Targeting S100B Protein As A Surrogate Biomarker And Its Role in Various Neurological Disorders Part 2

2. STRUCTURE OF THE S100B PROTEIN

S100B is zinc (Zn2+) and calcium (Ca2+) binding acidic protein confined to the nucleus and cytoplasm of a broad range of cells. S100 protein genes comprise 13 members present as a cluster on chromosome 1q21 [45, 46]. 

Acidic proteins are one of the important components of chromatin in the nucleus of nerve cells. Its content and distribution are closely related to memory.

Scientific research shows that there are two types of memory in the human brain: short-term memory and long-term memory. Long-term memory can last for days, months, or even longer, and is the memory method needed in people's lives.

In the brain, long-term memory is achieved through synaptic connections between neurons. When memory is stored, acidic proteins in neurons are expressed, which can increase the durability of synapses and maintain long-term memory.

At the same time, overexpression of acidic proteins can lead to synaptic abnormalities, memory decline, and even brain diseases. Therefore, it is also very important to keep acidic proteins at an appropriate level.

To protect and improve memory, the following measures can be taken:

1. Exercise. Appropriate exercise can promote the connection between neurons, increase the production of acidic proteins, and thus enhance memory.

2. Improve living habits. Pay attention to rest, and avoid staying up late and overusing electronic devices, which will help maintain a good working state of the nervous system.

3. Eat more memory foods. For example, fish, walnuts, dark chocolate, eggs, etc. These foods are rich in Omega-3 fatty acids, zinc, copper, folic acid, and other ingredients, which help improve memory.

In short, the relationship between acidic protein and memory is very important. Through scientific methods, we can ensure the reasonable distribution of acidic protein, thereby improving memory. At the same time, we should also pay attention to maintaining a positive attitude and maintaining a good physical and mental state to better have an excellent memory. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because Cistanche is a traditional Chinese medicine with many unique effects, one of which is to improve memory. The efficacy of Cistanche comes from the various active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health in many ways.

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Structurally, it contains two binding regions of EF-hand type, known as a helix-loop-helix motif joined by a central hinge region [27]. 

The protein is composed of 2 identical chains of the 91-amino acid polypeptide, which contains two proposed EF-hands helix-loop-helix calcium-binding regions. Every subunit of S100B incorporates four helixes (helix 1, E2-R20; helix 2, K29-N38; helix 3, Q50-D61; and helix 4, F70-A83) and one antiparallel beta-sheet (strand 1, K26- K28; and strand 2, E67-D69). These helices and sheets form normal and pseudo-EF hands jointly [47, 48]. 

The C-terminal domain contains canonical 12 amino acid-binding loops with classical EF-hand and the N-terminal domain contains the 14 S100B specific amino acid–binding loops [49, 50]. 

The amino acid sequence has been found to have areas of an intense grouping of lipophilic, basic, and acidic amino acids and a calcium-binding region in the acidic portion [51]. 

S100b with a moderate affinity (2-20M) binds with two calcium ions per subunit [27] (Fig. 1). Moreover, calcium-binding to EF-hand initiates structural changes that permit the target proteins interactions. S100B proteins are differentiated from other helix-loop-helix EF-hand proteins by their distinct ability to bind Ca2+ ions in their amino-terminal binding sites which is a peculiar diametric building design. 

Moreover, there is a potential for transition metal binding such as copper, zinc, and manganese at dimer interface at histidine-rich binding sites [27]. In the existence of magnesium and potassium, the affinity of the protein for Ca2+ ions is decreased. 

The calcium-binding occurs possibly at two sites that are alpha and beta, which are strongly antagonized by potassium [52]. 

This protein interacts with the target proteins shown to bring in cysteine residues (one on S100A1 and two in S100B); moreover at a stretch of 13 amino acids, a linker region in the middle joins two EF-hand calcium-binding domains [53].

