Antioxidant Potential of Phycobiliproteins: Role in Anti-Aging Research

Aging research has made significant progress over recent years, particularly after the formulation of ‘Oxidative stress theory of aging’. According to this theory, aging and its associated abnormalities may be prevented, at least to some extent, by application of certain antioxidants. Cyanobacterial phycobiliproteins (PBPs), the major light harvesting pigment proteins are widely characterized for their in vivo and in vitro antioxidant activity. Since, reactive oxygen species (ROS) are considered as important factors to cause aging, PBPs can be used as an effective free radical scavengers and be a potent candidate to develop the anti-aging drug. The use of PBPs in preventing the oxidative stress mediated abnormalities or aging is rationally debated. The present review enlightens the recent advances in the field of antioxidant function of PBPs and major challenges in the application of these pigment proteins in anti-aging research. Also included is the possible mechanism behind the anti-aging capacity of these ecologically as well as economically important biomolecules.


Introduction
In contemporary scenario, oxidative stress and its effect on life attracts attention of many researchers due to substantial increase in the occurrence of oxidative stress related diseases such as atherosclerosis, arthritis, diabetes and cancer [1]. Many researchers have studied various oxidative stress mediated health disorders, which led to the invention of various antioxidants. Antioxidant can enhance the life-saving process by reducing the detrimental effects of oxidative stress caused by various means. Numerous natural and synthetic antioxidants are recognized; however, the natural antioxidants have become more popular because of their great advantages with no or little side effects [2].
Marine cyanobacteria are considered as emerging source of several compounds including fatty acids, carotenoids, polysaccharides and pigment proteins showing various biological activities. Specifically, protein and peptides of marine origin are widely studied and found to have potential biomedical applications [3]. Such wide range of application is credited in account of plenty of essential amino acids present in their constitution [4]. Phycobiliproteins (PBPs) are found in very high abundance (around 60% of the total protein content and 20% of the dry cell weight) in cyanobacteria [5]. They have been considered as a potent pharmacological and medicinal agent due to their antioxidant capacity [5,6]. On the other hand, PBPs are widely exploited as natural colorants in various food products including dairy products, chewing gums, jellies and in cosmetic industries due to their unique fluorescent properties [7].
In recent time, various biomolecules having potent antioxidant capacity are being used in anti-aging research. Aging is defined as systematic decline in physiological as well as biochemical functions that affects most living organisms. Isolation of first long-lived Caenorhabditis elegans mutant has raised the curiosity among researchers and ignited the aging research over past 30 years [8]. The 'Oxidative stress theory of aging' postulated the 'free radicals' as major cause of aging. Couple of evidences in favour of this postulation has provided the platform to fight against aging by application of various antioxidants. The present review highlights the recent updates on antioxidant assets of PBPs and its most significant role in anti-aging research.

The phycobiliproteins (PBPs)
PBPs are composed of apoproteins providing scaffold and chromophores giving unique spectral features to them. PBPs apoprotein is consist of two dis-similar peptides namely α-and βsubunits ( Figure 1A) [6]. The heterodimer (αβ), made of α-and βsubunits is further associates to form trimmers (α3β3) and hexamers ((α3β3)2). Trimmers and hexamers are stacked sequentially to construct the giant light harvesting complex -phycobilisome (PBS) ( Figure 1B) [9]. PBPs α-and β-subunits are uniform in length, with 160 to 180 amino acid residues, respectively [10]. X-ray crystallographic studies have shown that the fold of α-and β-subunit of PBPs are a well-defined helical globin-like domain with seven helices (A, B, E, F', F, G and H) and helical hairpin domains (X and Y) at the N-teminus. Of which, helical hairpin domains are responsible for providing the stability to αβ monomers [11]. Despite of having considerable divergence in amino acid sequences of PBPs apoproteins, the tertiary structure of all PBPs are highly similar [12]. Chromophore, covalently attached to the cysteinyl residue of PBPs apoproteins via thioether linkage, is open-chain tetrapyrrole pigments. Chromophore acts as cofactor in the PBPs with light-harvesting and transferring properties [13]. Conjugated double bond system is the heart of chromophore functioning. Chromophore-chromophore and chromophore-protein interaction and different degree of conjugation facilitates the diversity in PBPs spectral phenotypes among various cyanobacterial species [14]. Chromophores are classified in various sub-classes on the basis of their spectral characteristic. Phycocyanobilin (PCB), phycoviobilin (PVB), phycoerythrobilin (PEB) and phycourobilin (PUB) are the majorly found chromophores [15]. PBPs are also sub-divided into 3 major families according to the type of their attached chromophore and consequent spectral (absorption and fluorescence) characteristics: allophycocyanin (APC, λA max = 650-655 nm; λF max = 657-660), phycocyanin (PC, λA max = 610-620 nm; λF max = 645-653) and phycoerythrin (PE, λA max = 540-570 nm; λF max = 575-585) ( Figure 1C-D) [6]. APC and PC contain PCB and PVB whereas PE contains PEB and PUB as a cofactor with their protein scaffold. PBPs are widely studied by many research due to their unique and bright colour, non-toxic and proteineous nature. PBPs have been reported to express pharmacological and health beneficial effects including antioxidant, anticancerous, neuroprotective, antiinflammatory, hepatoprotective and hypocholesterolemic. Numbers of physiologically abnormalities have been proven to be averted in various experimental animals by PBPs administration [16][17][18][19][20].

