TTRMDB: A database for structural and functional analysis on the impact of SNPs over transthyretin (TTR) using bioinformatic tools
Graphical abstract
Introduction
TTR gene is located on chromosome 18 in humans which contains 4 exons and 5 introns. It encodes 127 amino acid long homotetrameric carrier protein, Transthyretin. Transthyretin is a highly conserved protein that functions as a thyroid hormone-binding protein and transports thyroxine in the plasma to cerebrospinal fluid and brain. It also plays a role in retinol (vitamin A) transport through its association with retinol-binding protein (Buxbaum and Reixach, 2009). Native TTR has a globular shape with eight antiparallel β-strands that make up each monomer. Two four-stranded β-sheets (DAGH and CBEF) form the antiparallel strands. Further, the β-strand E has a short α-helix. Hydrogen interaction of the β-strands, F and H of each subunit results in the formation of a dimer. Also, the interaction of the residues of the loops that join β-strands, G to H and A to B lead to the formation of tetramer. Synthesized by the liver, kidney, pancreas and choroid plexus, transthyretin has emerged to be involved in several functions apart from being a carrier protein (Vieira and Saraiva, 2014). Liver and choroid plexus are the well- known major sites in TTR synthesis (Vieira and Saraiva, 2014; Herbert et al., 1986). Additionally, TTR is also synthesized by cellular components of the peripheral nerve(axons), vascular smooth muscle cells, Schwann cells and neurons (Murakami et al., 2010; Gonçalves et al., 2017).
Mutations in TTR gene generally result in amyloidosis. TTR amyloidosis is characterized by the deposition of amyloid fibrils usually in the peripheral nerves or the heart and also, present in the retinal epithelium, leptomeningeal epithelium of the eye and choroid plexus of the brain (Herbert et al., 1986; Liepnieks et al., 2006). The process of amyloidogenesis involves the dissociation of tetrameric TTR, misfolding of the protein, followed by aggregation into amyloid fibrils (Ando et al., 2013). Thermodynamic studies have concluded that the dissociation of tetramer is the rate-limiting step (Kelly, 2000). Heritable mutations of TTR are inherited in an autosomal dominant manner (Ando et al., 2005). Familial amyloid cardiomyopathy (FAC) or familial amyloidotic polyneuropathy (FAP) occur due to these mutations in TTR protein. However, TTR amyloidosis can also result from the misaggregation of wild-type TTR, causing senile systemic amyloidosis (SSA), a sporadic non-inheritable disease (Ruberg and Berk, 2012).
ATTR has been found to be the most common form of autosomal dominant hereditary neuropathy (Shin and Robinson-Papp, 2012). It is an irreversible sensorimotor and autonomic neuropathy. Specifically, the three stages manifested by TTR-FAP includes sensory polyneuropathy, progressive walking disability and wheelchair bound or bedridden. It is a rapidly progressive disease with a life expectancy of 7.3–11 years from onset (Parman et al., 2016). The current therapeutic approaches to polyneuropathy include prevention of amyloids followed by symptomatic therapy for the affected areas and finally, the treatment of end-stage organ failure (Adams, 2013).
Though liver transplantation remains the ‘gold standard’ for the treatment of FAP, various drugs are being developed to slow or halt the progress of the disease. Tafamidis is a drug that prevents the dissociation of transthyretin tetramer and binds to one of the two thyroxine binding sites of tetramer to stabilize the correctly folded form, which is used as a treatment for FAP and FAC (Maurer et al., 2018). The drugs, Patisiran and Inotersen, both inhibit the synthesis of transthyretin in the liver. While Patisiran acts as an RNA interference therapeutic agent and Inotersen is a 2′-O-methoxyethyl-modified antisense oligonucleotide respectively (Mathew and Wang, 2019; Adams et al., 2018). However, for the specific therapy, the familial screening of individuals affected by TTR mutation, as well as the nature of these mutations, how they affect the individual and the stability of the protein produced, need to be studied (Adams, 2013). On the other hand, Antisense Oligonucleotides (ASO) and small interfering RNA (siRNA) are used as therapeutic oligonucleotides. These oligonucleotides degrade and block the mutated mRNA, help to treat various types of TTR mutations. Currently, this process of RNA interference (RNAi) that inhibits the formation of protein aggregation plays an vital role in the treatment of TTR (Mathew and Wang, 2019).
More than 100 mutations of TTR have been identified from different ethnic groups that lead to different phenotypes (Rowczenio et al., 2014). It has been found that when mutants are more destabilizing, the onset of amyloidosis is earlier. However, two mutations D18G and A25T, though found to be the most destabilizing, are not the most pathogenic, which attributed to the fact that factors like cellular secretion efficiency of the protein, along with instability, also influence pathogenicity (Johnson et al., 2012a). The most common amyloidogenic mutation is V30M. This mutation is found to have different clinical manifestations for patients from different geographic areas. Studies conducted on affected individuals conclude that there could be a common founder for this mutation of patients from Japan and Portugal (Ohmori, 2004). Moreover, the reports from the clinical data have suggested that V30 M mutations are also endemic in other countries such as Sweden, French and Brazil (Norgren et al., 2012; Zaros et al., 2008). Other common mutations include T60A and V122I. T60A is implicated in both, cardiomyopathy and neuropathy which is found in the affected population in north-west Ireland (Reilly et al., 1995). V122I is a predominant mutation found in patients affected by cardiomyopathy. It is especially prevalent among African-American patients (Buxbaum et al., 2010).
