Proteomics And System Biology Modeling

A cell continuously senses environmental stimuli and relays that information inside across membranes to trigger a response. The process of signal transduction occurs by sequential transfer of information via protein-protein interaction. Take the example of a yeast cell growing in a glucose-rich environment suddenly encountering galactose. It senses the change in composition of the carbon source, shuts off the expression of genes required for glucose metabolism and turns on the genes required for galactose utilization. What is the mechanism by which the cell makes necessary adjustments? It is known that galactose itself can induce changes in gene expression within the yeast cells. The mechanism of regulation of gene expression by galactose is mediated by altering the interaction between GAL4p and GAL80p. As shown in Figure 6.16, in the absence of galactose GAL4p is in complex with GAL80p and is functionally inactive as a transcription factor. However, the presence of galactose induces recruitment of GAL3p to GAL4-GAL80 complex. This

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Galactose

Figure 6.16 Transcriptional regulation of galactose-inducible genes is mediated by protein-protein interaction. In the absence of galactose, GALA transcription factor is kept transcriptionally silent as a result of interaction with the negative regulator GAL80 protein. This inhibition is relieved in the presence of galactose by GAL3 protein, which binds GAL80 and displaces it from the GALA transcription activation domain.

Galactose

Figure 6.16 Transcriptional regulation of galactose-inducible genes is mediated by protein-protein interaction. In the absence of galactose, GALA transcription factor is kept transcriptionally silent as a result of interaction with the negative regulator GAL80 protein. This inhibition is relieved in the presence of galactose by GAL3 protein, which binds GAL80 and displaces it from the GALA transcription activation domain.

binding revives the transcriptional competence of GAL4p by relieving the inhibition caused by GAL80p. As a consequence, GAL4 induces transcription of genes required for galactose metabolism.

Identifying the interacting partners of every protein in a cell is paramount to the understanding of how cells initiate and coordinate myriad functions to maintain homeostasis. Stanley Fields devised a yeast two-hybrid method of discovering protein-protein interaction (Fields and Song, 1989). Briefly, the two-hybrid method detects protein-protein interaction by transcriptional induction of reporter genes. Two separate reporter systems are used, a nutritional marker that allows growth of yeast strains on a selective medium and an enzyme (beta-galactosidase), whose activity can be measured by colorimetric assay. The transcriptional induction of the reporter genes is dependent on the availability of a functional GALA protein. GAL4p is a modular transcription factor with a distinct DNA-binding and transcription activation domain. Each domain retains its function in the absence of the other. In the two-hybrid method, the DNA-encoding the test proteins are fused in vivo either to the DNA-binding domain or to the activation domain of the GALA transcription factor by homologous recombination in yeast. By themselves, the fusion proteins are incapable of inducing transcription. However, if the two test proteins interact with each other, the two domains of the transcription factor will be brought close together to reconstitute a functional GAL4 protein. As a result, the reporter gene under the control of GALA promoter will be transcriptionally induced. Positive interactors are selected by monitoring the expression of beta-galactosidase using a synthetic substrate that turns blue following the action of beta-galactosidase. Yeast cells containing positive interactors grow into colonies that appear bluish when grown on selective plates. The two-hybrid method is shown in Figure 6.17. Yeast two-hybrid libraries are constructed by fusing cDNAs (obtained from mRNA) with the activation domain of GALA protein (AD-fusion) and cloned in the prey plasmid. The genes of interest are fused to the DNA binding domain of GALA protein (BD-fusion) and cloned in a bait plasmid.

The two-hybrid technology was employed for the first time to initiate a genome wide interaction screen in S. cerevisiae. The complete sequence information of the yeast genome permitted cloning of all the roughly 6200 open reading frames (ORFs). Each gene (ORF) was screened against a yeast two-hybrid library and their interactors were identified. Since the first high throughput screen (Uetz et al., 2001) that detected around 1000 interactions, the yeast interaction database today holds about 5000 unique interactions. A

Figure 6.17 Yeast two-hybrid system. Yeast cells are transformed with plasmids expressing two separate fusion proteins, one containing the GAL4 DNA-binding domain (bait plasmid) and the other with the GALA activation domain (prey plasmid). The DNA-binding and the activation domains are brought closer to each other into a stable complex if the fusion proteins interact with each other, turning on the expression of the reporter gene. Positive interactors show growth on selective plates.

Figure 6.17 Yeast two-hybrid system. Yeast cells are transformed with plasmids expressing two separate fusion proteins, one containing the GAL4 DNA-binding domain (bait plasmid) and the other with the GALA activation domain (prey plasmid). The DNA-binding and the activation domains are brought closer to each other into a stable complex if the fusion proteins interact with each other, turning on the expression of the reporter gene. Positive interactors show growth on selective plates.

recent analysis of the interactions has revealed many biologically relevant protein complexes associated with distinct cellular processes (Bader et al., 2004).

A second approach of studying protein-protein interaction is by mass spectrometry. The major impetus of using a yeast system to analyze protein complexes by this method was the fact that the data generated using this developing technology can be cross-validated easily with the vast amount of genetic, biochemical and molecular biology information already available in this model organism. Two drug discovery companies have tagged about 1900 yeast proteins with an epitope tag and expressed them in yeast under the control of their native promoter. Protein complexes were purified from yeast lysates by affinity chromatography using an antibody against the tag. After separating individual proteins in the complex by denaturing gel electrophoresis, each protein band was excised from the gel, digested with trypsin and identified by Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). Together, the two studies identified 3018 interacting proteins (about half of all yeast proteins) distributed in a variety of biologically relevant complexes (Ho et al., 2002; Gavin et al., 2002).

High throughput protein-protein interaction analysis generates huge amount of data that is not easy to tease apart to extract biologically meaningful interactions from the noise. A straightforward approach to assess the quality of any interaction is to determine whether the interacting proteins are expressed together at any given time and also whether they localize within the same cellular compartment. S. cerevisiae was used to examine the expression and localization of the yeast proteome.

Erin K. O'Shea and Jonathan Weissman carried out a global analysis of protein localization in yeast (Ghaemmaghami et al., 2003; Huh et al., 2003). The objective of this experiment was two-fold: first, to localize proteins into specific cellular compartment, using a GFP tag; and second, to determine quantitatively the steady state level of all proteins in a yeast cell by western blot using a myc-tag. The researchers therefore generated two different strains of yeast in which 6109 of the 6243 predicted yeast-ORFs were fused either with a myc-tag or with a GFP-tag, providing for the first time a comprehensive view of the expressed proteome and its subcellular localization in a eukaryotic cell. Expression of proteins by western blot and protein fluorescence in yeast cells under normal growth conditions revealed that around 80% of the proteome is expressed. The abundance of individual proteins ranged from 50 to 1,000,000 molecules under normal growth conditions. The examination of protein localization at high resolution and sensitivity was achieved by GFP fluorescence. The study was able to localize (two-thirds) of the previously unlocalized proteins into 12 subcellular categories. A future goal is to understand how localization of proteins within a cell changes as a result of cellular signaling.

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