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Author ORCID Identifier

https://orcid.org/0000-0003-0230-7308

Document Type

Campus-Only Access for One (1) Year

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded

2019

Month Degree Awarded

September

First Advisor

Richard W. Vachet

Subject Categories

Analytical Chemistry | Biochemistry | Biophysics

Abstract

In dialysis patients, β-2 microglobulin (β2m) can aggregate and eventually form amyloid fibrils in a condition known as dialysis-related amyloidosis, which deleteriously affects joint, bone, and organ function, and eventually causes organ failure. To understand the early stages of the amyloid assembly process, we have employed a series of biophysical tools including chromatography, spectroscopy, and most especially, native electrospray ionization (ESI) together with ion mobility mass spectrometry (IM-MS) to study soluble pre-amyloid oligomeric species. We have also collaborated and integrated computational modeling to help better understand and rationalize the structural basis behind oligomerization.

Recently, several small molecules have been identified as potential inhibitors of β2m amyloid formation in vitro. In two chapters of this dissertation, we investigate if these molecules are more broadly applicable inhibitors of β2m amyloid formation by studying their effect on Cu(II)-induced β2m amyloid formation and examine their inhibitory mechanisms. We found that three molecules (doxycycline, rifamycin SV, and epigallocatechin gallate) can inhibit β2m amyloid formation in vitroby causing the formation of amorphous, re-dissolvable aggregates. Rather than interfering with β2m amyloid formation at the monomer stage, we found that doxycycline and rifamycin SV exert their effect by binding to oligomeric species both in solution and in gas phase. Their binding results in a diversion of the expected Cu(II)-induced progression of oligomers toward a heterogeneous collection of oligomers, including trimers and pentamers, that ultimately matures into amorphous aggregates. EGCG is similar, generating a separate set of new oligomeric species that are ultimately off-pathway and distinctly non-fibrillar.

Using IM-MS, we show doxycycline and rifamycin promote the compaction of the initially formed β2m dimer, which causes the formation of other off-pathway and amyloid-incompetent oligomers that are isomeric with amyloid-competent oligomers in some cases. Epigallocatechin gallate appears to deplete an important tetrameric conformer. Overall, our results suggest that doxycycline, rifamycin SV, and epigallocatechin gallate are general inhibitors of Cu(II)-induced β2m amyloid formation. Interestingly, the putative mechanism of their activity is different depending on how amyloid formation is initiated with β2m which underscores the complexity of how these structures assemble with different methods in vitro.

With our ESI-IM-MS measurements, we revealed the presence of multiple conformers for the dimer, tetramer, and hexamer that precede the Cu(II)-induced amyloid assembly process, which is a brand new observation for this system. Experimental and computational results indicate that the predominant dimer is a Cu(II)-bound structure with an antiparallel side-by-side configuration. In contrast, tetramers exist in solution in both Cu(II)-bound and Cu(II)-free forms. Selective depletion of Cu(II)-bound species results in two primary conformers – one that is compact and another that is more expanded.

Molecular modeling and molecular dynamics simulations identify models for these two tetrameric conformers with unique interactions and interfaces that enthalpically compensate for the loss of Cu(II). Unlike with other amyloid systems, conformational heterogeneity seems to be an essential aspect of Cu(II)-induced amyloid formation by β2m. Moreover, the Cu(II)-free models represent a new advance in our understanding of this critical event in Cu(II)-induced amyloid formation, laying a foundation for further mechanistic studies as well as development of new inhibition strategies.

Finally, we end by presenting preliminary data on efforts that we have made in the lab to begin to better characterize the oligomeric conformers that we have detected. This is achieved primarily by performing tandem mass spectrometry experiments to study unfolding behavior/pathways, or by using solution phase labeling (i.e. deuterium) to enhance IM-MS.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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