Antioxidant activity of PBPs
During last few decades, the PBPs have been exclusively investigated for their antioxidant or free radical scavenging potential by various in vitro and in vivo experimental systems (Table 1). Recently, Sonani et al. [6] have described the in vitro antioxidant and radical scavenging activity of three major PBPs -PE, PC and APC purified from the marine cyanobacterium Lyngbya sp. A09DM. We have established that PE exhibits its antioxidant activity mainly via primary route i.e., by scavenging the already produced reactive oxygen species (ROS) through redox reaction. On the contrary, the PC and APC work via both primary as well as secondary route (by chelating the metal ion which produces ROS) to express their antioxidant activities [21]. Antioxidant action of PBPs is hypothesized to differ due to different mechanisms associated with side chains of various constituting amino acids [6]. Variation in amino acids distribution on outer surface of PBPs may favour one mechanism over other and thus, PBPs express diverse antioxidant activity. For example, amino acid with hydrophobic side chain is good proton donor and metal ion chelator. Similarly, acidic, basic and aromatic amino acids are supposed to sequesters metal ion [2]. PBPs are reported to chelate as well as reduce the ferrous ion very efficiently [6]. This indicated the combined involvement of both electron donating and metal ion chelating ability of the PBP-constituting amino acids in expressing antioxidant activity. Specifically, PE showed lower chelating ability and higher reducing power in comparison with PC and APC. This indicated the leading role of redox potential of PE in its antioxidant activity. Contrary to PE, the antioxidant activity of PC and APC was contributed equally by both reducing ability and chelating ability [6]. The variation in mode of action might be due to variation in surface distribution of hydrophobic and hydrophilic amino acid. It signified the presence of more amino acids having reduction ability than that having chelating potential on outer surface of PE whereas both are equally distributed on outer surface of PC and APC in their nativefolded state [6]. Establishment of antioxidant nature of PBPs will be of great importance in therapeutics of ROS-associated disorders.

Oxidative stress, antioxidants and aging
Oxygen molecule is the final electron acceptor in mitochondrial oxidative phosphorylation. With increasing age, the mitochondria produce various reactive oxygen species (ROS) as by product of oxidative phosphorylation. Generally, in early life, ROS and their effect is abolished by inherent antioxidant defence of the body. But in the later part of life, the imbalance between ROS and antioxidant defence occurs due to the increase in either ROS production rate, decline in antioxidant defence or both [22]. Such imbalance will cause ROSaccumulation leading to damage of the life indispensible macromolecules [22]. Accumulated ROS damages the mitochondrial molecules and thus, also impairs healthy metabolic activity. Accumulation of ROS damages gives rise to systematic dysfunction of almost all organs, which ultimately figures in aging [23]. This 'oxidative stress theory of aging' has been greatly supported on the basis of some solid evidences. First, ROS accumulation is found to accelerate within eukaryotic cells mainly as by-products of mitochondrial respiration. Second, ROS has been proved to damage the life obligatory macromolecules. Third, it has been well established that the long lives animals (worms, flies and mice) are associated with reduced oxidative stress and/or increased antioxidant defence activity. Forth, transgenic animal models, engineered with increased antioxidant defence have been confirmed by longevity phenotype. Fifth, effectiveness of some exogenous antioxidant supplements like ascorbic acid, vitamin E (α-tocopherol) [24], trolox [25] and α -lipoic acid [26,27] in moderating aging and associated abnormalities.
The 'oxidative stress theory for aging' has generated the curiosity among researchers to find the anti-aging dietary supplements. In light of this theory, various natural antioxidants like resveratrol, quercetin, curcumin, mycosporine-like amino acids and green tea extract have been studied in great details by using various model organisms [23].

