Browsing by Subject "Intrinsically Disordered Proteins"
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Item Characterization of the Roles of Intrinsically Disordered Regions from RNA-Binding Proteins in Phase Separation(2016-05-27) Lin, Yuan; Ross, Elliott M.; Liu, Qinghua; Tu, Benjamin; Rosen, Michael K.Eukaryotic cells organize complex biochemical reactions through compartmentalization. While many intracellular compartments are enclosed by membranes, others are not. Messenger ribonucleoprotein (mRNP) granules are membrane-less organelles that enrich RNA and RNA-binding proteins containing intrinsically disordered regions (IDRs). I demonstrate that IDRs, coupled with RNA binding domain and RNA, can phase separate in vitro, producing dynamic liquid droplets. Over time, these droplets mature into more stable states, as assessed by slowed fluorescence recovery after photobleaching and resistance to salt. Maturation often coincides with the formation of fibrous structures. Pathological mutation within IDRs leads to the acceleration of maturation. Different disordered domains can co-assemble into phase-separated droplets. In the case of the IDR from FUS (fused in sarcoma), I show that tyrosine residues are important in promoting phase separation. Either mutation of these aromatic residues or phosphorylation of the IDR disassembles liquid droplets. I further discover that the disassembly is due to the disruption of aromatic interactions mediated by critical tyrosine residues and therefore an increase in the overall solubility of proteins. Taken together, these studies demonstrate a plausible mechanism by which interactions between IDRs, coupled with RNA binding, could contribute to mRNP granule assembly in vivo by promoting phase separation. Progression from dynamic liquids to stable fibers may be regulated to produce cellular structures with diverse physiochemical properties and functions. Misregulation of maturation could contribute to diseases that are associated with aberrant mRNP granules. Posttranslational modifications of IDRs could modulate the assembly and disassembly of mRNP granules by altering the solubility of IDRs.Item Modulation of Nocturnin Phosphatase Activity through the Disordered Amino Terminus(August 2021) Wickramaratne, Anushka Christobel; Hibbs, Ryan E.; Green, Carla B.; Takahashi, Joseph; Conrad, NicholasThe endogenous circadian clock controls the rhythmicity of behavioral and physiological processes and this is entrained by the daily fluctuations in light and dark. Nocturnin (Noct) is a rhythmically expressed gene regulated by the circadian clock that belongs to the CCR4 family of endonuclease-exonuclease-phosphatase (EEP) enzymes. Its expression is induced by acute stimuli and loss of Nocturnin (Noct-/-) in mice results in resistance to diet-induced obesity on a high fat diet and confers a protective effect to oxidative stress in HEK cells. Modeling of full-length Nocturnin reveals a partially structured amino terminus that is disparate from its CCR4 family members. I show that Nocturnin functions as a phosphatase, catalyzing the removal of the 2′-phosphate from NADP(H). High sequence conservation of the leucine zipper (LZ)-like motif, the only structural element in the amino terminus, highlights the potential importance of this domain in modulating phosphatase activity. I use in vitro biochemical and biophysical techniques to demonstrate that the amino terminus and the LZ-like domain are necessary for preserving the active site cleft in an optimal conformation to promote efficient turnover of the substrate. This modulation occurs in cis and is additionally pivotal in maintaining the stability and conformational integrity of the enzyme. These new findings suggest an additional layer of modulating the activity of Nocturnin in addition to its rhythmicity in order to provide fine-tuned control over cellular levels of NADPH. This lays the essential groundwork necessary to further understand the role of the partially structured amino terminus in metabolism and the oxidative stress response through regulation of NADP(H) and NAD(H) levels.