 Welcome to the MOOC course on Interactomics. Proteins are dynamic molecules which interact with a wide variety of biomolecules such as lipids, nucleic acid and various small drug inhibitors, metabolites and many other biomolecules to provide different type of significant information for physiological action. Proteins also interact with one another to form the large complexes. These complexes regulate various fundamental processes such as signal transduction and gene regulation. A detailed understanding of protein interactions provides an opportunity to understand the protein functional behavior. Today in this lecture I will describe what is interactomics and a historic perspective of the conventional techniques involved in studying interactions which includes yeast to hybrid and immunoprecipitation techniques. Let us first start with interactomics. Interactomic comprises the study of interactions and their consequences between various proteins and other cellular components. The network of all such interactions known as interactome which aims to provide better understanding of genome and proteome functions. Interactions can have different kinds of impacts. It could be positive as well as negative. The one which is shown here in the slide shows the negative impact. Biology has evolved several mechanisms that regulate interactions such as post-translation modifications and presence or absence of activator or inhibitor molecules. Interactions can also be modulated by altering the expression level of proteins. The aberrant interactions such as the one shown in the slide can lead to the dysregulation of cellular functions and ultimately diseases such as cancer may happen. Proteins interact with a variety of biomolecules. The interaction of proteins with another protein or other biomolecules help to perform these functions. These proteins after interactions form complexes that modify the proteins help in protein transportation as well as many properties in which they are involved. The field of interactomics aims to identify the function of uncharacterized proteins so that one can assign the new role of various proteins. The mechanism to regulate protein activity can also be understood by studying the interactions. The network of protein interactions provide very valuable information for the processes such as signal transduction and various type of pathways in which these proteins can be involved. There are different methods of studying interactions because interactions occur in various ways. There are few interactions which are very transient that will be very short duration whereas other interactions could be permanent which may alter the activity. Few interactions are very weak whereas other interactions could be very strong. Interactions can also be obligate or non-obligate. They can form homo oligomers as well as different kinds of oligomeric units which could combine to form the hetero oligomer. This just gives you a glimpse of the complexities involved in the interactions because studying the interaction require various type of technologies just because of the sheer diversity of the interactions as well as different type of complexities involved. So, what are the physical reasons for these interactions? It could be electrostatic, hydrophobic, hysteric or hydrogen bond etc. Protein-protein interactions have various potential effects. Few effects are described in the slide such as catalysis, protein inactivation, altered kinetics, alteration of substrate specificity for substrate binding, new binding site information etc. These are just a few examples of potential effects of protein-protein interactions but they alter magnitude of effects which ultimately lead to either positive effects or dysregulation which may result in various diseases. These proteins are involved in many binary interactions and understanding of the underlying biochemistry of proteins and biological interactions is critical element for the development of novel therapeutics and diagnostics. Protein-protein interactions have various potential effects. Some of these effects are described in the slide. They alter magnitude of effects which ultimately lead to either positive effects or dysregulation which may result into various diseases. So, protein interactions are very essential for any cellular mechanism whether to discuss about signal transduction, DNA replication, translation, cell cycle control, how the metabolic processes are governed, the modality, how the growth and morphology are altered, splicing, transportation etc. I am sure if you think about your biological questions of interest you will be able to add many more examples here how protein interactions are essential for the activity of the cell. So what is significance of studying protein interactions? These are very dynamic which interact with a wide variety of biomolecules as I discussed previously such as lipids, nucleic acids, small drug inhibitors and many other biomolecules. Proteins also interact with one another to form the large complexes which regulates signal transduction and gene regulation. Let me now describe you few concepts involved in the intractomics field by showing you this animation. Interaction studies of proteins with various biomolecules help in deciphering and understanding the functions of various proteins in the complex network of cellular pathways. Proteins interact with other biomolecules such as nucleic acids, lipids, hormones etc. to execute a multitude of functions in living organisms such as signal transduction, growth and regulation and metabolism. These are few examples there are many other cellular processes and functions in which these are also involved. Protein interactions with other biomolecules can be of several different types. They may be weak or strong, obligate or non-obligate, transient or permanent. The physical basis for these interactions include electrostatic, hydrophobic, steric interactions, hydrogen bonds etc. So now let us talk about different methods to