Aging associated abnormalities
Aging is systematic and progressive decline in physiological as well as biochemical integrity raising the susceptibility to death [28]. Accumulation of such health defects increases the risk for the aging associated disease including arthritis, atherosclerosis, muscular dystrophy, cataracts, pulmonary dysfunctions, cancer, diabetes, cardiovascular and neurodegenerative disorders [29,30]. López-Otín et al. has reviewed nine major molecular and cellular hallmarks of aging includes: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication [28]. Due to the oxidative microenvironment in mitochondria and nucleus, occurrence of somatic mutation becomes highly prone in aged individual [31]. Other abnormalities linked with DNA damage, such as chromosomal aneuploidies and copy number variation, increased clonal mosaicism also found to associate with aging [32][33][34][35]. Increasing mutational load with age causes dysfunction of various tissues, majorly by impairing the respiratory functionality.
Loss of proteostasis is established as the chief mediator for oxidative stress caused age associated disease [36]. Generally, oxidative stress caused the denaturation of native folding of proteins, which resulted in unfolded proteins (Figure 2). The unfolded proteins are generally refolded by molecular chaperons or destructed by either the ubiquitinproteasomal or lysosomal (autophagic) pathways (Figure 2). The unfolded proteins fail to undergo either of these phenomenon lead to protein aggregation, which ultimately resulted in proteo-toxicity ( Figure 2). Such disruption in proteostasis causes mismanagement in cellular traffic, premature protein degradation, and formation of toxic folds [37,38]. All of these are recognized as common molecular events for a large number of disorders including Huntington's disease (HD, CAG-repeat/polyglutamine aggregation), Parkinson's disease (nonCAG repeat aggregation disease), Alzheimer's disease (Aβ peptide oligomerization), Kennedy's disease, Spinocerebellar ataxias, Amyotrophic lateral sclerosis (ALS) and Prion disease [39].  Unfolded or misfolded proteins are generally processed by two pathways. In first way, they are either undergoes proteins degradation by various pathways in cluding lysosomal/cheparon -mediated autophagy and ubiquitin -proteosomal degradation. Otherwise, they are refolded by folding chaperons like heat shock proteins. If the protein fails two undergo either of process, it tries to aggregate in haphazard manner, leading to proteotoxicity and thus, aging.

PBPs as anti-aging molecules
Enhancement of endogenous antioxidant system or provision of exogenous antioxidant supplement was believed to alleviate the aging and associated abnormalities. Currently, our research group is working on the topic -'Is PBP's antioxidant virtue can be explored to alleviate aging and associated abnormalities? ' We have reported that the PE, one of the PBPs having antioxidant nature, efficiently moderates the progression in physiological dysfunctions and molecular mechanism associated with aging in Caenorhabditis elegans (C. elegans) [6,40]. PE feeding also confers the better stress tolerance in C. elegans under thermo-as well as oxidative stress [40]. Moreover, PE was found to inhibit the development of Aβ plaque and thus, Alzheimer's disease in C. elegan CL4176 (Alzheimer's disease model) ( Figure 3) [40]. Nevertheless, PE-mediated life span expansion of C. elegans null mutants (e1370, mu86, hx546) for up-and down-stream components of insulin signalling pathways (IIS pathway) suggested that the PE's anti-aging action is independent of IIS pathway [40]. Better survival of C. elegans upon PE treatment in paraquat sensitivity assay has confirmed the in vivo potential of PE's antioxidant activity in moderating aging ( Figure 4A) [6]. Inefficiency of PE in expanding life span of hsf-1 knockout C. elegans has first time evidenced the dependency of PE's life expanding action on heat shock response transcription factor (HSF-1) ( Figure 4B) [40].  Figure 3A and 3B are adapted from Sonani et al. [40].
Moreover, Singh et al. has reported the crystal structure and in silico affinity of PC αβ dimer with β-secretase ( Figure 5), the potential drug target for Alzheimer's disease [41]. The same was also evidenced experimentally by using the Alzheimer model C. elegans CL4176 [41].
These couple of reports have greatly enlightened the possibility to explore the PBP's antioxidant nature in the invention of the natural drug that can delay the onset of aging process.

Conclusions and Future Perspective
Since the amino acid sequences of PBPs vary among different species/strains of cyanobacteria, an extensive research is needed to explore the various chemical natures of different PBPs from different cyanobacteria. However, the question 'How does exactly the PBPs works against aging?' is still remains elusive. Further research on antiaging potential of PBPs may reveal the better understanding about their mode of action in averting the aging process. Scarcity in the reports describing the PBP's anti-aging potential also propose to employ various eukaryotic experimental systems including rat, drosophila, hamsters, human cell lines to verify the in vivo antioxidant activity and thus, to establish consistence anti-aging virtue of PBPs. Overall, the present review may attest the opening of new horizons for putative role of PBPs in moderation of aging and associated life threating diseases.  Figure 4A and 4B are adapted from Sonani et al. [6] and Sonani et al. [40], respectively) Figure 5: Protein-protein docking between PC αβ-Dimer and β-Secretase. PC αβ-Dimer were docked with two different β-secretase (PDB IDs: 1FKN, yellow, and, 3UQP, green) structures. 1FKN is found to dock in close proximity of the β-subunit while the 3UQP is found to dock in an α-subunit proximal location. (Adapted from Singh et al. [41].