Parkinson's Disease (PD) is a neurodegenerative disorder linked to the loss of dopaminergic neurons in the midbrain. A key pathological marker of PD is the presence of Lewy bodies, which are mainly composed of misfolded α‐synuclein protein. α‐synuclein is a highly post‐translationally modified protein. While phosphorylation and nitration of α‐synuclein are well studied in the context of PD pathology, less is known about sumoylation, which is proposed to be neuroprotective based on limited studies. The majority of sumoylation takes place on the lysine‐96 and lysine‐102 sites of α‐synuclein, and it increases the protein's solubility. The goal of this research was to better understand the role of sumoylation in regulating α‐synuclein toxicity, and we performed four studies towards it. First, we evaluated the effects of blocking sumoylation on α‐synuclein in the well‐established budding yeast model for PD and found that α‐synuclein becomes more aggregated, gains toxicity, and loses localization at the plasma membrane. Second, we evaluated the effects of altering sumoylation pathways by using yeast strains with reduced (ulp1ts) or excessive sumoylation (smt3ts), and found that α‐synuclein aggregates more with reduced sumoylation, but becomes less toxic with increased sumoylation. Third, we asked how altering phosphorylation of α‐synuclein would alter sumoylation's protective role and found that blocking phosphorylation (in α‐synuclein already blocking sumoylation) reduced the protein's toxicity. Finally, we began evaluating whether blocking sumoylation and altering phosphorylation on familial PD mutant versions of α‐synuclein would exacerbate its toxicity. We found preliminary evidence that the toxicity of the A53T mutation increases when sumoylation is blocked. In the future, we will conduct further studies to understand how sumoylation affects other variants and modifications of α‐synuclein.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Colorectal cancer (CRC) is a common, and often incurable, form of cancer. Gene silencing by CpG island hypermethylation often plays a role in CRC progression. Certain regions of the genome, called high confidence differentially‐methylated regions (DMRs), are consistently hypermethylated across numerous patient samples. In this study, we used bisulfite PCR sequencing to investigate methylation levels at DMRs in the promoter region of CDKN2A, DKK3, EN1, MiR34b, SPG20, and TLX1 in HCT‐116 CRC cells. We observed that for all DMRs except CDKN2A, the demethylating agent decitabine significantly reduced CpG methylation. Using ENCODE project data, we observed that transcriptional activator binding inversely correlates with DNA methylation at all of these sites across diverse cancers and cell types. Our data increase resolution of the methylation status at the above DMRs, show the reversibility of methylation at these sites by decitabine, and the likely role of hypermethylation at these sites in gene silencing. In the future, we plan to test if DMR any specific gene silencing protects HCT116 cells.Support or Funding InformationThis work is supported by the Lake Forest College biochemistry and molecular biology program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background: Gene silencing by CpG island hypermethylation often plays a role in colorectal cancer (CRC) progression. Certain regions of the genome, called high confidence differentially-methylated regions (DMRs), are consistently hypermethylated across numerous patient samples. Methods: In this study, we used bioinformatics and bisulfite PCR sequencing of HCT-116 cells to investigate methylation levels at DMRs in the promoters of six genes: DKK3, EN1, MiR34b, SDC2, SPG20, and TLX1. We then investigated whether the anti-cancer drug decitabine, had a demethylating effect at these promoter regions. Results: We found that hypermethylation correlated with lack of transcriptional enhancer binding in these six regions. Importantly, we observed that for all DMRs, decitabine significantly reduced CpG methylation. Decitabine also reduced clonogenic survival, suggesting that there is a correlation between lower CpG island methylation levels and reduced cancerous properties. Conclusions: Our study provided single-nucleotide resolution and revealed hypermethylated CpG sites not shown by previous genome-wide methylation studies. In the future, we plan to perform experiments that demonstrate a causal link between promoter hypermethylation and carcinogenesis and that more accurately model treatments in CRC patients.

Parkinson’s Disease (PD) is a neurodegenerative disorder linked to the loss of dopaminergic neurons in the midbrain. A key pathological marker of PD is the presence of Lewy bodies, which are composed of misfolded α‐synuclein protein. α‐Synuclein is highly post‐translationally modified in healthy and diseased states. While phosphorylation and nitration of α‐synuclein are well‐studied as contributors to PD pathology, less is known about SUMOylation, acetylation, and glycation. Also, the combined effects of these modifications remain largely unclear, on both wildtype α‐synuclein (linked with sporadic PD) and the six mutant forms (A30P, E46K, H50Q, G51D, A53T, A53E) linked with early‐onset familial PD. We first evaluated the effects of blocking SUMOylation on α‐synuclein in the well‐established budding yeast model for PD and found that α‐synuclein becomes more toxic and aggregated, losing its membrane localization. Second, we found that SUMOylation and phosphorylation counteract each other in toxicity and localization. Third, we expanded our investigation to two newly reported modifications – acetylation and glycation. We found that acetylation is protective and glycation is harmful. When we combined acetylation or glycation manipulations with SUMOylation and phosphorylation alterations on the α‐synuclein level, we found that the effects of these modifications are not additive – the impacts of acetylation and glycation depends on phosphorylation status. Furthermore, we found that decreased nitration protects against toxicity and reduces α‐syn aggregation in the mutants modifying SUMOylation and phosphorylation. Finally, we investigated the effects of the familial mutants in tandem with altered acetylation, glycation, or nitration. We report two familial mutant‐specific effects: H50Q is surprisingly sensitive and non‐toxic in a hyper‐acetylation environment, while A53E is highly toxic with hypo‐glycation. We also found that too much nitration exacerbates toxicity of the familial mutants relative to low nitration. Specifically, mutant A53T is highly sensitive to excess nitration while H50Q is non‐toxic in such environment. These studies show the relevance of covalent modifications in sporadic and familial PD, and their in‐tandem effects that underlie toxicity mechanisms. Support or Funding Information YG= supported by Parkinson's foundation‐American Parkinson's disease association, A.B,C.M, A.R and P.J: Research grants from Neurosci