Journal of In Silico & In Vitro Pharmacology

Keywords

Ganoderma lucidum (GL); Hypothetical proteins (HPs); Bioinformatics tools

Introduction

Proteins arbitrate multitude of essential housekeeping functions critical for cellular survival and cell integrity. Even little aberration in protein misfolding and aggregation can compromise normal functioning, engendering neurodegeneration and necessitating therapeutic intervention [1] Proteins exhibit indistinguishable activities providing additional edges to immune function, in addition, to possessing anti-cancer potential. Moreover, the discovery of sundry other medicinal values in natural products instigated the researchers to sequence the genome and focus on diverse aspects of proteomics. The genome sequencing laid the foundation for rapid accumulation of different characterized as well as uncharacterized genes in Gene bank. Genome sequencing has achieved the functional annotation only to 50-60% of genes; therefore making functional annotation of remaining uncharacterized proteins a challenging task [2]. The introduction of bioinformatics tools has resulted in the inception of a new era [3] results in solving and annotating function to proteins through less time-consuming means.

A commonly known basidiomycetes fungus, Ganoderma, importantly indicated for cancer and neurodegenerative diseases, has been indisputably claimed to be an inexhaustible resource of cardinal myco-constituents including terpenoids, polysaccharides, and proteins [4,5]. Mitochondrial genome sequencing of Ganoderma lucidum revealed a total of 57 protein-coding genes, 2 rRNA and 26 tRNA [6]. Another species, Ganoderma sinensis, has not been still sequenced, but UniProt confirms the presence of 25 hypothetical proteins (HPs) [7]. The sequence retrieval from UniProt disclosed that genus Ganoderma have 33 hypothetical proteins. Proteins isolated from genus Ganoderma exhibit plethora of bioactivities via the various adapter proteins dominant at different stages of immune modulation [8,9], inflammation, and cancer signaling [10,11]. In addition to these, there are some other proteins that are predicted by gene prediction software, without in-vivo demonstration, known as hypothetical proteins (HPs)/non-characterized/unknown proteins, which play a decisive role in signaling mechanism. Annotating functions to these uncharacterized proteins can help in fathoming the various mechanisms fundamental to the adaptation of this fungus to a various stress condition. In silico studies have been found to be reliable enough to reveal crucial and lucid information about the biological functionality and comparative genomics in lesser time as compared to an experimental characterization which is more time-consuming [12]. This research work was carried out with an impetus to fathom the genomics and to understand the functionality, of the Ganoderma hypothetical proteins, uncharacterized until now [13].

Sequence analysis and comparison forms the prime step in identifying homologues and sequence similarity for characterization of the protein. BLAST proved to be the most reliable method for comparing the query sequences with the database for the prediction of proteins. Multiple sequence alignment of homologues in a family has been found to be a reliable method for functional annotation important in conserved domains. Motif analysis is a necessary step in the identification and characterization of HPs and detection of common motifs among proteins in sequence identities (e.g., less than 30%) may provide important clues for function or classification of HPs into appropriate families [14].

Various databases available freely for determining structure and function of motif include GenomeNet [15], PROSITE [16], PRINTS [17], Pfam [18], ProDom [19], BLOCKS [20], MEME [21] and InterPro [22] using InterProScan [23]. STRING database [24], is such software, pivotal in revealing the functionality of individual protein as well as the interaction with other factors. Moreover, it also determines the gene neighbourhood, gene fusion events and co-occurrence of specific subset of species which confer to the protein confidence score by direct (physical) and indirect (functional) associations, which in turn, are based on genomic context, high-throughput experiments, conserved co-expression and previous knowledge of the protein.

Materials and Methods

In order to predict the functionality of hypothetical proteins in Ganoderma species, various bio informational tools were used, each equipped to highlight a different parameter of protein (Table 1). Sequence retrieval from UniProt, a database of the protein sequence, was the primary step in the process of annotation. This was followed by the functional annotation of the hypothetical protein which involved prediction of the functional aspect of proteome using the bioinformatics tool, by analyzing the sequence, conserved domain, analyzing motif, phylogenetic relationship, and protein interaction.

Table 1: List of bioinformatics tools and databases used for annotating function.

