Screening of Human Proteins for Fluoride and Aluminum Binding

Previous studies showed that prolonged exposure to fluoride (F-) and aluminum (Al3+) ions is associated with numerous diseases including neurological disorders. They don't have any known biological function. But they can bind with proteins that interact with ions similar to them. Such unwanted interactions affect the normal biological function of the target proteins, as well as their downstream protein-protein interactions. Several studies show the detrimental effects posed by them including Alzheimer's disease. However, their target proteins have never been reported. Here, we have screened for the human protein targets subjected to F- and Al3+ interactions by using data-driven prediction tools. We have identified 20 different proteins that directly bind with them (10 interact with fluoride and 10 with aluminum). In addition, protein-protein interaction has been explored to find the proteins that indirectly interact with F- and Al3+. We have found 86 indirect targets for F- and 90 for Al3+. Furthermore, 19 common protein targets have been identified, including proteins (9 out of 19) associated with neurodegenerative disorders. However, wet lab experiments are beyond our scopes to validate the binding networks. Additional studies must be warranted.

in all types of products including corn, salt, herbs, spices, tea, yellow cheese, cosmetics, and aluminium ware and containers. Compounds of aluminium are used to purify drinking water and to treat wastewater plants [5]. Aluminium compounds are used in many medicines and pharmaceuticals (antacids, analgesics, antiperspirants). Presently, there is much concern about the presence of aluminium in drinking water [7,8] and its employment in water treatment processes (as coagulant) and as metal in consumer products [5,6]. In addition, aluminium can be leached from cooking vessels in case of storage of acidic foods [9].
There is evidence that fluoride and aluminum individually contribute to the development of different disorders. Fluoride causes detrimental effects on proteins expressed in soft tissues including the blood, brain, and liver [10] by penetrating the cell membrane [11]. Fluoride buildup in bone tissues disrupts the hematopoiesis in the bone marrow [12]. Numerous studies have demonstrated that high concentrations of aluminum are present

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in the brains of amyotrophic lateral sclerosis and senile dementia patients [13,14]. The excess of both fluoride and aluminum may cause excitotoxicity through microglial activation and induce neurotoxicity [15]. The Blood Brain Barrier (BBB)-inhibits passive diffusion-is susceptible to fluoride and aluminum. Aluminum, as well as aluminum fluoride (AlF3), can react with oxygen to form aluminum oxide (Al2O3), which is found in significant amount in the brain specimen of Alzheimer patients [16]. AlF3 also substantiates osteoporosis, osteomalacia, spontaneous bone fractures, and dementia [17]. Identified protein targets for fluoride and aluminum in this analysis are involved in various important biological functions, for instance, PPP3CC has a role in the apoptotic signaling pathway, FGF12 is involved in nervous system development, etc. Data-driven prediction tools are used to screen for the targets. However, In vitro experiments are warranted to validate the finding in this study.

Methodology:
Network analysis/ retrieval: The first step in the prediction of the potential protein targets of fluoride and aluminum is to find the network of interacting proteins with Fand Al 3+ ions in human. We have used the STITCH 5.0 (Search Tool for Interacting Chemicals) (http://stitch.embl.de/) web server to find the protein targets. The STITCH database aims to incorporate dispersed databases, texts, and prediction methods into a single, easy-to-use resource. The STITCH offers a comprehensive overview of chemical and protein interaction network for numerous organisms including binding affinities between them. The predicted interaction is global for an individual organism and does not distinguish tissue specificity [18]. Target proteins from the STITCH were then subjected to the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis to identify protein-protein association network. The STRING 10.5 (https://stringdb.org/) database goals to combine physical and functional interactions between proteins from a diverse source, including literature mining, experimental data, and computational prediction [19].

Retrieving the protein sequences:
The protein sequences identified with the STRING 10.5 analysis were downloaded in FASTA format from NCBI protein database (http://www.ncbi.nlm.nih.gov/protein). Two FASTA files were downloaded, containing 86 and 90 proteins for fluoride and aluminum, respectively. Additionally, numerous data-driven tools further analyzed these proteins.

