continue. The use of inorganic bone substitutes will continue to have its limitation, particularly in type II wounds, which we as plastic surgeons see in the craniofacial region. As we redefine our approach to skeletal repair, we may look back and find solutions to some of the major problems we have had. The rapid stretch of soft tissue after facial advancement or structural alteration that is accompanied by a relapse due to the elastic recoil of the soft tissue could be eliminated by gradual distraction. The bone will undergo better functional adaptation when it has a gradual change in structure based on adjustment and molding in a gradual fashion. The problem of donor site morbidity and a prediction formula for bone could be resolved with new bone formation in situ by mineralization of the area under repair. Bone healing enhancers are here to stay and their clinical application will produce a far-reaching better final outcome (Fig. 11).     

Identification of viral mutants by mass spectrometry

A method to identify mutations of virus proteins by using protein mass mapping is described. Comparative mass mapping was applied to a structural protein of the human rhinovirus Cys1199 --> Tyr mutant and to genetically engineered mutants of tobacco mosaic virus. The information generated from this approach can rapidly identify the peptide or protein containing the mutation and, in cases when nucleic acid sequencing is required, significantly narrows the region of the genome that must be sequenced. High-resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and tandem mass spectrometry were used to identify amino acid substitutions. This method provides valuable information for those analyzing viral variants and, in some cases, offers a rapid and accurate alternative to nucleotide sequencing.     

Mapping protein-ligand interactions by footprinting, a radical idea

PURPOSE: This study investigated the functional significance of perineal complications after ileal pouch-anal anastomosis. METHODS: Review of a prospective registry of 628 patients was undertaken. Bowel function was assessed by detailed functional questionnaire. Statistical analyses were performed using chi-squared and Fisher's exact probability tests. RESULTS: Of 628 patients, 153 (24.4 percent) had 171 perineal complications. The 277 control patients had no complications. Complications included 66 (10.5 percent) anastomotic strictures, 28 (4.5 percent) anastomotic separations, 36 (5.7 percent) pouch fistulas, 41 (6.5 percent) episodes of pelvic sepsis, and 18 (2.9 percent) patients with multiple complications. After these complications were addressed, the pouch failure rate was low (10 percent); in 90 percent of patients, the pouch could be salvaged. Most pouch failures were the result of pouch fistulas, and most occurred in patients ultimately diagnosed with Crohn's disease. Functional results after cure of these perineal complications revealed no significant functional differences between control patients and those cured of anastomotic separations, anastomotic strictures, and pouch fistulas. Only a few minor differences were demonstrated in function after an episode of pelvic sepsis. The major deterioration in function occurred after treatment for multiple perineal complications. CONCLUSIONS: An appreciable number of perineal complications occur after ileal pouch-anal anastomosis. Pouch-perineal fistulas are associated with the highest pouch failure rate. The majority of these fistulas occur in patients ultimately diagnosed with Crohn's disease or indeterminate colitis. Although there is no substitute for good technique and sound clinical judgment in the success of ileal pouch-anal anastomosis, if perineal complications are successfully treated, functional outcome is equivalent to that in patients without perineal complications.     

Green fluorescent protein as a signal for protein-protein interactions

Green fluorescent protein (GFP) is autofluorescent. This property has made GFP useful in monitoring in vivo activities such as gene expression and protein localization. We find that GFP can be used in vitro to reveal and characterize protein-protein interactions. The interaction between the S-peptide and S-protein fragments of ribonuclease A was chosen as a model system. GFP-tagged S-peptide was produced, and the interaction of this fusion protein with S-protein was analyzed by two distinct methods: fluorescence gel retardation and fluorescence polarization. The fluorescence gel retardation assay is a rapid method to demonstrate the existence of a protein-protein interaction and to estimate the dissociation constant (Kd) of the resulting complex. The fluorescence polarization assay is an accurate method to evaluate Kd in a specified homogeneous solution and can be adapted for the high-throughput screening of protein or peptide libraries. These two methods are powerful new tools to probe protein-protein interactions.     

Higher order iminodiacetic acid libraries for probing protein-protein interactions

Higher order iminodiacetic acid diamide trimer (560 compounds) and tetramer libraries (1260 compounds) are described and were assembled in a convergent multistep solution-phase synthesis for use in studying protein-protein interactions.     

Higher order iminodiacetic acid libraries for probing protein-protein interactions

Full details of the preparation of iminodiacetic acid diamide dimer (2040 compounds), trimer (560 compounds), and tetramer (1596 compounds) libraries by multistep convergent solution-phase synthesis for studying protein-protein interactions are provided. The libraries were assembled in a format providing small 8-10 compound mixtures and the deconvolution of many of the small mixtures to identify screening leads by resynthesis of the individual components have been conducted for 320 of the individual compounds to date. A representative example of the subsequent exploration of the structure-activity relationships for an identified receptor binding antagonist (200 additional individual compounds) and steps taken for potential elaboration to a receptor dimerization agonist are defined with preparation of representative linked dimers (70 compounds).     

Characterization of Aspergillus isolates by pyrolysis mass spectrometry

Pyrolysis mass spectrometry (PYMS) is a useful typing method for many bacterial and Candida species. We attempted to type Aspergillus spp. by PYMS. Four distinct A. fumigatus isolates could not be distinguished from each other, whereas one A. niger and one A. terreus could. Poor reproducibility was shown using multiple identical cultures of a single A. fumigatus isolate and several isolates of the same DNA type. PYMS is obviously an unsuitable typing method for Aspergillus spp.