I. What problems does the Y2H method face when screening protein interactions? How to solve the problems of false negatives and false positives ?

The Y2H method faces the following problems when screening protein interactions: False negatives: False negatives in the Y2H method refer to protein interactions that cannot be detected due to the limitations of the screening method. For example, in the classic Y2H method, protein interactions involving membrane proteins are mostly undetectable. To solve this problem, it is necessary to select an appropriate Y2H strategy based on the cellular subproteinome of interest. In addition, the interaction between two proteins detected in Y2H is usually not symmetrical, which means that it depends on the fusion of a given protein in the bait or prey construct. The fused yeast reporter protein or anchor point may cause steric hindrance, resulting in false negatives. Other causes of false negatives may be different or absent post-transcriptional modifications of proteins in the yeast system. In this case, the modifying enzyme can be co-expressed in yeast with the bait and prey. In addition, very transient interactions may not be detected. Therefore, to solve the false negative problem, substrate trap mutants that lack phosphatase activity but retain affinity for the substrate can be used to identify the substrate of the phosphatase.

II. When performing two-hybrid screening, choosing the most appropriate vector system is an important decision. What are the advantages and disadvantages of the GAL4 and LexA systems? How to avoid common pitfalls when performing screening?

GAL4 and LexA are two commonly used two-hybrid systems, each with its own advantages and disadvantages. The GAL4 system is one of the most commonly used systems because it was the first commercially available system. Its advantages are ease of operation and wide application, but its disadvantages are the potential for background signals and false positive results. On the other hand, the LexA system uses the DNA binding domain of the bacterial LexA protein and the transcriptional activation domain of VP16 or B42AD. Its advantage is that it can avoid interference from endogenous GAL4 and GAL80 proteins in the GAL4 system, but its disadvantages are that the operation is relatively complicated and requires the use of additional selection markers. Therefore, when choosing a vector system, it is necessary to weigh the advantages and disadvantages of the two systems and make a choice based on the specific experimental requirements. Common pitfalls when performing two-hybrid screening include the following. First, because the two-hybrid system is a comprehensive screening method, false positive results may occur, that is, the detected interactions may be artificial or have no biological significance. Secondly, due to time and space constraints, the two interacting proteins may never be close to each other in the cell, or they may be located in different subcellular structures in the cell. In addition, the interacting proteins may be expressed at different stages of embryonic development or the cell cycle. Therefore, after the interacting proteins are identified, their biological significance needs to be further determined.

III. Interaction inhibition is one of the few techniques to evaluate the biological significance of the interaction between two proteins. How to screen out mutants that affect the binding of the target protein to its partner through the two-hybrid technology? How to restore the phenotypic changes of the mutants by finding binding inhibitors ?

First, the mutants that affect the binding of the target protein to its partner are screened through the two-hybrid technology. Once the mutant is found, the effect of this mutation on the phenotype can be studied. However, if it is found that the mutant of protein A does not interact with protein B, it cannot be ruled out that the interaction with the unknown protein C is also abolished and causes phenotypic changes. Therefore, in the second step, it is necessary to find a binding inhibitor, that is, to make a mutant B that can interact with mutant A and restore the phenotypic changes. This method has been used to study the Ras/Raf signaling pathway. In addition to confirming the results of the two-hybrid method, people usually want to confirm the interaction outside of yeast cells. Therefore, confirmation can be performed using a variety of methods, such as in vitro "pull-down" experiments and co-immunoprecipitation. Most experiments use epitope-tagged versions of the proteins. Alternatively, colocalization experiments can be used to verify two-hybrid interactions. In most cases, interactions found in two-hybrids can be confirmed, although it may take time to find the best conditions because most experimental protocols include empirical steps that need to be optimized for each pair of interactions. The most convincing experiments may be performed by co-immunoprecipitation of endogenous proteins.

IV. What are the advantages of two-hybrid technology?

1. It embodies an in vivo technology that uses yeast host cells as a living test tube. This yeast system is closer to higher eukaryotic reality than most in vitro methods or technologies based on bacterial expression. An attractive feature of this system is the minimal requirements for starting a screen. Compared with traditional biochemical methods that sometimes require large amounts of purified protein or high-quality antibodies, only cDNA, full-length or even partial genes of interest are required; 

2. Weak and transient interactions are more easily detected in two-hybrids because genetic reporter strategies lead to significant amplification. In the process of screening, there is a correlation between the identification of weak interactions and the number of false positives encountered, indicating that it is useful. In addition to the ability to screen libraries, two-hybrid systems can also be used to analyze known interactions. This can be achieved by pinpointing key residues that interact or by functional characterization of entire subdomains. By performing semi-quantitative experiments, one can even interpret affinity relationships from two-hybrid experiments. Studies have shown that the strength of interactions predicted by the two-hybrid approach is often correlated with the strength of interactions determined in vitro, allowing for the distinction between high, medium, and low affinity interactions. Furthermore, the binding affinity of peptides to retinoblastoma (Rb), as determined by surface plasmon resonance, correlated with the results of the two-hybrid assay.

V. In addition to the Y2H approach, what other methods can be used to validate high-throughput screening results? What role does computational biology play in addressing this issue?

In addition to the Y2H approach, there are other methods that can be used to validate high-throughput screening results. According to the reference information, the following methods can be used for validation: 1. Biochemical methods: including pull down assays, immunoprecipitation, and Biacore surface plasmon resonance. These methods can study physical protein interactions, but pull down assays require that the protein complexes are relatively stable, while Biacore requires purified interacting partners. These methods may be difficult for transient protein interactions or interactions with transmembrane proteins. 2. Intracellular/in situ methods: including methods such as colocalization, immunohistochemistry, and in situ hybridization. These methods can provide information on the co-expression and colocalization of two proteins, but usually cannot provide conclusive evidence of direct interaction. 3. FRET method: by studying the distance between two different fluorescent substances to study the spatiotemporal occurrence and physiological significance of the interaction. FRET can only occur when the distance between two directly interacting proteins is within a few nanometers. However, these methods are relatively cumbersome and can only be applied to a few interactions detected in larger screening. Computational biology plays an important role in validating high-throughput screening results. By integrating a large amount of bioinformatics data and computational models, high-throughput screening results can be validated and analyzed. For example, protein interaction network analysis methods can be used to evaluate the accuracy and reliability of screening results.