Protein S‐homocysteinylation: Identification of S‐linked Protein Targets in the Blood Based on In‐silico Study and Investigation of the Effect of Homocysteine on the Structural and Functional Integrity of the Potential Target Proteins
Authors: Bhattacharya Reshmee , Singh Laishram Rajendrakumar 
Published: April 2020

Homocysteine (Hcy) is a sulphur containing non‐protein, intermediary amino acid produced from the metabolism of essential amino acid methionine. The prime toxicity of Hcy is believed to be due to the adduct formation with the free cysteine residues of the proteins (known as S‐homocysteinylation). Another cause of Hcy‐induced toxicity is believed to be due to the adduct formation of its cyclic thioester form, called Homocysteine‐thiolactone (HTL) with the lysine residues of the protein (known as ‘N‐homocysteinylation’). The condition of elevated Hcy levels in human blood is called as Hyperhomocysteinemia and is believed to be associated with a wide spectrum of diseases including neurodegenerative and cardiovascular complications. Binding of Hcy to proteins can potentially alter the structure and function of proteins. Till date there have been innumerable work done on understanding the reactivity of HTL and identifying different cellular as well as serum proteins modified upon N‐homocysteinylation. However, there has been no attempt made in understanding S‐homocysteinylation biology. Till date only 21 proteins have been experimentally shown to bind Hcy.

Since Hcy levels elevate in the blood, blood proteins could be the prime targets of S‐homocysteinylation thereby modulating their structural and functional integrity. In this study we have attempted to identify blood proteins that could preferentially bind with Hcy using in‐silico approach. This present study also aims at understanding the effect of Hcy on the structural and functional integrity of the blood proteins that could potentially bind with Hcy.

Methodology

In‐silico studies were carried out to identify positive target proteins based on cysteine content, solvent accessibility of cysteine residues, pKa values of these cysteine residues and high dihedral strain energy of cysteine bridges. Out of the positive targets identified by computational approach, five proteins were selected as models and in‐vitro studies were carried out in order to understand the effects of Hcy on their structural and functional integrity.

Results

The analysis led us to the identification of ~ 40 blood proteins that could potentially bind with Hcy. Further, interestingly our sulfhydryl estimation results proved that different proteins show different reactivity towards Hcy. Our structural and functional analysis showed significant differential behaviour in the native state conformation of proteins due to Hcy‐modification.

Conclusions

Using different web servers, ~ 40 positive protein targets were identified that could potentially bind Hcy. In‐vitro screening also suggested that different proteins show different behaviour with Hcy. Hcy‐induced modification also results in significant alteration in protein structural conformation that could be crucial for its functional activity. The study may provide an insight into the new pathological consequences associated with Hyperhomocysteinemia.