Most studies use direct DNA sequencing to identify such mutations in specific patients. These mutations need to be studied, not only to assess their pathogenic impact, but also to check how they affect the stability of the protein. In the biochemistry perspective, the stability of a protein is a key characteristic feature that affects function, regulation and activity of the protein. Thus, studying the impact of these mutations helps gain a better understanding of the affected protein in finding therapeutic design solutions for the disease-causing variants. However, performing extensive wet-lab experiments to study the pathogenicity and functional impacts of each would be a labor-intensive and cumbersome task. Thus, the use of the bioinformatics tools could provide a secure and better understanding of the stability and pathogenicity of the protein upon various notable disease-causing missense mutations. Generally, homologous and distantly related sequences are aligned with amino acid sequences to obtain information on conservation. Moreover, the conservation across various species, the physicochemical properties of these amino acids, the potential protein structural changes and the database annotations are the most common criteria considered in many bioinformatics programs for predicting the functional effect of an amino acid substitution (Seifi and Walter, 2018). In this study, the effects of different mutations on the stability and the pathogenic effects of transthyretin were studied using different tools, such as, iMutant 3.0, STRUM, Provean, PredictSNP, PhD-SNP, PolyPhen-2, SIFT, FATHMM, iStable, mCSM, SDM, DUET, DynaMut, FoldX, ENCoM, CUPSAT, TANGO, WALTZ and LIMBO.
Section snippets
Data collection
Initially, the sequence and the structure of Human Transthyretin were retrieved from RCSB PDB (id: 3A4D). In our study, the first 20 sequence of the amino acids were neglected in order to maintain the consistency with the experimental studies. Further, the list of mutations of TTR was obtained from the database, ‘Mutations in Hereditary Amyloidosis’ (Rowczenio et al., 2014).
Identification and selection of tools
Various bioinformatics tools were chosen based on their relevance and the reported accuracy of their predictions.
Overview of database
TTRM database provides information on TTR, its mutations, the tools used to predict the impact of mutations and the prediction results for each mutation. Specifically, the database was created to study how changes in the sequence of TTR affect the structural and functional properties of transthyretin.
Home
TTRM database was created to provide a better understanding of how a mutation in the sequence of TTR gene can affect the structure and function of protein. The ‘Home’ page provides necessary
Conclusion
Due to advances in sequencing technology, SNPs are being identified at a high rate, but because carrying out wet-lab studies for each of these mutations is a cumbersome task, various bioinformatics tools have also been developed to predict the effect of these mutations on the affected protein. However, the accuracy of prediction varies for each tool and thus, many tools are used in combination to increase the accuracy of the prediction. In this study, the structural and functional impact of
CRediT authorship contribution statement
E. Srinivasan: Methodology, Investigation, Software. Nandhini Natarajan: Data curation, Writing - original draft. R. Rajasekaran: Conceptualization, Writing - review & editing, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
The author (E. Srinivasan) thank CSIR for providing Senior Research Fellowship for carrying out this research work. The authors also thank the Vellore Institute of Technology (Deemed to be University) providing computational facilities to carry out this research work.
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Theoretical investigations of TTR derived aggregation-prone peptides’ potential to biochemically attenuate the amyloidogenic propensities of V30 M TTR amyloid fibrils
2023, Journal of the Indian Chemical SocietySmall molecule protein binding to correct cellular folding or stabilize the native state against misfolding and aggregation
2022, Current Opinion in Structural BiologyCitation Excerpt :The native state of TTR is a tetramer comprising β-sheet-rich 127-residue subunits and featuring two binding sites for thyroxine at the dimer-to-dimer interface (Figure 2). Over 130 TTR mutations have been described that cause familial ATTR with autosomal dominant inheritance [15]. Disease-associated mutations lead to kinetic and thermodynamic destabilization of the natively folded tetramer, resulting in TTR aggregation [16].
Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation
2021, Journal of Biological ChemistryCitation Excerpt :This dynamic signature of the DE loop was not observable in static-X ray structure, which might respond to the drastically different environment that the system experiences in crystallographic experiments with respect to MD simulations, where the system is in solution and not conformationally constrained in crystal form. Notably, the relative destabilizing effect of the different 34 and 35 variants cannot be read directly from the static TTR structure at physiological pH without considering dynamics since structure-based computational tools wrongly classified K34T-TTR to be the most destabilized of the mutants (43). Overall, our data indicate that the specific residue at position 34 or 35 determines the BC-loop flexibility.
Chlorinated Naringenin Analogues as Potential Inhibitors of Transthyretin Amyloidogenesis
2022, Journal of Medicinal Chemistry