study the protein-protein interactions. Understanding the protein-protein interactions provides important clues to the function of proteins. The identification of interactions with known proteins may suggest the functional role played by a novel protein. There are two broad ways of studying this protein-protein interaction. One is the traditional approach and then there are few high throughput approaches. The traditional approaches rely heavily on yeast to hybrid, affinity chromatography and immunoprecipitation. The more recent studies include high throughput approaches such as protein microarrays and different type of label-free biosensors. The traditional way of studying protein-protein interactions involved yeast to hybrid and immunoprecipitation. These are two widely recognized technologies which have been used to map the protein-protein interactions on a large scale. These are yeast to hybrid also known as YTH and IP with MS also known as IP MS technique. Both of these approaches have been used to identify thousands of novel interactors in different organisms including human, C. elegans, drosophila, etc. Let us first talk about immunoprecipitation technique. IP or tandem affinity purification also known as TAP is a technique used to purify the protein complexes and study the protein-protein interactions. Depending on the protein that need to be purified, different types of tags could be attached to the bait proteins. Through the immunoprecipitation method, the purification of protein complexes by IP or tandem affinity purification method is performed. The target proteins and its interacting partners are isolated from a given complex sample and then by using different kinds of tags, these proteins can be isolated from a mixture and then further processed which I will describe in the next slide. In IP method, the antibody specific to the bait is attached to the whole cell extract which forms the complex. Remember, this step is performed in the native or non-dening condition. The protein-protein complex is immobilized on protein A or protein G c-pherose beats. Protein complex is eluted and further analyzed on SDS pages. As you can see on the slide, it shows that the antibody is used which binds with the antigens and the unknown ligand and this complex is separated on SDS pages. This condition is done under denaturing condition whereas the first step was performed under non-denaturing conditions. What are the merits of using IP method? In this method, proteins are isolated in the native state. Why native state is important? The native state will allow the native complexes to be formed. It also allows the formation of post-translational modifications. It is essential to perform these steps in the native or non-denaturing conditions. Interactions by using the IP method are natural and by using these methods, large-order complexes can be observed because the native state will promote the native complex formation. What are the different demerits of using IP method? It has been reported that frequently, sticky proteins may be picked up. The proteins which are not specific, they could also be eluted out because they are sticky on the other protein surface. It means few proteins that are specifically interacting with target of interest will be isolated along with non-specific sticky proteins. It is unclear whether this interaction is direct or indirect because the proteins which are directly interacting will bind along with those indirectly bound interacting proteins will also be eluted. This method is also quite expensive because it needs a good equipment setup and different type of analytical and computational analysis. So, let us discuss the immunoprecipitation method, how the mechanism occurs in more detail by showing you this animation. In immunoprecipitation, the protein of interest is fused with a tab-tag which contains a Cal-modulin binding peptide, a TeV cleavage site and protein A. Depending upon the proteins to be studied, this tag can be modified. The tag is then bound to a column through affinity interactions between the protein A and IgG. The protein mixture whose interactions with the braid protein are to be studied is then added. Some of the proteins form a complex with the braid protein through specific binding interactions. The remaining unbound proteins are then washed away. It is followed by cleavage at the TeV site by the TeV protease to release only the protein complex bound to CBP. These reactions constitute the first affinity step. In the second affinity purification step, the braid pre-complex is bound via the CBP domain to a Cal-modulin functionalized column in the presence of calcium ions. The column is washed to remove any other unwanted contaminants after which the keratin agent is added which complexes the calcium ions. Once these are removed, the CBP-Cal-modulin interaction is weakened and leads to the release of purified protein complex. Once the protein complex has been purified, the components of the complex are separated by electrophoresis under reducing conditions. The LGS gel is then analyzed and the protein components evaluated thereby providing an understanding about the interactions with the braid protein of interest. So we have discussed the traditional ways of studying protein-protein interactions. First one was immunoprecipitation. Now let us move on to the next method which is yeast to hybrid. In yeast to hybrid, two types of proteins are involved, a braid protein and a pre-protein. Bade protein is protein of interest whose interaction is to be studied. The Bade protein is fused with the binding domain of the transcriptional activator by inserting it and expressing along with the binding domain in a suitable manner. The pre-protein is a protein whose interactions with the Bade needs to be determined and that is also known as pre-protein. The pre-protein is fused with the activation domain of the transcriptional activator. So the successful interactions of the Bade and the pre-protein activates the