3. THE RECEPTOR OF S100B PROTEIN

S100 proteins act on RAGE and TLR-4 receptors. RAGE is a cell surface receptor of immunoglobulin present in various cell types like mononuclear phagocytes, tissue macrophages, cardiac myocytes, lungs, fibroblasts, epithelial cells, endothelial cells, neurons, and smooth muscle cells [54]. 

They are also described as a pattern recognition receptor. The activation of extracellular RAGE domains by S100 proteins initiates multiple intracellular signaling pathways and various transcription factors such as NF-kB, AP-1, and STAT3 (Signal transducer and activator of transcription 3), which leads to increased expression of proinflammatory cytokines and cellular adhesion molecules. 

S100 proteins interfere with diverse RAGE domains such as the V domain, C1 domain, and C2 domain [55]. The V domain is located at N- the terminal at the most lateral position from the plasma membrane, whereas the C2 domain is located near the membrane. The two adjacent domains V and C1 join together and build a little bent elongated structure [56, 57] (Fig. 2). 

S100B protein interferes with the V domain whereas other proteins like S100A12 and S100A6 are known to interact with the V and C domains. S100A12 interacts via V and C1 domain and S100A6 interacts via V and C2 domain. The exact binding site of S100A8/A9 in the RAGE domain has not been confirmed yet. 

The RAGE domain is mainly activated by different S100 proteins such as S100B, S100A12, S100A8/A9, etc., and further they can lead to the MAPK pathway activation and NF-Kb translocation from the cytosol to the nucleus, which results in the cell survival and proliferation as well as gene upregulation [55]. 

4. S100B IN ALZHEIMER`S DISEASE

AD is an irreversible, progressive neurodegenerative disorder that slowly shatters thinking, memory ability, and the potential to carry out easy tasks [58]. The initial histopathological markers of AD are neurofibrillary tangles found intracellularly and amyloid plaques found extracellularly that are well-known to degenerate hippocampal neurons [59]. 

In AD patients, the level of S100B protein in the frontotemporal lobe promotes inflammatory cascade, and oxidative stress and alters Ca2+homeostasis [60]. Oxidative stress initiates mitochondrial DNA damage, leading to the propagation and/or destruction of neurons, which has been a concern in the pathogenesis of neurological disorders. 

Various reports have hypothesized that an imbalance between highly cytotoxic ONOO− and protective NO could be crucial for several vascular and neuronal diseases including AD [5]. 

The production of NO occurs due to the increase in the production of Aβ either through disruption of Ca+ homeostasis and subsequent increase in intracellular Ca+ (eNOS and nNOS mediated NO release) or through interactions with glial cells (iNOS-mediated NO release) [61]. NO is a free radical with the potential to produce peroxynitrite. 

These ROS induce various mechanisms of neurotoxicity, including alteration of protein/DNA, mitochondria dysfunction, lipid peroxidation, neuro-inflammation, and apoptosis, inducing integrity of the cellular membrane, which leads to further Ca+ influx and NO release [62]. 

These mechanisms are known to be implicated in cell death and observed cognitive impairments in AD [63]. There is a distinct interaction between extracellular beta-amyloid and S100B protein expression which is chronically elevated in AD and is associated with senile plaques. 

This biomarker may play a role in amyloid aggregation and help determine brain distress [64]. Activated astrocytes are well-recognized components of Aβ plaques in AD. 

These activated astrocytes found in the AD patient brain markedly over-express S100B which is reflected by its increased brain tissue level. Most of these activated astrocytes and S100B overexpression in the brain of AD patients are closely associated with either diffuse or neuritic Aβ plaques. Distribution of these astrocytes across brain regions recognized distribution patterns for Aβ plaques. 

These topographical associations between S100B overexpression-activated astrocytes and Aβ plaques in AD together with the known neurotrophic effects of S100B suggest that S100B overexpression may be an important pathogenic factor responsible for the genesis and evolution of neuritic plaques in AD. 

Astrocytes activation and overexpression of S100B are prominent and consistent features of AD conditions that confer increased risk for AD. S100B has both trophic and potentially toxic effects on neurons and neurites which suggests that S100B overexpression plays an important role in the genesis of neuritic changes in Aβ plaques. 

