Determining the Effect of Catechins on SOD1 Conformation and Aggregation by Ion Mobility Mass Spectrometry Combined with Optical Spectroscopy

Abstract

The aggregation of Cu,Zn-superoxide dismutase (SOD1) plays an important role in the etiology of amyotrophic lateral sclerosis (ALS). For the disruption of ALS progression, discovering new drugs or compounds that can prevent SOD1 aggregation is important. In this study, ESI-MS was used to investigate the interaction of catechins and SOD1. The noncovalent complex of catechins that interact with SOD1 was found and retained in the gas phase under native ESI-MS condition. The conformation changes of SOD1 after binding with catechins were also explored via traveling wave ion mobility (IM) spectrometry. Epigallocatechin gallate (EGCG) can stabilize SOD1 conformation against unfolding in three catechins. To further evaluate the efficacy of EGCG, we monitored the fluorescence changes of dimer E,E,-SOD1(apo-SOD1, E:empty) with and without ligands under denaturation conditions, and found that EGCG can inhibit apo-SOD1 aggregation. In addition, the circular dichroism spectra of the samples showed that EGCG can decrease the β-sheet content of SOD1, which can produce aggregates. These results indicated that orthogonal separation dimension in the gas-phase IM coupled with ESI-MS (ESI-IM-MS) can potentially provide insight into the interaction between SOD1 and small molecules. The advantage is that it dramatically decreases the analysis time. Meantime, optical spectroscopy techniques can be used to confirm ESI-IM-MS results. Graphical Abstract ᅟ.

CIUSuite 2: Next-Generation Software for the Analysis of Gas-Phase Protein Unfolding Data

“…Many reports have described using CIU to assess ligand binding to a variety of protein targets , in an effort to build information-rich small-molecule screening platforms. CIU can be used as an analogue to stability shift assays commonly carried out in solution, as differences in gas-phase stabilities can offer similar information for unpurified samples at lower concentrations and potentially resolve intermediate transitions that may be missed in low-resolution binding assays. − Others have leveraged the detailed information provided by CIU to characterize the cooperativity, binding location, and the allostery of ligand binding events within proteins. , CIU has also been developed into a versatile tool for the characterization of biotherapeutic antibodies . For example, CIU has proven to be highly sensitive to the presence of immunoglobulin isoforms, differences in glycosylation and disulfide bonding patterns, antibody–drug conjugation patterns, and different binding epitopes .…”

“…One of the most common outputs of CIU experiments is the accelerating voltage necessary to convert 50 percent of a compact protein form into an energetically adjacent extended state, sometimes termed a “CIU50” value. CIU50s have been used extensively to assess protein–ligand binding − and the stability of domains within larger protein constructs. ,,,,, The Pulsar software package uses feature models to fit CIU50 values but requires manual annotation of the features prior to analysis. Other packages, including the original CIUSuite and Benthesikyme, annotate CIU features but lack an automated method to fit CIU50s.…”

Therapeutic Potential of Polyphenols in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia

“…A molecular docking study showed the potential of EGCG to reduce mutant SOD1 aggregates [ 123 ]. In vitro studies confirmed an inhibitory effect on apo-SOD1 aggregation [ 124 ]. It has also been shown that the addition of EGCG induces oligomerisation of TDP-43 and inhibits its degradation into toxic aggregation-prone fragments [ 125 ].…”

Antioxidant Therapy in Oxidative Stress-Induced Neurodegenerative Diseases: Role of Nanoparticle-Based Drug Delivery Systems in Clinical Translation

“…In a mouse model of ALS, treatment with anthocyanin-enriched extracts from strawberries was found to delay the disease onset, improve grip strength, reduce spinal motor neuron death, and preserve neuromuscular junctions (NMJs) [ 222 ]. Zhao et al discovered that EGCG treatment stabilizes SOD1 conformation against misfolding and inhibits apo-SOD1 aggregation [ 223 ]. EGCG was found to have a substantial binding affinity for mutant SOD1, which reduces its toxic aggregate formation [ 224 ].…”