transcription of reported genes. The yeast to hybrid system was first demonstrated by scientists Fields & Song in 1989 for studying the protein interactions. Since then, this approach has been widely used in different organisms for different type of biological questions to be addressed. In general, the yeast to hybrid system uses the Bade binding protein and the pre-activation domain. These hybrid proteins are jointly expressed in the yeast nucleus. If the protein interactions established between the Bade and the pre which are coupled with BD and AD, then transcription will occur. As you can see the slide, the transcriptional activation for the two hybrid system consists of two protein domains. One is DNA binding domain which remains bound to the promoter region and is fused with a suitable Bade protein whose interaction with another protein is required for the study. The activation domain is the other domain of the transcriptional activator which is fused with the pre-protein. This activation domain when bound to the DNA binding domain forms the functional transcriptional B-activator and brings the expression of reporter genes. As you can see in the slide, the Bade protein binds with DNA binding domain and the partner protein or the pre with transcribe activator domain or AD domain. Once the BD binds with DNA, while the AD activates the transcription when both of these are associated, the Bade with the BD and pre with AD when they bind together the transcription event occurs. What are the different advantages of using yeast to hybrid system? As I mentioned, since 1989 when it was shown first time studying the protein interactions using yeast to hybrid, it has been used for various type of interactions including protein RNA, protein DNA, analysis of particular complexes as well as studying the large protein interaction networks. The protocols for yeast to hybrid is quite simple. Unlike the other approaches, there is no requirement for heavy instrumentation here. The method also allows to screen the large libraries very rapidly. What are different disadvantages of using yeast to hybrid system? It provides very high positive and negative rates. So high false positive rate has been one of the major limitation of this approach. The protein must localize and interact in the nucleus. So that is the limitation of the approach because it is restricted to the binary interactions or if you compare with immunoprecipitation, the complexes cannot be formed. Soplication in a non-east environment is also questionable because the system mostly used here is yeast. The protein folding if you are aiming for studying the mammalian system is not guaranteed. It is also not quite sensitive to the toxic genes. Finally, it is limited to the pairwise interactions. If you have fair idea about the molecules which are potentially interacting then you can use the pairwise study by using yeast to hybrid. But also if you just want to generate a list of potential interactors which may or may not be true then those can be quickly screened by using different libraries by using yeast to hybrid method. Once you have established the list of potential interactors then you can use different type of validation approaches for really establishing how many of these proteins are interacting well and how many of these are false positives. So the yeast to hybrid screening remains as a novel biological technique which can be used for screening and the discovery of protein-protein and protein other biomolecular interactions. Let me show you how yeast to hybrid works in the following animation. We will not talk about yeast to hybrid. In yeast to hybrid screening the binding of the transcriptional activator protein composed of binding domain and activation domain to the promoter region is essential for expression of the corresponding reporter genes located downstream of the promoter. The binding domain is fused with the bait protein while the activation domain is fused with the prey protein. Binding of either one of the fusion proteins to the promoter is not sufficient to bring about transcription of the gene. The two hybrid screening protocol uses this interaction as a basis for the screening protein interactions. When the bait protein bound with the binding domain interacts with the prey protein fused with the activation domain there will be expression of reporter gene which can be easily detected. LAXZ is a commonly used reporter gene whose protein product beta galactosidase leaves the substrate at scale resulting in blue color colonies. Let me show you the yeast to hybrid concept applied in one of the studies by showing this interaction. To study the protein-protein interaction between the vaccinia virus and human proteins in this study authors used the virus bait proteins fused with the C-terminus of Gal 4 binding domain BD while the prey human proteins were fused with Gal 4 activation domain of the transcriptional activator. These interactions were further validated by other techniques which gave a successful validation rate of 63%. Let me show you by this interaction how this yeast to hybrid screening can be used. If you drag and drop the components such as the transcription will be carried out you can see the first Gal 4 domain are here to the promoter region and now as soon as the activation domain binds their transcription events happen. So in summary today we talked about significance of studying interactions in the field known as intractomics. We discussed different types of traditional approaches which have been used for studying the protein interactions such as yeast to hybrid and immunoprecipitation assays. In the next lecture we will discuss a variety of platforms currently being used for studying the proteins in the high throughput manner by using the exciting field of protein microarrays. I will describe to you how different contents such as antibodies, verified proteins, peptides or even cell-free expression could be performed to make the protein microarrays. Thank you.