In the late AD stage, the progression of non-fibrillary amyloid deposits as neurite forms consequently leads to the progression of the disease itself [65]. It has been evidenced that the synthesis of both S100B protein mRNA and S100B protein in the culture of astrocytes is stimulated by beta-amyloid [14]. The accumulation and influx of alpha-beta (α-β) in the brain are mediated by RAGE. 

Directly or indirectly α-β initiates calcium homeostasis dysregulation which leads to activation of S100B protein. Glial cell activation by RAGE leads to NF-kB beta activation that causes gene transcription and inflammatory cytokines release [66]. 

In AD patients, the brain is the intense site of inflammation and oxidative stress that brings about AGE formation. S100B protein, alpha-beta, AGEs, and other RAGE ligands such as HMGB1, TTR, S100A6, S100A8/A9, and S100A12 concentrate in the brain during the disease course. 

Excessive release of S100B and RAGE also initiates neuropathological changes in the brain through activation of microglia, neurite degeneration, neuronal apoptosis, and formation of NFT which finally leads to impairment of memory [27]. 

Excessive AGE formation by protein modification also triggers RAGE-dependent oxidative stress and NF-kβ. The activated NF-kB leads to the elevated expression of RAGE because of the NF-kB response element present within the RAGE promoter region [65]. 

Activation of both RAGE and NF-kB leads to alteration in neuronal redox potential and neuroinflammation. At the site of inflammation, high levels of AGEs, NFT, and senile plaques are localized in the brains of AD patients [67]. 

Amyloid-forming proteins such as amyloid-- beta peptides and TTR initiate the formation of the second group of RAGE ligands. The APP processing by beta and gamma-secretase leads to amyloid beta-peptide production. 

Accumulated amyloid beta proteins in the AD patient's brain are important in disease pathogenesis. The transport of amyloid beta through the cell membrane of neurons and BBB is also evidenced to be mediated by RAGE. Furthermore, in AD patients, TTR is shown to have a protective effect by binding to amyloid beta in a chaperone-like manner [55].

5. S100B IN PARKINSON'S DISEASE

Parkinson's disease (PD) is a prevalent progressive neurodegenerative disorder that is described by aggregation of α-synuclein in cortical or brain stem region [68]. 

The first and most prominent physical disabilities due to these variations include motor incoordination which is collectively called Parkinsonism. These include insufficiency and slow movement that is akinesia, bradykinesia, rigidity, and tremors produced at rest [69]. 

Pathogenesis of PD focuses on ROS, the initiation of oxidative stress that results in oxidative damage to substantia nigra pars compacta. Free radical species being the cause of the death of a dopaminergic cell in PD is unclear, but some data have suggested that hydroxyl radical (OH'), NO, and peroxynitrite are involved [70]. 

Nitric oxide synthase (NOS) activation produces NO, which reacts with superoxide to form peroxynitrite. This molecule modifies nucleic acid, protein, and lipid, in an oxidative manner, resulting in nuclear damage, proteasome inhibition, mitochondrial damage, and endoplasmic reticulum stress (ER). 

Excessive level of nitrosative stress leads to the hyperactivation of glutamate receptor group N-methyl-D-aspartate (NMDA), mitochondrial dysfunction, and cell aging. Excessive free radicals and NO species were reported to activate the pathological mechanism including abnormal mitochondrial dynamics, misfolded proteins, and apoptotic pathways in dopaminergic cells [71]. 

Some studies have suggested that excessive production of NO may contribute to these pathological processes, mainly by S-nitrosylation of specific target proteins, such as ubiquitin-protein ligase, parkin, protein disulfide isomerases (PDI), and mitochondrial degradation by ß-amyloid-related S-nitrosylation of dynamin-related protein-1. 

