Designing a Research Project to Facilitate Further Understanding of the Development of Melanocytes in Relation to Delocalized Cytoplasmic Pirin

Throughout the course of the last several months I have utilized several biology databases and much scientific literature to investigate the novel protein pirin and the PIR gene that encodes it. This week will be the apotheosis of my efforts, in which I will design a research project to conduct upon pirin that will, hopefully, help us to develop a better understanding of the protein. The question as to what type of experiment to design for pirin is an extremely broad one; over the course of this blog I have discussed pirin’s role as a transcriptional cofactor, its correlation with the maturation of myeloid hematopoietic cells and thereby its possible role in the development of leukemia, the potential for pirin to play a role in programmed cell death, and many other aspects of the protein that warrant further research (The UniProt Consortium, 2021). However, the research I wish to engage in revolves around pirin's potential to contribute to melanoma when decentralized from the cell nucleus (Licciulli, Luise, Zanardi, et al., 2010) (Madeira, Park, Lee, et al., 2019).

So the first question is where to start. Shocking, right? However, for someone such as myself whose lab experience goes no further than high school chemistry and a couple of digital lab simulations in college, the question is quite daunting. Let’s start with the use of immunohistochemical analysis as discussed in my blog post titled Using the European Bioinformatics Institute's Database to Determine Pirin Pathology. To summarize: A 2010 study (Licciulli, Luise, Zanardi, et al., 2010) found correlation between the presence of pirin in melanocyte cells' cytoplasm and the progression of melanoma using immunohistochemical analysis, a process in which, to quote myself, “Proteins are located in the body by inducing catabolic reactions that break a given substrate down into a colored product, with a higher absorbance of a corresponding wavelength of light indicating a higher concentration of the colored product and by extension the substrate that was catabolized to make it” (Abendroth & Dabbs, 1995).  Immunohistochemical analysis is a common method of detecting cancer, and essentially allows us to see what antigens antibodies are binding to (Carneiro, Barbosa, Takemura, et al., 2018). 

With further experimentation, the process of utilizing immunohistochemical analysis to detect pirin might allow us to develop new technique to detect melanoma in high risk individuals, which would enable much earlier preventative measures than diagnoses via the biopsy of visible lesions. Whereas the aforementioned experiment’s methodology involved forming microarrays composed of metastatic and benign melonomic cells as well as a control group, I am interested in a methodology that will allow us to utilize the detection of delocalized pirin as a preliminary source of detection. The methodology I propose would involve collecting and testing for delocalized pirin in epidermal tissue samples from individuals with no visible signs of melanoma, but who are simultaneously at a higher risk of melanoma, and then testing positive samples against externally visible metastatic and benign melanoma tissues as well as tissue from low risk and asymptomatic individuals.

The ideal candidate for this experiment is caucasian men, ages 60-65 with work histories involving long term UV exposure (Rastrelli, Tropea, Rossi, et al., 2014). Ideally, the candidate should be a long term resident of Australia or New Zealand, given the countries’ statistically high amount of melanoma diagnoses per capita (Matthews, Li, Qureshi, et al., 2017), though for practical reasons this may prove unfeasible. The tissue collected from candidates who give their fully informed written consent should undergo Immunohistochemical staining overnight in a controlled temperature environment and introduced with polyclonal rabbit PIR antibodies to be detected with a peroxidase substrate kit and transformed via an E. coli expression system, as was used in previous studies into pirin-melanoma relationships (Licciulli, Luise, Zanardi, et al., 2010). Growth and incubation of plated cell lines is an area in which, frankly, the research that I have attempted is far too technical for me to understand at my current ability level. However, once the slides have been properly prepared and incubated, staining can be obtained by utilizing DAPI staining on each slide for 5 minutes and observed with a fluorescent microscope (Ibid).

As previously discussed, the pirin encoding PIR gene is subject to a wealth of regulation via transcription factor binding sites (Kent, Sugnet, Furey, et al., 2002) as well as alterations in expression by interaction with proteins that are associated with phosphorylated growth factor receptors and responsible for DNA repair and cytoplasmic organization (NCK1 and NCKAP1, WASF2; Via the STRING protein-protein interaction database) (Szklarczyk, Gable, Nastou, et al., 2021). Furthermore, pirin itself has been demonstrated to display upregulation under microdoses of curcumin which have lead to increased oxidative regulation of the protein complex and transcription factor NF-kappaB and decreases in apoptosis (Panchal, Vranizan, Lee, et al., 2008), both of which have been linked to, though not confirmed to, play roles in the development of cancers (Karin, 2006) (Licciulli, Cambiaghi,  Scafetta, et al., 2009). These statements lead me to believe that UV radiation may very possibly affect how pirin is expressed, and thus, via alterations in the transcription of kappa light-chain genes, may contribute to melanoma. While the mechanisms by which pirin becomes delocalized may involve interactions with the aforementioned structural cytoplasmic proteins, this is ultimately outside the scope of this experiment and warrants further research. I hypothesize that this experiment will yield data suggesting pirin delocalization begins to occur in affected individuals prior to visible epidermal symptoms of melanoma occur. Specifically, high risk individuals will show a level of pirin delocalization in greater quantities than low risk individuals but less concentration that confirmed melanoma tissues, both metastatic and benign. The implications of this experiment may eventually result in the development of further immunohistochemical staining techniques capable of detecting melanocytes earlier in melanoma's pathology than existing methodologies.

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