Olx

membranes are washed in distilled water and 50% alcohol, exposed to a phosphor-imager screen for 4 h to overnight and scanned to identify proteins binding calcium. Using this approach, many of the bacterially expressed proteins we tested were positive for calcium-binding under the conditions used (Figure 43.3B). This experiment demonstrates the feasibility of using bacterially produced protein to investigate Ca2+-binding. Two types of protein microarrays could be used to identify calcium-binding proteins. To verify the calcium binding of all the identified putative calcium-binding proteins, a chip could be constructed with the specific proteins identified in the database search and then probed with 45Ca2+ (Figure 43.3C). The binding conditions, especially Ca2+ concentration, could be varied as calcium-binding conditions differ for different calcium sensors. The second type of assay would involve complete proteome chips. Probing these chips would identify calcium sensors that do not have a conserved calcium-binding domain as well as those whose calcium binding has been predicted. In this case a protein chip with proteins representing the proteome of an organism could be probed with the 45Ca2+ under varied conditions (Figure 43.3C). These chip assays would not only identify calcium sensors but would also differentiate sensor affinity to Ca2+.

Identification of Targets of Calcium Sensors

The diverse range of calcium sensors suggests that they are likely to interact with a large group of targets. Several approaches are available for identification of target proteins. Some of these have been used successfully to identify targets of some calcium sensors, especially CaM and CBLs.

Screening of Expression Libraries. Radioactive and nonradioactive methods for isolation of cDNAs encoding CBPs have been developed and successfully used for screening many expression libraries [11, 12]. Expression libraries can be plated and screened with either biotinylated CaM or 35S-CaM (see Figure 43.4A for flow sheet). Positive colonies can then be plaque-purified using two more rounds of screening (Figure 43.4B) and the plasmid DNA is sequenced. Using this method, nearly 150 CBPs have been identified [12-15].

Y2H Screens. Y2H screens are another method for identifying calcium sensor targets by PPI. Individual Y2H assays have identified many target proteins. The Y2H system was used to identify CBL-interacting proteins and their interactions with the 18 kinases that are their targets [16]. However, screens with one clone/protein at a time are time-consuming and laborious. Global Y2H assays have been attempted using proteins from organisms ranging from yeast to humans [17, 18]. Complete pro-teomes have been used to construct all ORFs in bait and prey constructs to be used in genome-wide large-scale binary screens conducted using automated, robotic platforms. These screens have generated a large body of interaction data. Using binary interactive Y2H assays for a subset of the proteome such as the calcium sensors and their targets could identify interaction networks among these proteins. High-throughput Y2H assays with the calcium sensors as bait against all Arabidopsis ORFs as prey should

Plate plaque expression library

Incubate at 42°C until plaques appear

Place filters pre-soaked in 10 mM IPTG on plates

Incubate at 37°C overnight

CaM+ Ca2+

Remove filters and wash in Ca-containing buffer

Incubate in35S-calmodulin 1

Wash 1

Expose filters to X-ray film or phosphoimager screen

Incubate in avidin followed by biotin

Rinse filters in Ca2+-buffer

Incubate in biotinylated calmodulin

Incubate in ABC-HRP (Vectastain)

Detect with DAB

Identify positive clones and purify by secondary screens

FIGURE 43.4. Screening of expression libraries for calcium sensor targets. (A) Flow sheet for screening an expression library with a labeled sensor. (B) An example of a filter from a screen showing binding of labeled CaM to a target protein in the expression library.

identify many sensor targets. However, the Ca2+ requirement of the bait proteins needs to be met in some way for these assays. This problem was overcome for a Y2H assay using a CDPK (AtCPK11) as bait [19]. The bait protein in this case was expressed as either constitutively active or catalytically inactive overriding the requirement for Ca2+. Novel approaches like this need to be developed for effective use of calcium sensors as bait.

