currents
An Affinity forSignaling Specific protein−protein interactions that often involve the assembly of large protein complexes containing cytoskeletal proteins as well as protein kinases and phosphatases and their substrates control cell signaling pathways. One way to study these interactions is by affinity purification using a bait molecule—typically one member of the complex. Recently, Matthias Mann and colleagues at the University of Southern Denmark (Odense) combined an affinity purification strategy with stable isotope labeling to identify proteins involved in epidermal growth factor receptor-mediated (EGFR) signaling (Nat. Biotechnol. 2003, 21, 315–318). The researchers cultured HeLa cells in media containing either 12C- or 13C-labeled arginine, and after 8 h of serum starvation, stimulated the labeled cells with EGF for 10 min. They then harvested and lysed the cells from each culture and combined the lysates at a 1:1 ratio based on protein concentrations. The researchers incubated the combined lysates for 4 h with Grb2GST fusion protein bound to glutathione-Sepharose beads, washed the beads extensively, and eluted the bound proteins from the beads, separating them by SDS-PAGE. Finally, the researchers excised the gel lanes in 10 bands and digested the proteins with trypsin before analyzing them by LC-MS/MS. From a starting pool of 228 proteins expressed by the HeLa cells, the researchers identified 28 proteins that were specifically enriched more than 1.3-fold by EGF stimulation. Upon sequencing the proteins, the researchers divided them into four categories. Some proteins, such as SOS-1 and Vav-2, were involved directly in signaling, while other proteins, such as the four subunits of AP-2, were involved in signal attenuation. Another group of proteins, including actin and keratin-8, were involved in
Sidechain Pattern M atching Although various protein structure initiatives have led to a dramatic expansion of the protein database (PDB), often all that researchers know about a new protein is the relative orientation of a few key residues in or near an active site. If these residues are consecutive in the protein’s primary sequence, standard sequence algorithms can easily scan for similar proteins in the database. Typically, however, these amino acids are sequentially distant, complicating database searching. To address this issue, in 1993 Peter Willett and colleagues at the Krebs Institute for Biomolecular Research (Sheffield, © 2003 American Chemical Society
cytoskeletal function, while a final group included proteins not previously associated with EGFR signaling. The researchers used confocal immmunofluorescence microscopy to confirm some of the associations identified by affinity chromatography. The researchers believe that their system allows for the identification of functional protein−protein associations while“avoiding the trade-off between false-positive binding and the ability to detect Com paratively speaking.Researchers couweakinteractions.”Furpled stable isotopic labeling and affinity purifithermore, by studying cation to identify proteins involved in EGFR endogenous protein signaling. (Reproduced with permission from complex formation, Blagoev, B.; et al. Nat. Biotechnol. 2003,, 21, the researchers reduce 315−318.) the likelihood of artifacts caused by overexpression. Finally, they are confident that if they perform the experiment at different time points, they should be able to study the kinetics of complex formation.
Although ASSAM England) developed works, Willett’s group ASSAM. This program looked for ways to furuses computational ther refine or expand techniques common the search method. to chemical structure Recently they added matching algorithms, features that would such as pharmaallow users to define cophore mapping, to the sidechains in probe protein strucgeneric terms such as tures for similar 3-D Taking a vector.Sheffield researchers used the serine proaromatic, acidic, or residue patterns. In ASSAM, amino tease catalytic triad from a-chymotrypsin as a database hydrophobic (J. Chem. query pattern, with arrows showing the position and direcInf. Comput. Sci. 2003, acid sidechains are tion of each computational vector. (Reproduced with perASAP). Similarly, they represented as vecmission from Spriggs, R. V.; et al. J. Chem. Inf. Comput. introduced features tors, where the main Sci. 2003,ASAP.) that would define a functional compogiven sidechain by its nents of a residue distances between the respecsecondary structure environserve as the vector’s start or tive starts, middles, and ends ment, distance from a known end. For example, a serine of the vectors of the sidevector might start at the backbinding site, and solvent chains being examined, and bone Cα and end at the accessibility. Also, because uses this matrix to probe the sidechain hydroxyl. The promain chain atoms can play gram then creates a matrix of PDB. significant roles in protein JournalofProteom e Research •Vol. 2, No. 2, 2003
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currents activity, the researchers incorporated main chain vectors into ASSAM. To test the newly refined ASSAM, the researchers performed various searches on almost 10,000 PDB entries. In one search of the catalytic Asp-His-Ser triad from α-chymotrypsin, ASSAM identified 360 of the 450 expected hits. Of the remaining 90, reexamination showed that the original prediction had been incorrect in 37 cases and mutations or covalent modifications caused ASSAM to miss another 48. Thus, ASSAM was able to correctly identify almost 99% of the expected proteins in the PDB that carry the catalytic triad. Having demonstrated the effectiveness of their system, the researchers are now developing a Web interface to provide remote access.