PDI is responsible for the normal folding of proteins in the ER, among these proteins [72]. In addition, No mediated effects on dopaminergic neuron cells can include the inhibition of mitochondria complexes I, II, and IV, cytochrome oxidase, ribonucleotide reductase, glyceraldehyde-3-phosphate dehydrogenase, superoxide dismutase, lipid peroxidation, activation or initiation of DNA strand breakage, protein oxidation and increased production of toxic free radicals including hydroxyl radicals and peroxynitrite. 

Evidence has suggested that excessive RNS/ROS may lead to UPS impairment and misfolding of protein molecules, resulting in aggregation of protein and dopaminergic neuronal death [73]. 

The low expression S100B protein results in neuroprotection due to decreased microgliosis, AGEs, and TNF-alpha expression. There are increased indications that S100B is not only involved in inflammation but also neurodegenerative disease activates proinflammatory cytokine release and leads to damage to dopaminergic neurons. 

The increased level of S100B proteins in post-mortem substantia nigra of PD patients has been reported as compared with the normal tissue group in the CSF [74]. 

Furthermore, S100B shows dual action at low concentration (nanomolar), activates neurotrophic factor, and promotes neuronal survival as well as the growth of neurites during the development phase [75]. It also initiates neuronal apoptosis at micromolar concentrations both by direct action on neurons and microglia activation [76]. 

To some range, these effects may be mediated by an iNOS enzyme which increases nitric oxide production, intracellular calcium levels, and activation of caspase-3 [24]. 

Further, it has been reported that the treatment of astrocyte culture with S100B protein leads to iNOS activation and nitric oxide production. Nitric oxide produced in response to S100B can cause astrocytes to undergo apoptotic cell death. 

Nitric oxide-mediated excitotoxicity, inflammation, oxidative stress, mitochondrial function impairment, DNA damage, and S-nitrosylation of various proteins lead finally to neuronal death [47] (Fig. 3). 

This indicates that S100B could be a promising marker for the degree of disease severity during the beginning of the disease. PD patients have lower levels of S100B and individuals with reduced S100B levels could be more vulnerable to neurological problems. 

These findings suggest that S100B may have a possible role in either the underlying PD development mechanism or in the assessment of disease [14] Furthermore, astroglial C6 and oligodendroglial OLN-93 cells treatment with haloperidol and clozapine at a concentration corresponding to the therapeutic dose range of these drugs decreases the S100B release in vitro [77].

6. S100B IN MULTIPLE SCLEROSIS

Multiple sclerosis (MS) is an autoimmune disease of CNS caused by chronic inflammatory demyelination of neurons, affecting young people [78]. In early disease stages, it is characterized by the T-cell activation, infiltration, and accumulation of monocyte macrophages that promote damage to the myelin sheath which further leads to the formation of focal demyelinated lesions [79]. 

Moreover, a higher S100B level triggers the activation of astrocytes and microglial promoting the NO release [80]. NO is a free radical that is found at a higher concentration than the normal in inflammatory lesions of MS. This increased concentration occurs because of the appearance of iNOS in astrocytes and macrophages. 

Markers of NO production such as nitrite and nitrate concentrations are increased in the blood, CSF, and urine of MS patients. Also, evidence suggests the function of NO in various disease features such as BBB damage, injury of oligodendrocyte, demyelination, and degeneration of axon and it further contributes to functional loss due to axonal conduction impairment [81]. 

Elevated S100B level was first detected in the cerebrospinal fluid of acute-phase MS patients [82]. In the diagnosis of relapsing-remitting MS patients, elevated levels of S100B in the CSF or serum were detected, which decreased after therapy with immunosuppressive or natalizumab [83]. 

During the injury, an increased level of S100B may induce glial reactivity, aggravating tissue damage or delaying remyelination. Increased S100B levels were detected in the CSF of relapsing-remitting MS patients after diagnosis [84].

Active demyelinating MS lesions showed an elevated level of S100B and its receptor, RAGE in the lesion area while chronic active lesions showed raised S100B levels in demyelinated areas with lower expression of RAGE receptors in the rim [85].

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