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Interactions of matrix metalloprotease-1 with water-insoluble aggregates of alpha-synuclein and amyloid-beta peptide

Kamboj, Sumaer
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Abstract
Alzheimer’s Disease (AD), Parkinson’s Disease (PD), and Lewy body dementia (LBD) are the leading causes of neurodegeneration [1]. Currently, the cost of PD patient care is $52 billion per year and the cost of AD patient care is expected to reach $1.1 trillion per year by 2050. Irrespective of the debate about whether protein aggregates are the cause or consequence [2-4], ~50% of patients with AD [5], most patients with PD [6], and all patients with LBD [7] show intraneuronal cytoplasmic protein aggregates (primarily aSyn) that are toxic to cells [8]. In the context of AD, most patients also show extracellular aggregates with amyloid-beta peptide (Aβ), where Aβ is partially cleaved Aβ precursor protein (APP). As aggregates accumulate within or on neurons, the neurons are no longer able to function properly, leading to the symptoms associated with AD, PD, and LBD [9]. However, there is currently no drug to dissolve or degrade protein aggregates of neurodegeneration. Our central hypothesis is that it may be possible to degrade aggregates by leveraging broad-spectrum protease activity of matrix metalloproteases (MMPs). MMPs have diverse functions in the human body and are known to degrade extracellular matrix and non-matrix proteins [10, 11] and have intracellular functions [12]. Tetracycline and its derivatives, well-known inhibitors of MMPs [13], have shown therapeutic potential in AD [14] and PD [15]. MMPs can partially cleave aSyn [16-20], amyloid-beta precursor protein (APP) [21, 22], and Aβ [23-26]. This thesis takes the first step and presents experimental and computational results to show how MMP1, a collagenase in the 23-member human MMP family, interacts with aggregates of aSyn and Aβ at the single-molecule level. We have discussed how molecular insights into MMP1 interactions with aggregates can inform virtual screening of drugs for potential targeting of MMPs for a specific substrate and function. Briefly, we report quantification of allostery (communication between distant locations in the protein) using single-molecule measurements of MMP1 dynamics on aSyn- and Aβ-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. While MMP1 prefers open conformations with its two domains well-separated on aSyn, MMP1 prefers close conformations with the domains closer on Aβ. For both aSyn and Aβ, a two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since crystal structures of aSyn- and Aβ-bound MMP1 are not available, we performed molecular docking of MMP1 with aSyn and Aβ using ClusPro. We simulated MMP1 dynamics using different docking poses and matched the experimental and simulated interdomain dynamics to identify the appropriate poses. We used experimentally validated simulations to define conformational changes at the catalytic site and identify allosteric residues in the hemopexin domain having strong correlations with the residues at the catalytic site. We defined Shannon entropy to quantify MMP1 conformational fluctuations. We performed virtual screening of drugs against a site on selected aSyn- and Aβ-MMP1 binding poses and showed that lead molecules differ between free MMP1 and substrate-bound MMP1. In other words, virtual screening needs to take substrates into account for substrate-specific control of MMP1 activity. Molecular understanding of MMP1 interactions with aSyn- and Aβ-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs. We have organized the thesis into five chapters. Chapter one provides an overview of the current literature and places the thesis in the context of neurodegeneration. Chapter two describes our novel and patented method for purifying recombinant aSyn in E. coli and forming aSyn-induced protein aggregates without using chromatography. Chapter three presents interactions of MMP1 with aSyn-induced aggregates at the single-molecule level. Chapter four presents interactions of MMP1 with Aβ-induced aggregates at the single-molecule level. At last, Chapter five summarizes the results in the thesis and proposes a framework for testing the relevance of the results in C. elegans.
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