Protein Function

National Human Genome Research Institute   (comparison of genomes of other species with our species to determine protein function)

Although genomics hold much promise, it is clear that analysis of DNA or RNA content alone is not sufficient to understand cell biology and disease. Furthemore, the estimated 30,000-40,000 protein-encoding genes in the human genome could result in 10-100 times this number of unique proteins through post-transcriptional and post-translational processing and modification.

Although the sequencing of complete genomes provides a list that includes the proteins responsible for cellular regulation, this does not reveal what these proteins do, nor how they are assembled into the molecular machines and functional networks that control cellular behavior. With the genomes of so many organisms completely sequenced, science and its new biomedical discipline of functional genomics, are faced with understanding the function of these newly discovered genes.

The regulation of many different cellular processes requires the use of protein interaction domains to direct the association of polypeptides with one another and with phospholipids, small molecules, or nucleic acids. Interaction domains can target proteins to a specific subcellular location, provide a means for recognition of protein posttranslational modifications or chemical second messengers, nucleate the formation of multiprotein signaling complexes, and control the conformation, activity, and substrate specificity of enzymes.

As an example, enzymes like kinases often generate modified amino acids on their substrates that are then recognized by interaction modules in signal transduction. For example, phosphotyrosine (pTyr) sites formed by the actions of tyrosine kinases bind effectors with pTyr recognition domains (i.e., Src homology 2 (SH2), whereas phosphoinositides produced by phospoinositide kinases recruit pleckstrin homology domains.

Mutant cellular proteins that cause inherited disorders can exert their effects through the loss of protein-protein interactions, or conversely, by the creation of aberrant protein complexes. This suggests that rewiring of protein-protein interactions could be used experimentally to alter cellular function. Understanding the network of cellular protein interactions should expand the scope for creating novel biological responses through engineered proteins or small molecules.

It is becoming increasingly clear that an important level of organization is provided by multi protein complexes because instead of proteins and substrates colliding in diffusion-dependent manner, proteins generally interact with each other and form larger assemblages in a time and space dependent manner.

The importance of studying complexes is that it allows to place proteins with unknown roles into a functional context that is provided by their associated partners, some of which may have a known function.

Analysis of protein complexes has some special challenges in that more than 10k different genes might be expressed at the same time in a single cell or tissue and diversity on the protein level is much higher. In addition diversity on the level of primary protein sequence and the presence of modificaitons, complexity is further increased when considering the dynamic range of expression levels of individual proteins. While some proteins are present thousand copies per cell, others are just represented by a few molecules.

Protein-Protein Interactions

Technical Approaches toward identifying Novel Protein Interactions

1. Reporter Assays: These assays have a variety of uses such as determining protein-protein interactions:

• CAT Assay: This assay uses a reporter gene that encodes for the enzyme chloramphenicol acetyltransferase (CAT) which transfers the acetyl group from acetyl-CoA to the antibiotic chloramphenicol. CAT assays have been instrumental in determining promoter activity. For example, a promoter of interest can be attached to the gene that encodes CAT. The construct can then be transfected into eukaryotic cells. If the promoter is active, the CAT gene will be transcribed.  The presence of the CAT can easily be detected by lysing the cell and incubating the lysate with [¹⁴C]chloramphenicol and acetyl-CoA. CAT will transfer the acetyl group from acetyl-CoA to the chloramphenicol forming acetylated chloramphenicol which can be easily detected by thin-layer chromatography.

• Yeast Two-Hybrid System: was originally developed by Fields and Song in 1989 and takes advantage of the finding that many eukaryotic transcription factors can be divided into two functionally distinct domains that mediate DNA binding and transcriptional activation. Since this assay has the advantage of being both rapid and easy to use, it has become the most frequently used assay to detect novel protein-protein interactions.

This assay uses a reporter gene to detect the physical interaction of a pair of proteins inside a yeast cell nucleus. It is used when you want to determine other proteins that bind to your target protein of interest. (1) Your DNA sequence that codes for your target protein is fused with DNA that encodes the DNA binding domain (DBD) of a gene activator protein (transcription factor) and introduced into yeast. The yeast cells will produce your target protein attached to this DNA binding domain. This construct will bind to the regulatory region of the reporter gene where it serves as "bait" to fish out proteins that interact with your target protein. (2) Now you just prepare the "prey" or the proteins that may potentially interact with your target gene of interest. To do this, you ligate DNA encoding the activation domain of a gene activator protein to a large mixture of DNA fragments from a cDNA library and introduce these constructs into yeast cells containing the bait. If the yeast cell has received a DNA clone that expresses a prey partner for the bait protein, the two halves of a transcriptional activator are united, switching on the reporter gene.

Some possible pitfalls with the yeast two-hybrid system is that 1) interactions in the yeast have to take place in the nucleus. Consequently, proteins that possess hydrophobic transmembrane domains will be unable to reach the nucleus. (This might be avoided by using libraries that express protein fragments). 2) Another class of troublesome proteins are those that interact with DNA or the transcription machinery, because they will activate the reporter genes in the absence of any protein-protein interaction when expressed as AD fusions and give false positives.

Another application of the Yeast Two-Hybrid System is the reverse two-hybrid system which is used to identify compounds in yeast that prevent protein-protein interactions. In other words, this is a negative rather than a positive selection process. The way this works is that yeast cells expressing different DB-X: AD-Y interactions and exhibiting a similar sensitive phenotype are seeded on the surface of a solid agar medium containing the medium for this phenotype. Compounds are then spotted onto the plate. A ring of growth is expected around non-toxic compounds that are able to penetrate the cells and dissociate one of the interactions expressed by the pool of cells (rescue).