Using Protein Chips to Identify Calcium Sensor Targets and Interaction Networks between Calcium Sensors and Targets. Proteome chips are potentially the most useful method for identification of calcium sensor targets. The protein chips used for screening for calcium binding can be used for PPI screens and activity screens for enzymes (Figure 43.5B, C). Protein chips have been used for the global analysis of protein phosphorylation in yeast [20] and protein modification that involved covalent attachment of a small Ub-like modifier protein to target proteins [21]. A protein array containing 1690 proteins from Arabidopsis was used for identifying phosphorylation candidates for the protein kinases MPK3 and MPK6 [22]. From this small portion of the proteome (6.7%), 48 and 39 candidates respectively were identified. Because as there are 34 CDPKs in Arabidopsis, studying phosphorylation of

FIGURE 43.5. Proteome chip assays. Chips on the left contain proteins expressed from each ORF from Arabidopsis. Possible probes are given in the middle lane and mock detection chips on the right show some positive spots. The possible information gained is listed in the last column. (A) Probing with the GST antibody is for quality control, showing that the protein is on the chip and that there are equal amounts of each protein. (B) Known calcium sensors can be used as probes with varying concentrations of Ca2+ to identify targets and affinity to the targets at each Ca2+ concentration. (C) Sensors that have possible enzymatic functions can be used in enzymatic assays by providing the necessary conditions—that is, labeled ATP for CPK and kinase assays. Phosphorylated targets on the chip could be identified. For phosphatase assays, the proteins on the chip would need to be phosphorylated first and then incubated with the phosphatase. (D) Known sensor targets could be used as a probe with varying concentrations of Ca2+ to identify other sensors that might interact with the target and the affinity of the interaction. Or an activated target (in the presence of the interacting sensor and Ca2+) could be used as a probe to identify proteins (other than the sensor) that interact with it.

FIGURE 43.5. Proteome chip assays. Chips on the left contain proteins expressed from each ORF from Arabidopsis. Possible probes are given in the middle lane and mock detection chips on the right show some positive spots. The possible information gained is listed in the last column. (A) Probing with the GST antibody is for quality control, showing that the protein is on the chip and that there are equal amounts of each protein. (B) Known calcium sensors can be used as probes with varying concentrations of Ca2+ to identify targets and affinity to the targets at each Ca2+ concentration. (C) Sensors that have possible enzymatic functions can be used in enzymatic assays by providing the necessary conditions—that is, labeled ATP for CPK and kinase assays. Phosphorylated targets on the chip could be identified. For phosphatase assays, the proteins on the chip would need to be phosphorylated first and then incubated with the phosphatase. (D) Known sensor targets could be used as a probe with varying concentrations of Ca2+ to identify other sensors that might interact with the target and the affinity of the interaction. Or an activated target (in the presence of the interacting sensor and Ca2+) could be used as a probe to identify proteins (other than the sensor) that interact with it.

target proteins using protein chips would provide valuable information about autophos-phorylation and the targets of the individual CDPKs. Phosphorylation assays could be performed with each CDPK using Arabidopsis proteomic chips, which should be available in the near future (Figure 43.5C). The analysis of the EF-hand-containing proteins in Arabidopsis also identified other putative enzymes as calcium sensors that could be used in enzymatic reactions using the protein chip platform [6] (Figure 43.5C).

A proteome chip study relevant to calcium signaling was done as a proof of concept for assaying protein chips. A yeast proteome was expressed, purified, and printed on chips, and the chips were then probed with biotinylated CaM in the presence of calcium [23]. Six known CaM targets were detected along with 33 additional partners including many types that were consistent with a role for CaM. This type of assay should be useful for detecting the targets of the many other calcium sensors, and analysis of the target proteins can lead to identification of domains that bind the sensors. The chips could also be probed with target proteins to identify sensors and activated targets to identify secondary targets involved in a signaling network (Figure 43.5D). A major advantage of the high-throughput protein chips is that complete proteomes can be screened in one assay, and multiple assays changing the concentrations of Ca2+, sensor, or target can be performed yielding not only PPI pairs but also the conditions required for interaction. Overall, whole proteome chips will be useful in identifying (i) all calcium-binding proteins, (ii) targets of calcium sensors, (iii) differences in calcium affinity of sensors, and (iv) global calcium signaling networks.

