12 Jun Protein Functions of DISC1 Gene Linked to Schizophrenia Identified
MedicalResearch.com Interview with:
Marcelo Pablo Coba PhD
Assistant Professor of Psychiatry
Zilkha Neurogenetic Institute
Keck School of Medicine of USC
MedicalResearch.com: What is the background for this study? What are the main findings?
Response: Psychiatric diseases such as schizophrenia (SCZ) are complex brain disorders where a multitude or risk factors have been implicated in contributing to the disease, with a low number of genes that have been strongly implicated in a very low number of cases.
One of these genes is Disrupted in schizophrenia 1 (DISC1), which was first described in 2000 as a balanced translocation that segregates with schizophrenia and related psychiatric disorders in a large Scottish family. Because DISC1 does not have an identified protein function such as enzymatic, channel, transporter, etc… the field moved to try to understand what proteins are associated (physically connected) to DISC1 and to try to explain DISC1 function through the function of its protein interactors. This means that if DISC1 binds proteins X, Y and Z, then mutations in the DISC1 gene should affect the functions of these proteins. Therefore, there has been much effort in trying to identify DISC1 protein interactors. However this task has not been straightforward.
One of the most used methods to study protein interactions is the immunoisolation of target proteins (like DISC1) by specific antibodies directed to the target. Then different assays such as western blotting or mass spectrometry can be used to identify what proteins are associated to the target. One of the critical steps in these methods is to have specific antibodies that are able to recognize and isolate the target. This was a critical issue during some time as there has been controversy about the specificity of antibodies used against DISC1.
However, many DISC1 protein interactors have been identified by a variety of different methods. Almost all these methods used conditions that do not represent the cellular environment. For example these include in vitro systems or assays where the intracellular protein concentration of DISC1 was altered, thus not representing the real stoichiometry relationship of DISC1/interactor within cells.
In this way, it was postulated that DISC1 works as a scaffold protein, this means a protein that helps to localize and cluster a number of other proteins in what it is believed to be large-macromolecular protein complexes.
However, we were puzzled that DISC1 peptides were almost absent from Mass Spectrometry databases in particular from assays corresponding to neuronal tissues or neuronal samples. We also were unable to isolate DISC1 with a number of interactors that were reported to be associated to DISC1 in our own assays. Thus, we decided to insert a specific tag (3X-FLAG) coding sequence at the C-terminal end of the endogenous DISC1 gene using CRISPR/Cas9 genome engineering in human iPSCs. This allowed us to differentiate iPSCs into hNPCs and astrocytes. Then, the endogenous DISC1 was specifically isolated and DISC1 binding partners were determined by immunoisolation of DISC1-FLAG followed by HPLC-MS/MS in a cell type-specific manner. We were able to identify a number of novel DISC1 interactors that allowed us to define in a more precise manner the protein interaction networks that might be relevant for DISC1 function and showed that DISC1 is expressed at sub-stoichiometric levels when compared to its binding partners.
MedicalResearch.com: What should readers take away from your report?
Response: It is important to highlight the importance to consider protein stoichiometry when analyzing the role of protein-protein interactions. For example, DISC1 is usually described as a scaffold protein that interacts with a number of proteins. This gives way to the idea that mutations affecting DISC1 structure of protein concentration will translate into changes in the function of all their interactors. This is a misleading interpretation for any protein-protein interaction, but it’s particularly important for DISC1. We found that the protein levels for DISC1 are very low when compared with its interactors, therefore only a minimal number of each interactor might associate with DISC1 molecules. Thus it is dangerous to infer DISC1 functions through the analysis of its protein interactors. This is more important, if interactions are obtained in conditions that do not consider the stoichiometry ratios of proteins. It is probable that DISC1 might affect very specific functions from its interactors. This include specific cell types, developmental stages, and protein localization. These functions are generally more difficult to be spotted when using mouse genetics models.
It is also important to highlight that a number of functions that were previously associated to DISC1 might also be relevant at very early stages of development, including fetal brain and not only pools of neuronal progenitor but also astrocytes.
MedicalResearch.com: What recommendations do you have for future research as a result of this work?
Response: First I want to highlight that in studies such as the one that we discuss here, we are not defining direct protein interaction, or proteins that physically intereact. While this might be true for a number of the proteins identified in the interactomes, we don’t show a direct proof for that. There are a number of definitions that can be misleading, and we use the words interactomes, protein complexes, etc… While this might be clear for a number of readers, others might get a wrong interpretations. People using databases usually forget the conditions on how this so called “interactions” have been determined. I’m very concerned on how data coming from large-scale analysis is being interpreted and analyzed.
It is fundamental to recognize the specific conditions used to identify protein associations. They are useful as long as we recognize that they are highly dynamic and they represent associations in specific cell types, developmental stage and time/space conditions within the cell.
Unfortunately most of this information is missed when people uses databases or repositories. Therefore, it is common to find that many proteins are defined as scaffolds, just because there is data that point to a particular protein being associated to many protein “interactions”. In many cases this information is used to analyze and compare data obtained from human genetics to infer the role of proteins and their interactions in a number of human diseases. Unfortunately the misuse of data regarding protein interactions usually generates a variety of wrong interpretations and statements about the role of a number of proteins in disease.
Brent Wilkinson, Oleg Evgrafov, DongQing Zheng, Nicolas Hartel, James A. Knowles, Nicholas A. Graham, Justin Ichida, Marcelo P. Coba. Endogenous Cell Type-Specific DISC1 Interactomes Reveal Protein Networks Associated to Neurodevelopmental Disorders. Biological Psychiatry, 2018; DOI: 10.1016/j.biopsych.2018.05.009
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