Regulatory Phosphorylation In eukaryotic cells, signal transduction relies heavily upon the phosphorylation of regulatory proteins by kinases. This is especially important in the regulation of synaptic nerve signaling, where associated axo-axonic synapses can depress or enhance neurotransmitter release by inhibiting or facilitating a specific axonal terminal. These effects result from the flow of signals into the synaptic cleft that separates the synapse and axon, where changes in local calcium concentrations modulate the secretion of a specific transmitter from the axonal terminal; the activity of specific protein kinases affect this system. Thus, a key question arises about whether the phosphorylation events directed by these and other kinases act sequentially or independently to alter synaptic signal transmission. To study this question, Alain Robichon and colleagues at the CNRS (Dijon,
France) followed three major proteins from neurosecretory vesicles to determine the effects of the coincidental arrival of signal-mediating kinases on the phosphorylation patterns of defined protein residues (J. Cell. Biochem. 2003, 88, 589–598). The researchers used various combinations of commercial protein kinase A (PKA) and C (PKC) and affinity-purified rat Cam kinase II with γ32P-ATP to radiolabel rat crude synaptic vesicles. They used SDS-PAGE or immunoprecipitation to determine which proteins were phosphorylated. By treating the vesicles with the various kinases, either in combination or sequentially, the researchers found that the phosphorylation effects are not additive, but vary qualitatively—individual phosphorylation events can either mask or unmask sites and thereby block or enhance subsequent kinase or phosphatase activity. For example, the protein P-38 was phosphorylated intensely by the combination of Cam kinase II and PKA but only weakly with each kinase separately. Also, PKA and PKC activity on the vesicles increased the affinity of Cam kinase II for the proteins synaptophysin and synapsin I. Similarly, the type and sequence of kinases used modulated the degree to which calcium inhibited the synaptic vesicles’ uptake of glutamate. On the basis of their results, the researchers concluded that in regulatory systems like this one, multiple phosphorylation events at vesicular sites induce protein associations or conformational changes that synergistically promote the efficient phosphorylation of other sites. In turn, this effect increases the regulatory capabilities and responsiveness of these systems.
tional in solid-phase extractionenables a large diameter and short column length and facilitates the equal flow of analyte solution throughout the entire disk. Moreover, the perimeter of a vessel, such as a pipette tip, can hold the membrane in place without any additional support. This feature further minimizes flow irregularities and makes StageTip assembly very straightforward. Overall, fabrication simply involves corking out the disks from a commercially available C18 bead-embedded Teflon mesh and placing them into pipette tips. An occasional user can easily manufacture about 5 of these tips per minute, say the scientists, at a materials cost of ,0.1 cent/disk. Preparation tips.Reversed-phase resin beads The researchers are embedded in a Teflon mesh, which is held in place by the tapering of the vessel. (Reproduced evaluated the C18with permission from Rappsilber, J.; et al. Anal. StageTips with staChem. 2003,75, 663–670.) ble-isotope-labeled digests. They used small amounts of peptide BSA, digested with trypsin in material. To improve this proeither H216O or H218O to assess and quantify the effects tein digestion-MS interface, of flow rate during peptide scientists from the University loading and elution. They of Southern Denmark then adjusted both of these (Odense) have designed a variables over wide ranges novel microtip procedure, and showed that neither that using what they call Stop and nor analysis with MALDI-TOF Go Extraction tips, or MS had any detectable influStageTips, for sample preence on analyte binding to the treatment (Anal. Chem. 2003, chromatographic beads. 75, 663–670). The scientists also preCentral to the StageTip pared dilution series of these design is a Teflon matrix digests to determine the useembedded with C18 beads fulness of StageTips for low formed into a very small sample amounts. The mean (about 60 nL in volume) disk16O/18O peak ratio value indishaped membrane. According cated complete sample recovto the authors, the Teflon ery down to peptide concenmaterial and the minute disk trations of 0.5 fmol/µL. On the volume discourage irreopposite end of the spectrum, versible peptide absorption. the researchers judged the The fixed position of the device’s loading capacity beadsas opposed to the using a combination of mealoose packing material tradi-
M S M icro-Prep
Achieving microscale concentration and purification of proteolysis-generated peptides for MS analysis has generally run into some challenges involving retention by reversed-phase beads within the microcolumn, ease and reproducibility of column packing, robustness of the column, and recovery of very
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currents surements on the BSA digests and on digests of the soluble lysate from E. coli cells (analyzed by LC/MS). They ascertained a capacity value of 2–4 µg for one 0.4 × 0.5 mm disk, which corresponds to about 50 pmol of a proteinlarge compared to most proteomic MS analysis requirements. Researchers can increase the column length by simply stacking additional disks both for greater capacity and to extract more complex samples.