Sequence analysis and physiochemical properties

Sequence investigation of the Ganoderma mitochondrial genome disclosed a total of 33 hypothetical proteins (https:// www.ncbi.nlm.nih.gov/genome/) subsequently retrieved from UniProt (https://www.uniprot.org/). Primarily, Expasy’s ProtParam server [25] was employed to compute numerous theoretical physiochemical properties such as molecular weight, amino acid composition, theoretical isoelectric point, extinction coefficient, instability index, aliphatic index, estimated half-life, instability index, extinction coefficient, average hydropath city (GRAVY) to deduce the protein sequence and assign function to the protein (Table 2).

Table 2: List of predicted sub-cellular localization of HPs in Ganoderma.

Sub-cellular localization prediction

The role of protein sub-cellular localizations in cytology, proteomics, and drug design investigation is axiomatic. Determining the location of the protein and annotating the genome would help in strategizing the drug and vaccine delivery approach with enhanced selectivity and efficacy, a prerequisite for facilitating systemic pharmacokinetics. This also becomes important because the drug delivery system involves the absorption of the drug in the cytoplasm and vaccine in the surface membrane. In addition, UniProt provides information about various proteins which have still not been characterized, thus, making the prediction of their localizations an urgent need. Where experimental methods may prove to be time-consuming, numerous recently exploited in-silico tools have paved the way for quicker determination of such pivotal characteristics. Such bioinformatics tools that clearly demarcate the protein localizations include WoLF PSORT [26] and CELLO [27]. CELLO is a multi-class support vector machine classification system divulging critical factors like the amino acid composition, dipeptide composition, partitioned amino acid composition and sequence composition. WOLF PSORT is a similar protein subcellular location prediction tool which, based on sorting signals, amino acid composition and functional motifs, converts protein amino acid sequences into numerical localization features which are then predicted. Prediction of signal protein was done using the Signal P 4.1 software [28] whereas the SecretomeP server [29] determined the cleavage sites in amino acid sequences. In addition, TMHMM [30] and HMMTOP [31] calculated the transmembrane helices and topology of the protein, distinguishing between soluble and membrane proteins with high degree of accuracy Moreover, methods based on protein sorting signals, TMHMM and HMMTOP, function on the basis of transport of initial synthesis to its functional areas, depending on signals of N and C-terminal (Table 2). Another method for annotation is based on protein function domain and gene ontology. The former is based on evolutionary process, conserved and known functional domain, whereas the latter is concerned with labeling the gene product.

Hypothetical proteins: Comparing the sequences

The initial step involves the prediction of functionality by comparing the sequence of the concerned hypothetical proteins with the sequences retrieved from UniProt databases. BLASTp [32] and HHpred [33] computed the similarity in query sequence and homology, revealing a total of 33 HPs in Ganoderma (Hidden Markov models) against the non-redundant database which gave different hits with different E-values (Table 3).

Table 3: Functional prediction of HPs by BLASTp, HHpred, ProDom and BLOCK.

BLASTp is a commonly used algorithm, included under Basic Local Alignment Search Tool, for searching protein sequences against the non-redundant protein sequences (nr). For each HP queried, this database provides 100 homologs, excluding the proteins with low query coverage of 50% or low sequence identity, referred to as the remote homologues. On the contrary, proteins with sequence identities of 40% with an Evalue of 0.005 form the close homologs of HPs. Moreover, this search tool is also employed for examining the availability of structural homologs in Protein Data Bank (PDB). On the other hand, HHpred is utilized for detection of remote protein homology via pairwise comparison of profile hidden Markov models (HMMs), which it achieves by searching various protein databases like PDB, SCOP, CATH, etc. It is also employed for detection of structural homologs. In comparison to BLASTp, used for determining the sequence identity between two proteins sequences, PRALINE is used for multiple sequences comparisons.

ProtoNet 6.1 clustering based automatic hierarchical database of the SWISS-PROT protein relies on sequence similarity defined in BLAST. ProtoNet predicted superfamilies and subfamilies and large-scale protein annotation. Protein motif sequences are the signature of protein families predicting the function of HPs, particularly catalytic property of the enzyme. Motif known as a super secondary structure of a protein is useful for functional prediction of protein analyzed by the database including InterProScan, PANTHER, Pfam, SMART, ProSite, SUPERFAMILY, etc. InterProScan analyzed different recognition protein and predicted the results by analyzing various parameters related to protein family membership, domains and repeats, biological process and molecular function of HPs. MOTIF was used to assign the probable role of the HPs utilizing Pfam and PROSITE (Table 4).