2. Affinity Capture Techniques of Protein Complexes:

• Immunoprecipitation is perhaps the most widely used approach to isolating multiprotein complexes. Once putative partners of the target protein are identified, an antibody to one of the associated proteins can be used for another immunoprecipitation experiment. Such a series of immunoprecipitations of this type can be used to confirm the associations identified in the initial experiment.

• Affinity Capture  As an alternative to immunoprecipitation, the target protein can be fused to an epitope tag or a well characterized marker protein like the small enzyme glutathione S-transferase (GST). Commercially available antibodies directed against the epitope tag or the marker protein can then be used to precipitate the whole fusion protein, including any cellular proteins associated with it. If the protein is fused to GST, antibodies may not be needed at all since the hybrid and its binding partners can be readily selected on an affinity column with beads coated with glutathione.

Say you want to examine PKC multiprotein signaling complexes using glutathione S-transferase (GST). A procedure to do this might be (1) isolate cardiac mitchondria. (2) incubate the mitcohondrial lysates with recombinant GST-tagged PKC protein. (3) perform GST-based affinity pull-down assays to purify the PKC protein complexes. (4) separate the purified protein complexes by blue polyacrylamide gel electrophoresis (PAGE), a nondenaturing separation technique developed for the mitochondria that does not disrupt protein-protein interactions within the complexes. Parallel purification experiments are conducted using the GST epitope alone (i.e., GST-null proteins incubated with mitochondria) and solely the GST-PKC protein (without mitochondrial proteins) to serve as negative controls for the PAGE step. (4) excise from the gel the specific protein complexes and identify them using liquid chromatography-coupled tandem mass spectrometry analysis (ionization by electrospray).

The target protein of interest can also be immobilized on a solid support. One possible disadvantage is that the linkage of the target protein may interfere with a protein interaction that would otherwise occur. An advantage is that one need not be concerned about whether an antibody recognizes the appropriate epitope on the target protein in a complex. Covalent linkage can be achieved by incubation of the protein with an activated support, such as epoxyalkyl Sepharose, which reacts covalently with nucleophilic amine or thiol groups on the protein. Alternatively, recombinant proteins containing His-tag or FLAG-tag sequences can be generated. These then associate tightly with immobilized nickel resins or immobilized anti-FLAG antibodies. The bead linked protein is then incubated with a cell lysate or similar extract, which contains putative partners to form the multiprotein complex. The complex is then harvested by centrifugation of filtration and the associated proteins are dissociated form the complex, digested, and analyzed. As with immunoprecipitation, once one has identified putative partners, confirmation can be made by selecting one of the partners and preparing an immobilized version of that protein.

Tandem Affinity Purification (TAP): is similar to epitope tagging except that it involves the sequential utilization of 2 tags rather than one. This TAP tag consists of a (1) terminal protein A tag (which binds to IgG), (2) a tobacco etch virus ((TEV) cleavage site, and (3) a calmodulin-binding peptide tag (CB). This TAP tag is fused either to the N or C terminus of the target protein of interest. The TAP-tagged protein is expressed in cells to form a complex with endogenous binding partners. This tagged protein along with associated partners is then retrieved via binding of the protein A tag to immobilized IgG. The material is then eluted using the TEV protease. The eluate of this first affinity purification step is then incubated with Ca- calmodulin coated beads. The bound purified protein complex is then released for subsequent MS analysis by treatment with a Ca-chelator.

3. FRET (fluorescence resonance energy transfer): involves the indirect excitation of an acceptor, such as yellow fluorescent protein (YFP) by a directly excited donor, such as cyan fluorescent protein (CFP).

In FRET, two proteins of interest are labeled with a different fluorochrome such that the emission spectrum of one fluorochrome overlaps the absorption spectrum of the second fluorochrome. If the two proteins and their attached fluorochromes come very close to each other, the energy of the absorbed light will be transferred from one fluorochrome to the other. The energy transfer, called fluorescence resonance energy transfer (FRET) is determined by illuminating the first fluorochrome and measuring emission from the second. Thus protein interactions between the two proteins can be monitored in live cells.

4. Protein Chips:

Strenghts and weakness of commonly used affinity approaches for the retrieval of protein complexes.

        Approach                            Pros                                    Cons                                Application

Protein-DNA Interactions

Technical Approaches toward identifying Proteins with DNA binding activities

An important class of protein-protein interactions is that of proteins interacting with specific nucleic acid sequences, such as in the promoter elements of genes. These interactions involve not only interactions between several proteins associated with transcription and repair, but also critical interactions with defined nucleotide sequences.

1. Electrophoretic Mobility Shift Assay (EMSA): This technique has been instrumental in identifying binding sites for DNA binding proteins. Here, a radioactively labeled promoter or enhancer sequence is incubated with a nuclear extract containing a DNA binding protein. That DNA-protein complex is run on one lane of a gel and the DNA alone is run on another lane. The DNA-protein complex lane will have an extra slower running band (corresponding to the DNA-protein complex) in addition to the faster running free DNA band.

The DNA-binding proteins seen in EMSA can be identified using 2-D electrophoresis coupled with mass spectrometry. T

2. Chromatin immunoprecipitation (ChIP) is the best test to insure your protein binds with your promoter. In this assay you fix and lyse your cells, shear the DNA and then use an antibody which binds to your promoter to precipitate your DNA. For example, you might use the ChIP assay to assess histone H4 acetylation at the Il4 and IFNg regulatory regions. To do this you could purify T cells from the spleen and lympho nodes of mice. Chromatin complexes are then immunoprecipitated with antiboides to acetylated histones H4(+). PCR primers specific for the Il4 promoter is then used to amplify the precipitated DNA.

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