Analysis of Protein Complexes in Calcium Signaling Using TAP. Y2H

interaction studies and the PPI studies using proteome chips are very informative for direct interactions between proteins. However, many components of signaling networks form complexes of proteins that interact directly and indirectly with each other. In order to identify members of complexes, a complete complex needs to be isolated and analyzed. One method developed for this is TAP (Chapters 36 and 37). A cassette consisting of the protein of interest as a fusion to two affinity tags, protein A that binds IgG-sepharose and a CBP that binds CaM-sepharose, is introduced into an organism and expressed at a level equal to its endogenous level. Cell extracts are processed and purified by first using IgG-sepharose, eluting the bound proteins with TEV protease and then binding to CaM-sepharose. Eluted proteins are treated with trypsin, and the resulting peptides can be analyzed by LC-MALDI-TOF-MS and/or nESI-MS/MS. Proteins are then identified by peptide or spectra searches against protein databases such as Mascot (http://www.matrixscience.com) or Sequest (http://www.thermo.com). This method was used in a high-throughput manner for the yeast proteome. Over 4500 proteins were tagged and 2357 purifications were successful, yielding identification of 4087 proteins organized into 547 protein complexes averaging 4.9 subunits per complex [24].

A great advantage to this method is that it is in vivo where the appropriate conditions (i.e., Ca2+ levels, presence of all interacting proteins in appropriate concentrations and locations, etc.) are met. This is particularly important for identification of interactions between calcium-sensor targets and their interacting proteins because the activated sensor (or possibly, lack of activated sensor) is necessary for the interaction to occur. The TAP method has been modified for specific uses; in the case of proteins involved in calcium-binding protein networks, the calmodulin-binding protein tag would be inappropriate. In this case the secondary tag could be a His or GST tag and purification would be by anti-His- or GST-sepharose. The calcium sensors and sensor targets identified by bioinformatics, and PPI assays could be used as a subset of proteins to be TAP-tagged and used to isolate complexes for analysis.

Deciphering Calcium Signaling Networks using Protein Chips and Other High-Throughput Methods

The ultimate challenge is to decipher the calcium signaling network—to elucidate all events from a signal to a response and when and how the different components of the signaling pathway are involved. Computational approaches are being developed to analyze the data generated from the proteomics approaches and to integrate data from other high-throughput approaches [25]. The goal of these models is to take qualitative and temporal information and produce a model that explains the relationship between given proteins. These models could organize, display, and simulate the complex protein interaction networks involved in calcium signaling. This type of modeling was used to construct a model of guard cell ABA signaling [26]. In order to model the proteins and their interactions, we need to identify all individual PPIs between calcium sensors and targets and the targets of the sensor targets and their spatial and temporal information. Protein chip assays are a potential high-throughput approach to produce a large body of information for elucidating networks. Protein chips containing potentially all the ORFs for a species can be used in independent assays using a series of proteins and conditions.

Was this article helpful?

0 0
How To Win Your War Against Allergies

How To Win Your War Against Allergies

Not Able To Lead A Happy Life Because Of Excessive Allergies? Want To Badly Get Rid Of Your Allergy Problems, But Are Super Confused And Not Sure Where To Even Start? Don't Worry, Help Is Just Around The Corner Revealed The All-In-One Power Packed Manual Containing Ample Strategies And Little-Known Tips To Get Rid Of Any Allergy Problems That Are Ruining Your Life Learn How You Can Eliminate Allergies Completely Reclaim Your Life Once Again

Get My Free Ebook


Post a comment