M alarialProteom ics Of the three human malarial parasites, Plasmodium falciparum is the only one that causes the expression of unique adherent proteins on the surface of infected red blood cells (IRBCs). Significant to the etiology of the disease, these proteins trigger binding of the infected cells to a variety of endothelial surfaces, sequestering them in the microvascular capillaries of a variety of organs. Like small blood clots, IRBCs can accumulate in capillaries, damaging vital organs by blocking delivery of oxygen and nutrients and triggering production of toxic levels of proinflammatory cytokines. The adherent protein was previously shown to be a multidomain antigenic var gene family protein and was named the P. falciparum erythrocyte membrane protein 1 (PfEMP1). Using the suite of variants possible in the var gene, PfEMP1 has the ability to attach to different organs and to avoid the development of antibody defenses to all possible types. Over time, however, the body generally mounts a successful immune defense against whatever phenotypic forms of PfEMP1 are repeatedly expressed. However, because pregnancy is a unique condition that creates a hitherto unchallenged organ for infection in
individuals already harboring the parasite, the placenta becomes a new object of attack, which can result in potentially devastating effects on both mother and fetus. Only after several pregnancies cause the buildup of antibodies to PfEMP1 does it cease to be a significant danger. Previous research shows that the IRBCs bind condroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta. Recently, Rajeshwara Achur and colleagues at the Pennsylvania State College of Medicine (Hershey) further elucidated the nature of these placental binding proteins ( J. Biol. Chem. 2003, in press). The researchers isolated purified CSPGs for structural analysis using CsBr density gradient centrifugation of placental extracts followed by size-exclusion chromatography. They then used a combination of enzyme digestion, HPLC sizing, and carbohydrate composition analysis to demonstrate that the receptors consisted of a mixture of two major populations. Uronic acid content analysis using the carbazole−sulfuric acid method on the various column fractions demonstrated that these populations differed primarily in their sulfate contents2 to 3% and 9 to 14% of the chondrotin sufate chains are 4-sulfated (the remainder unsulfated) in the respective proteins. The researchers performed binding studies of IRBCs to purified CSPGs and assayed oligosaccharide fractions for their ability to inhibit adhesion, showing that the sulfate-clustered disaccharide chains in the placental CSPGs provided the molecular attachment site for the IRBCs. The researchers hope that knowledge of binding site architecture might lead to possible therapeutics, including potential vaccines.
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HKB11 or CHO cells with a Tat-oriP plasmid that proOne of the most challenging vided the transactivating Tat problems in the study of protein. After they had grown cloned genes is their inconsisthe cells for several days, the tent ability to efficiently scientists harvested and filexpress their proteins. Mamtered the tissue culture fluid malian cells such as Chinese (TCF) and stored it at –70 °C Hamster Ovary (CHO) cells for later screening. have long been the system of The researchers then perchoice for high-throughput formed ELISAs with antibodies against specific proteins to quantify the protein in the TCFs. In one experiment, they immobilized histagged V5 protein from the TCF in a multiwell plate, probed it with horseradish peroxidase-conjugated anti-V5 Protein production. Bayer researchers used antibodies, and ELISAs to determine the amount of protein secreted measured it colfrom HKB11 cells. (Reproduced with permission orimetrically. In a from Cho, M.-S.; et al. Biotechnol. Prog. 2003,19, second assay, they 229–232.) measured recombinant coagulascreening because they protion factor VIII (rFVIII) activity duce proteins that are corcolorimetrically using MEGA rectly folded and post-transla1, a U.S. working standard tionally modified. In 2001, antihemophilic factor. In a however, researchers at Bayer third test, the scientists added Corp. (Berkeley, CA) and the TCF to assay plates coated National Institute of Allergy with anti-interleukin-4 (IL-4) and Infectious Diseases antibodies. (Bethesda, MD) found that Using V5-ELISA, 85−92% huckebein (HKB11) cells were of the 230 cotransfected 10 times better at producing HKB11 cultures produced recombinant proteins than secreted proteins compared to CHO cells under similar cononly 60−70% of cells without ditions using a novel transthe transactivating Tat proactivator protein/trans-activatein. Also, three separate tion response (Tat/TAR-oriP) HKB11 cell lines produced 10 expression vector. times as much active rFVIII as More recently, Myungthe similar CHO cells. In sharp Sam Cho and colleagues at contrast, however, 3 to 4 times Bayer tested the ability of the more IL-4 was produced in Tat/TAR-oriP/HKB11 system CHO cells than in the HKB11 to produce secreted proteins cells. from a library of 230 genes Although the results indi(Biotechnol. Prog. 2003, 19, cate there is no optimal host 229–232). The researchers cell line for producing recomcloned the genes into a binant proteins, the authors p2TOP plasmid, which carried “believe that this higher suca TAR-oriP sequence that was cess rate of secretion results modified with a TOPO-V5-his from higher levels of protein element. They then transexpression using the Tat/TARfected these subclones into oriP expression vector.”
Recom binantProtein Production
