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(Above) ErbB4 Transmembrane Domain (2LCX) Source: RCSB Protein Data Bank
What is ALS?
What is ALS?
Amyotrophic lateral sclerosis is a debilitating illness characterized by sudden motor neuron death, resulting in widespread muscular atrophy. Ultimately, patients lose the strength to perform everyday functions, including "the ability to speak, eat, move and breathe" ("What is ALS?"). Most commonly, ALS appears in those "between the ages of 40 and 70," with a life expectancy ranging from about 2 to 5 years. There is no known cure for ALS, and only 4 drugs approved by the FDA to help treat the disease - and such drugs have varying degrees of effectiveness among patients. Furthermore, ALS is a difficult disease to recognize and can even take years to be formally diagnosed by doctors, at which point it is often too late to conduct efficient treatment ("What is ALS?").
Very little is known about ALS, including its cause. As someone who personally experienced ALS's far-reaching impact, I knew that any new information about this disease could not only aid future scientific pursuits and research, but also provide hope to other families impacted by ALS. I knew that any project focused on ALS could help make a difference, and I believe our project has done just that.
Establishing Connections
Establishing Connections
The initial focus of our experiment was ALS, and so we started by researching genetic and molecular connections to the disease. Using the DisGeNET database, we searched for genes with any correlations to ALS. At the same time, we scoured scientific papers for any information about associations to the disease that still needed to be explored. Multiple papers referred to ErbB4, a type of tyrosine kinase receptor protein which "plays an essential role in the function and viability of motor neurons" (Takahashi et al 1). One study in particular found that the immunoreactivity of certain ecto-ErbB4 fragments circulating within the cerebrospinal fluid of ALS patients was significantly lower than that of controls (Lopez-Font et al 1). This discovery is indicative of an impairment of the ErbB4 signaling pathway which could be "contribut[ing] to some of the complex processes involved in ALS-FTD pathology" (Lopez-Font et al 9). As a result, we decided to take a closer look at ErbB4, specifically the protein's proteolytic cleavage and its related enzymes to predict how they may impact the concentration and properties of the fragments produced.
To tie the project back to ALS, we identified two ALS-associated point mutations which are known to occur on the ErbB4 gene: one located at codon 927, and another located at codon 1275. In order to determine how the ALS mutations may be related to the ErbB4 pathway, we planned to compare the fragments produced through the proteolytic cleavage of wild-type ErbB4 and mutant ErbB4. There are many different proteolytic enzymes which interact with ErbB4, however the exact cut sites where fragments are cleaved are not clearly known. One of the enzymes we identified is TACE/ADAM17, which cleaves both ErbB4 and NRG1, and additionally serves as an inhibitor for Neuregulin-1, a ligand of ErbB4 (Yin et al). TACE was a relatively easy enzyme to acquire, and so we chose to conduct our experiment with TACE as the chosen proteolytic enzyme. In the end, we wanted our experiment to answer the question of how the two ALS-associated mutations on ErbB4 may alter or otherwise inhibit the protein's cleavage by TACE.
Originally, this experiment was going to be conducted in a lab, where we would measure the mass and concentrations of wild-type and mutant ErbB4 fragments produced through proteolytic cleavage by TACE. Our hope was to establish a connection between the TACE/ErbB4 pathway and the two specific ALS-associated mutations, so that we could identify whether or not those two mutations are to blame for faulty ErbB4 pathways in ALS patients. However, our project experienced many alterations in methodology as we encountered obstacles, and ultimately the project became a purely computational investigation. To adapt our purpose, we chose to use computational methods such as sequence alignment and PyMOL in order to answer our original intended research question.
Above: Location of ErbB4 mutationsSource: Human Protein Reference Database (edited by Kevin Collins and Sydney George)
--> Final Research Question <--
--> Final Research Question <--
Will mutations in the ErbB4 gene associated with amyotrophic lateral sclerosis (ALS) affect the proteolysis of the ErbB4 protein by TACE/ADAM17?
Will mutations in the ErbB4 gene associated with amyotrophic lateral sclerosis (ALS) affect the proteolysis of the ErbB4 protein by TACE/ADAM17?
Works Cited
Works Cited
Lopez-Font, Inmaculada, et al. “Decreased Circulating ErbB4 Ectodomain Fragments as a Read-out of Impaired Signaling Function in Amyotrophic Lateral Sclerosis.” Neurobiology of Disease, vol. 124, Apr. 2019, pp. 428–438., doi:10.1016/j.nbd.2018.12.021.
Takahashi Yuji, et al. “ERBB4 Mutations That Disrupt the Neuregulin-ErbB4 Pathway Cause Amyotrophic Lateral Sclerosis Type 19.” American Journal of Human Genetics, U.S. National Library of Medicine, pubmed.ncbi.nlm.nih.gov/24119685/.
“What is ALS?” The ALS Association, https://www.als.org/understanding-als/what-is-als.
Yin, D-M, et al. “Calcyon Stimulates Neuregulin 1 Maturation and Signaling.” Molecular Psychiatry, vol. 20, no. 10, 2014, pp. 1251–1260., doi:10.1038/mp.2014.131.
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