Cis-Specific Binding Partners Identify Critical Molecular Mechanisms Prominent in Biological Development and Disease

Gonzales, Josephine

Cis-Specific Binding Partners Identify Critical Molecular Mechanisms Prominent in Biological Development and Disease

Abstract

A growing body of literature recognizes the cis/trans isomerization of the peptidyl-prolyl bond as playing key roles in processes that are important for development and disease in humans and plants. The atomically identical cis and trans isomers are structurally distinct and may have different binding partners, thereby cis/trans isomerization can serve as a molecular switch. In this work, the impact of cis isomers in two different biological systems is investigated. In the first system, Oryza sativa (Asian rice) utilizes the phytohormone auxin to regulate the proteasomal degradation of a family of transcription repressors, the auxin/indoleacetic acid (Aux/IAA) proteins. The cis isomer of a well-conserved Trp-Pro peptide bond in Aux/IAA proteins is selectively recognized by the corresponding E3 ligase only in the presence of auxin. This leads to an auxin-dependent, cis-specific degradation of the transcription repressor and activation of its regulated genes including its own. One family member, OsIAA11, interacts with the peptidyl-prolyl isomerase (PPIase) LRT2, an enzyme shown to be essential for lateral root initiation. Quantitative studies in vivo of the rate of auxin-induced degradation of OsIAA11 and the dependence of this rate on LRT2 are necessary to mathematically model the auxin circuit. Levels of OsIAA11 and LRT2 were quantified using fluorescent protein tags and confocal microscopy. We found that detection of the degradation rate of OsIAA11 in vivo is dependent on both temperature and the elapsed time post-transfection. These studies confirm that the degradation rate of OsIAA11 is already optimally tuned by endogenous LRT2, and the results are consistent with a potential role of Hsp90 in the rice auxin circuit. Overall, these findings suggest a mechanism by which additional factors such as Hsp90 and co-chaperone SGT1 might be involved in LRT2-dependent proteasomal degradation of OsIAA11. In Alzheimer’s disease (AD), the amyloid precursor protein (APP) is cleaved at the _-site by APP Cleaving Enzyme-1 (BACE1). Cleavage is necessary for the amyloidogenic pathway to produce amyloid-beta (A_) peptides found in senile plaques. The cytoplasmic tail of APP contains a cis isomer of the phospho-Thr668-Pro669 motif correlated with an increase in amyloidogenic processing of A_PP and the production of A_. A novel cis-locked cyclic dipeptide pCDP-DB mimics the cis isomer of the prolyl peptide bond and is known to inhibit the generation of A_ and secretion of the large BACE1-generated APP fragment sAPP_. Here we have utilized H4 neuroglioma cell lines overexpressing APP695 (WT7) or BACE1 (B18) and confocal microscopy. We found that the treatment with pCDP-DB treatment does not alter colocalization of APP and BACE1. Importantly, treatment does not block the endocytosis of APP. Additionally, treatment reduces the colocalization of APP and BACE1 to early endosomes. Although point mutations made within the APP cytoplasmic tail significantly impacted the proteolytic processing, these mutations did not inhibit endocytosis. We performed proteomics to identify potential pCDP-DB interacting proteins and gain insight on the mechanism of pCDP-DB bioactivity. These findings point to a hypothesized mechanism by which pCDP-DB enhances the lysosomal targeting of endosomes containing APP and BACE1, resulting in the lysosomal degradation of sAPP_ and A_.

Description

163 pages

Date Issued

2020-12

Keywords

APP; Auxin; Colocalization; OsIAA11; Protoplast

Committee Chair

Nicholson, Linda K.

Committee Member

Holowka, David Allan
Fromme, Chris

Degree Discipline

Biochemistry, Molecular and Cell Biology

Degree Name

Ph. D., Biochemistry, Molecular and Cell Biology

Degree Level

Doctor of Philosophy

Rights

Attribution-NonCommercial-ShareAlike 4.0 International

Rights URI

https://creativecommons.org/licenses/by-nc-sa/4.0/

Types

dissertation or thesis