Table 4: List of different bioinformatics tools used to annotate domains of HPs in Ganoderma.

Function prediction

Functional prediction for the hypothetical proteins of Ganoderma retrieved from UniProt, was carried out with the help of various freely available databases such Pfam, SUPERFAMILY [34], CATH, PANTHER [35], SYSTERS [36], SVMProt [37], CDART [38], SMART [39] and ProtoNet [40]. Presently, BLASTp was used for searching SYSTERS database and the output was obtained in the form of clusters of functionally related proteins. The cluster sequences with an E-value of 0.005 were classified as HP (Table 5). SUPERFAMILY database provided structural and functional annotation based on hidden Markov models with the evolutionary relationship. CATH provided the hierarchical classification of protein domains including class, architecture, topology, homologous superfamily. Hierarchical classification of the protein sequence into superfamily and subfamily clusters was achieved by SYSTERS (SYSTEmatic Re-Searching) with the backing of BLASTp. SVMProt predicted the family of functional protein based on SVM-based classification [37] determining the function of proteins based on the primary structure and also classified the distantly related proteins and homologous proteins of different function [39]. CDART searches the NCBI Entrez Protein Database based on domain architecture, which, in turn, uses the evolutionary distances by direct sequence similarity. Furthermore, CDART found similarities between protein across significant evolutionary distances by utilizing sensitive protein domain profiles rather than using the direct sequence similarity, in addition providing the information about the domain [38]. SMART (Simple Modular Architecture Research Tool), based on profile hidden Markov models determined domains in protein sequences. Data obtained from SMART was used in creating the Conserved Domain Database collection which was also made available as part of the Interpro database. PANTHER is a (Protein Analysis through Evolutionary Relationships) comprehensively organized database that analyzed protein families and subfamilies. PANTHER 7.0 covers the whole genome of 48 organisms and gives the phylogenetics relationship of genes and families. Depending on Hidden Markov Models (HMM), various hits with an E-value less than 1e-3 acutely defined the functionality of HPs.

Table 5: Functional prediction of HPs in SYSTER, InterPro, Gene Ontology, Superfamily and STRING.

Associative network among HPs in Ganoderma

Proteins interact with a network which modulates the functionality of the related proteins. Associative networking among proteins is still to be explored completely. In addition, there is a necessity for experimental performance owing to the crucial role of interactive partners that participate in multimeric complex depicting its relation between protein and function [41]. Physical, functional, experimental, and coexpression are major forces in STRING database depicting interaction. Besides, it also furnishes information about the metabolic or epigenetic associative network related to these proteins. In present work, STRING 9.1 was used for the prediction of the associative network of HPs in Ganoderma, based on parameters comprising neighborhood, co-occurrence, co-expression, experiments, databases, and homology of predicted partners of HPs (Table 5).

Results and Discussion

Availability of Ganoderma commercial products has authenticated its medicinal value. Ganoderma protein sequence, retrieved from NCBI and UniProt, were annotated function to delve deeper in understanding the proteomics. In the present study, 33 HPs of genus Ganoderma were investigated and characterized by bioinformatics tools like BLAST, HHpred, Pfam, PANTHER, CATH, CDART, and SVMProt. Tools like InterProScan BLOCK, MOTIF were employed for discovering functional motifs in the HPs (Tables 3 and 6).

Table 6: List of HPs with gene and UNIPROT ID, SVM Prot with annotated function of HPs in Ganoderma lucidum.

The physicochemical properties of all 33 hypothetical proteins were determined by the ExPASy-ProtParam software (Table 2).

MOTIF predicted nature of Galu_Mp10 containing HupE/ UreJ motif, which was found to have similarity with piscicolin 126 immunity proteins as disclosed by HHpred software. TMHMM and HMMTOP give clue about four transmembrane proteins in the sequences which may contributes towards adaptability of Ganoderma in different environmental conditions. On the other hand, Gene Ontology provided information about the extrachromosomal plasmid, thus, verifying its relevancy as an immunological function [5]. In addition, BLOCK databases based on multiple alignments of conserved regions found Galu_Mp10 to be a fungal pheromone, STE3 G-PCR, which aids in detection and verification of protein sequence homology.