Gene ontology analysis:
Gene ontology (GO) of the retrieved protein sequences was analyzed by Blast 2 GO software (https://www.blast2go.com/) [20]. This tool provides a high-throughput functional annotation for gene product properties [21]. We have utilized the aforementioned tool to identify the association between the target proteins and with the ions in the biological processes, molecular function, and cellular component of the human.

Analysis of expression of common proteins:
The organs expressing the 19 common genes and their expression level have been determined from the NCBI (https://www.ncbi.nlm.nih.gov/gene/) database that uses RPKM (Reads Per Kilo-base per Million mapped reads) to quantify gene expression level.

Results: Interaction prediction:
The STITCH 5.0 predicts the association for ion-protein network and their binding affinities. The STITCH 5.0 has predicted 20 human proteins for fluoride and aluminum interaction (10 proteins for each) (Figure 1). The STRING 10.5 predicts the network for protein-protein interaction. The STRING 10.5 has found 86 human proteins that interact with proteins predicted by the STITCH for fluoride and 90 for aluminum (Table 1 and 2).

Pathway detection:
Through the Blast 2 GO software, we executed a series of steps for a list of target protein sequences of fluoride and aluminum, including protein Blast, mapping, and scanning motifs by Inter Pro Scan. The Gene Ontology (GO) has been performed to annotate the biological processes, molecular function, and cellular component for all the proteins found by the STITCH 5.0 and STRING 10.5 (Figure 2-4), and for 19 common proteins detected by the Venny web server ( Figure 6).

MYLK
Myosin light chain kinase Implicated in smooth muscle contraction via phosphorylation of myosin light chains. Involved in inflammatory response and cell migration. Pseudogene of MYLK found having carcinogenic effect. [23]

NOS1
Nitric oxide synthase 1 (neuronal) Process involved in neurotransmitter biosynthesis and secretion. Polymorphism in this gene has association with neurological disorder and asthma. [24]

NOS2
Nitric oxide synthase 2 (inducible) Nitric oxide-mediated cell signaling pathway and variation found in asthma patients.

[25]
NOS3 Nitric oxide synthase 3 (endothelial cell) Vascular smooth muscle relaxation through a cGMP-mediated signal transduction pathway has association with variation in this gene has association with asthma and Alzheimer disease.

Discussion:
Our findings suggest that fluoride and aluminum both have direct (ion-protein interaction) and indirect (protein-protein interaction) protein targets in the human. They directly bind with 20 proteins (both have 10 targets each), while the indirect binding network has 86 and 90 proteins for fluoride and aluminum, respectively. They have different binding affinities with their target, for instance, fluoride has a strong binding affinity with ENO2 compared to GAPDH and aluminum can strongly bind with GPX1 compared to SNCA. The GO annotation for the identified proteins demonstrates their association with numerous biological processes, cellular components, and molecular functions in the human. Both the ions have 19 common protein targets. We have further analyzed their expression site and expression level. RPKM data shows different expression level of the common target proteins of fluoride and aluminum in the human, for example, ALB (albumin) is highly expressed in the liver and SCN10A (Sodium Channel 10 Alpha subunit) is barely expressed in the testis. Most of the common target proteins are expressed in the brain. Fluoride and aluminum could have detrimental effects on many organs, including the spleen, liver, adrenal and prostate glands, small intestine, heart, and primarily the brain. The accumulation of aluminum-fluoride (AlF3) complex in the brain causes prolonged neurotoxicity, synaptic loss, and neurodegeneration [44]. AlF3 complex is able to impair the energy-producing enzymes; free radical balances, lipid peroxidation, and DNA repair mechanism [45,46]. The target proteins are involved in different important functions, including programmed cell death, nervous system development, osmotic blood pressure regulation, action potential generation, etc. More importantly, most of the target proteins are brain-specific; therefore, chronic exposure to these ions could lead to the development of neurological disorders. One recent study showed that Al 3+ could play a vital role as a mediator in the formation of fibrillary amyloid plaques in Alzheimer's disease [48].
Here, we find that APP or Amyloid beta (A4) precursor protein can interact with Fand Al 3+ . APP is a single-pass transmembrane protein highly expressed in the brain and is a key factor in the development of Alzheimer's disease [46]. It is possible that other target proteins of Fand Al 3+ are associated with different diseases. However, further studies must be warranted to validate the binding network and to distinguish the physiologically important and unimportant bindings.