The renin-angiotensin system (RAS) acts as a critical guard in the control of blood pressure and electrolyte homeostasis, through multiple enzymes mediated angiotensin peptides conversion network. Dysregulation of RAS causes hypertension and further cardiovascular disease, which is the leading cause of death worldwide. Recent studies have shown that angiotensin-converting enzyme（ACE）and angiotensin-converting enzyme 2 (ACE 2) composed two antagonistic axis in RAS, the traditional ACE/Ang II/ATR axis and the newly discovered ACE 2/Ang 1-7/MAS axis. The crosstalk effect of ACE 2 and ACE indeed has complex effects on the RAS, and thus might provide novel in-depth insights into the RAS regulation mechanism. However, the discovery of crosstalk effects on RAS is limited by the lack of approaches to quantitatively monitor, in real time, multiple angiotensin peptides with subtle differences and short half-lives.
Recently, Yi-Tao Long ’s group proposed a nanopore framework to quantitatively determine the effect of the hidden crosstalk between ACE and ACE 2 on RAS, based on the following three criteria: (1) sufficient resolution to identify angiotensin peptides with one-amino-acid difference; (2) high capture efficiency to ensure real-time analysis; (3) proper conditions for peptide detection that are compatible with the enzyme reaction environment. These features allow utilizing the engineered aerolysin platform to achieve real-time monitoring of the evolution of multiple substrates and also quantify instantaneous intermediates in a mixed enzyme reaction system. Using the developed aerolysin nanopore capable of single-amino-acid resolution, it is found that the ACE can be selectively inhibited by ACE2 to prevent cleavage of angiotensin I, that is, crosstalk effect.
Figure 1. Illustration of the aerolysin-based nanopore framework for quantitatively determine the effect of the hidden RAS crosstalk.
In this work, aerolysin nanopore single-molecule interface was precisely designed and engineered, capable of enhancing the detection efficiency and resolution ability of the neutral angiotensin peptides. With this engineered aerolysin nanopore, the single-molecule accurately identification and efficiently quantitation of a series of angiotensins in complex systems, including Ang I, Ang 1-9, Ang II, Ang 1-7, Ang III, was successfully realized.
Figure 2. Engineered aerolysin nanopore discrimination of Ang peptides with single-amino-acid resolution
Then, to achieve rapid quantification of angiotensin components, a novel effective time-based algorithm for nanopore sensing was constructed. Assisted with this fast quantification algorithm, the shearing process of Ang I by ACE and ACE2 enzyme was monitored in real time. The calculated kinetic parameters (Km and Kcat) of both ACE and ACE 2 were consistent with the results obtained by traditional mass spectrometry, confirming the reliability of the nanopore-based monitoring strategy.
Subsequently, the nanopore strategy was used to profile the Ang I peptide substrate transformation in the presence of both ACE and ACE2, and the dynamic evolution spectrum of angiotensin peptides was successfully mapped. The results indicate that ACE2 selectively inhibits the ACE activity for Ang I cleavage, but has almost no influence on ACE activity for degrading Ang 1–9, revealing the crosstalk effect between the two enzymes. This crosstalk effect was proved to be prevalent under different reaction conditions, and even physiological environments.
Given that ACE2 is the main receptor of the SARS-CoV-2 virus, the constructed nanopore strategy was employed to evaluate the effects of SARS-CoV-2 SP in RAS. The binding of the SARS-CoV-2 SP and ACE2 could weaken the crosstalk effect of ACE and ACE2, leading to the accumulation of vasoconstriction and the fibrosis factor Ang II. More importantly, the SARS-CoV-2 Delta variant can further inhibit the activity of ACE2. This phenomenon provided a new understanding for revealing the molecular mechanism between the new coronavirus and its induced diseases.
Figure 3. Revelation of ACE and ACE 2 crosstalk
This study provides a nanopore-based method to elucidate the temporal dynamic evolution of multi-component molecules in complex systems from the single-molecule level. With the single-molecule analysis, high-throughput, and label-free manner features, nanopore technology can intrinsically reflect the interaction of enzyme reactions without introducing additional interference, opening up a new direction of nanopore single-molecule omics.
The related paper entitled Protein nanopore reveals the renin–angiotensin system crosstalk with single-amino-acid resolution has been published online in Nature Chemistry (DOI： 10.1038/s41557-023-01139-8, paper link: https://www.nature.com/articles/s41557-023-01139-8). Dr. Jie Jiang and Dr. Meng-Yin Li are co-first authors, and Prof. Yi-Tao Long is the corresponding author of the paper. Prof. Huan-Xin Han at Naval Medical University gave important medical guidance to this work. This research was funded by National Natural Science Foundation of China (Grant No. 22027806, No. 21834001).