A novel method for nanopore protein sequencing reveals the enzyme crosstalk effect of the renin-angiotensin system

On February 21, 2023, Beijing time, Professor Long Yitao’s research group from the School of Chemistry and Chemical Engineering of Nanjing University published a research result entitled “Protein Nanopore Reveals the Renin-Angiotensin System Crosstalk with Single-Amino-Acid Resolution” in the journal Nature Chemistry.

This study reports a research strategy for molecular timing changes of complex systems based on nanopore single-molecule interface, using the precisely designed nanopore single-molecule interface, the constructed new method of sequencing single peptide molecules, the self-built weak current measurement device and the targeted development of single-molecule rapid quantification methods, sequencing angiotensin series peptides with individual amino acid differences in multi-component systems one by one, monitoring the dynamic evolution process of angiotensin polypeptides in real time, and elucidating their quantitative evolution pathways. The crosstalk effect in the renin-angiotensin system was revealed, and the influence model of SARS-CoV-2 spike protein and its variants on this system was further proposed.

The corresponding author of the paper is Professor Long Yitao, and the first authors are Dr. Jiang Jie and Dr. Li Mengyin.

The renin-angiotensin system (RAS) maintains the body’s blood pressure and electrolyte balance, and is deeply involved in complex interactions with the body’s central nervous system and immune system. Hypertension directly caused by RAS disorders, and the cardiovascular and cerebrovascular diseases caused by it, are currently one of the diseases that cause the most deaths in the world. Traditional clinical therapies for pathological RAS-activated elevated blood pressure have focused on interventions with the main effector peptide angiotensin II (Ang II), including the development of inhibitors of ACE against angiotensin-converting enzyme produced by Ang II, and antagonists against Ang II receptors. In recent years, studies have found that ACE2, another endogenous angiotensin-converting enzyme, can reduce the level of Ang II through direct degradation of Ang II, as well as the conversion of Ang I, the substrate upstream of Ang II. More importantly, recent experimental evidence shows that the downstream products of ACE2-mediated Ang II, Ang 1-7, play a completely opposite role than Ang II in the regulation of blood pressure regulation and immune response, showing the counter-regulatory effect of ACE2 in traditional RAS activation. Therefore, given the “cross-mediation” of the two enzymes in the transformation of angiotensin peptides in the RAS series, the study of the crosstalk effect on their interaction in multi-component systems will further provide new insights into the molecular mechanism of RAS regulation.

In addition, ACE2 is the main receptor of SARS-CoV-2 virus that has caused the new crown pandemic in recent years, and studying the interference of SARS-CoV-2 spike protein (SP) on ACE and ACE2 crosstalk systems will help to understand the pathogenic mechanism of new crown diseases at the molecular level. Although existing studies have preliminarily revealed the catalytic kinetics and substrate selectivity of ACE and ACE2, due to the differences in individual amino acids between angiotensin series peptides, the potential low half-life and large dynamic abundance changes, the crosstalk effect on ACE and ACE2 needs to overcome the current worldwide problems such as slow sequencing speed, high detection limit and small dynamic range of single-molecule polypeptides, and need to develop real-time accurate quantitative methods.

Professor Long Yitao’s research team of Nanjing University proposed a research strategy for the molecular sequence evolution of complex systems based on biological nanopores, including three central principles: designing high-resolution single-molecule interfaces that identify single constituent units/groups, developing high-read-efficient methods for real-time quantification, and constructing a physiological environment suitable for enzyme-catalyzed reactions. Through the precise design and engineering of the single-molecule interface of Aerolysin nanopores, the detection efficiency of electroneutral angiotensin series peptides and the ability to distinguish individual amino acid differences between them are enhanced, and a series of angiotensins in complex systems can be accurately identified and efficiently quantified at the single-molecule level, including Ang I, Ang 1-9, Ang II, Ang 1-7, Ang III, etc. At the same time, combined with the constructed effective time-based rapid quantitative algorithm, the strategy monitors the shearing process of ACE and ACE2 on Ang I in real time, calculates the kinetic parameters of the two against Ang I, and evaluates the selectivity of the enzyme for substrates, which compares the results obtained by traditional mass spectrometry to confirm the reliability of the nanopore-based strategy.

Figure 1: Schematic diagram of nanopore detection and its single-molecule resolution of angiotensin.

Figure 2: Real-time monitoring of enzyme sequential cleavage process and calculation of enzyme catalytic kinetic parameters.

Through the one-by-one and high-throughput characterization of individual angiotensin polypeptide molecules in the ACE and ACE2 mixed enzyme systems, the real-time and dynamic evolution path of Ang I when ACE and ACE2 exist at the same time is described, and the crosstalk effect of the two is revealed: that is, ACE2 selectively inhibits the degradation of Ang I by ACE without affecting the shearing of other polypeptides (such as Ang 1-9, Ang1-7, etc.) by ACE. This crosstalk effect has been shown to exist at different ACEs, ACE2 concentration ratios, different reaction salt concentrations and even physiological environment salt concentrations, which can significantly affect the transformation path of Ang I.

Figure 3: Crosstalk effects of ACE and ACE2.

Figure 4: Impact of SARS-CoV-2 and its variants on crosstalk.

Given that ACE2 is the main receptor for SARS-CoV-2 SP, the research group evaluated the changes in the crosstalk effect of ACE and ACE2 in the presence of spike protein SP. The results showed that SARS-CoV-2 SP could significantly reduce the selective inhibition of ACE2 on ACE and increase the production and accumulation of Ang II. More importantly, the SARS-CoV-2 Delta variant, which is considered to cause the most serious new coronavirus disease so far, can further inhibit the activity of ACE2: compared with wild-type SP, Ang II has become the main product in various time periods in the presence of Delta SP, and its production and accumulation have increased by 3 times and 4 times, respectively. Considering the additional role of Ang II in promoting cellular fibrosis and enhancing the inflammatory response, this phenomenon provides a new understanding of the molecular mechanism between the novel coronavirus strain and the conditions it induces.

This study provides a new method to elucidate the temporal dynamic evolution of multi-component molecules in complex systems from the single-molecule level, revealing the crosstalk effect between ACE and ACE2 in RAS. With its single-molecule, high-throughput and label-free characteristics, nanopore technology can “intrinsicly” reflect the interaction of enzyme reactions without introducing additional interference, opening up a new direction of nanopore monomolecular omics.

Director Han Huanxing of Changzheng Hospital of Naval Medical University jointly participated in the research of this project, which was supported by the National Natural Science Foundation of China (approval numbers: 22027806, 21834001). (Source: Science Network)

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