Another stable HP was predicted to be Galu_Mp16, on the basis of similarity and homology of sequence by BLASTp and HHpred tool. Subsequent analysis revealed the motif to be similar to Bunyavirus glycoprotein G2 (309.371) as deduced by Pfam-based on annotations and multiple sequence alignments in MOTIF. SYSTERS database, based on hierarchical partitioning, found Galu_Mp16 to be similar in sequence to integrase by in InterPro (IPR), whereas, Gene Ontology indicated it to possess DNA recombination function. STRING database found integrase to be present in the superfamily of Galu_Mp16.

Galu_Mp17 was suggested by HHpred to possess the prefoldin function evidenced throughout the literature for its fundamental role in protein folding. These proteins exhibit protein-folding activity synergistically combining with other protein. SYSTERS conferred Galu_Mp 17 to be similar to SMC proteins (Structural Maintenance Chromosome) which have ATPase family with a role in DNA recombination and repair. BLOCK further conferred it to possess thyroid hormone-inducible hepatic Spot 1 having the ability to regulate lipogenesis especially synthesis of fatty acids [8,9]. Motif by PROSITE which is a database of protein domains, families, and functional sites predicted hypothetical protein to play a role as tubulin-beta mRNA autoregulation signal, helper component proteinase, Mer2 whereas Pfam database based on multiple sequence alignments predicted it as MetRS-N binding domain.

BLASTp and HHpred predicted Galu_Mp19 to be homing endonuclease LAGLIDADG [42]. ProDom database consisting of an automatic compilation of homologous domains found Galu_Mp19 to be endonuclease LAGLIDADG, which was also confirmed by BLOCK. InterProScan identifies it as LAGLIDADG based on a prediction of protein families, domains and functional sites of the Galu_Mp19. SUPERFAMILY verified its evolutionary domain as an endonuclease later verified by CATH database. Motif annotated the protein to be LAGLIDADG by Pfam. SVMProt predicted the functional family based on a primary structure having the tendency for the classification of homologous proteins and distantly related proteins of different function and predicted it as glycosyltransferase. CDART and ProtoNet 6.1 predict and confirmed homing endonuclease LAGLIDADG-like domain. Gene Ontology predicted Galu_Mp19 as homing endonuclease LAGLIDADG, which is a rare cutting enzyme encoded by introns and inteins. Homing endonuclease is highly invasive elements which promote recombination by breakage of the double strand and facilitates repair system [42]. In spite of all, STRING does not exhibit any interaction with other.

Galu_Mp20 is another hypothetical protein portraying NADPH dehydrogenase function, a key enzyme for oxidative phosphorylation in the mitochondria, a crucial role in triggering apoptosis in addition to correlating mitochondrial activities and programmed cell death [10]. BLASTp found the hypothetical protein Galu_Mp20 to possess NADH dehydrogenase activity by sequence similarity, while the conserved sequence in BLOCK related this protein with flagellar basal body-associated protein FliL. SUPERFAMILY predicted it to possess Hect, E3 ligase catalytic domain whereas InterPro (IPR) prediction of protein families, domains, and functional sites revealed it to be lipoprotein, type 6Ferritin/ribonucleotide reductase-like. Gene Ontology recognized Galu_Mp20 to exhibit defense/immunity protein activity

Similarly, the hypothetical protein Galu_Mp21 was observed to possess DNA-dependent RNA polymerases like activity as indicated by BLASTp and HHpred bioinformatics tool. ProDom found DNA-directed RNA polymerase whereas conserved sequences in BLOCK database revealed it to have a frizzled protein signature, further confirmed by InterProScan and SUPERFAMILY prediction tools. Motif by Pfam revealed the N-terminal of the DNA-directed RNA polymerase, whereas SVMProt indicates the primary structure to be consisting of transferring phosphorus-containing groups. CDART and ProtoNet 6.1 determine the similarities across significant evolutionary distances of proteins in Galu_Mp21 to have an activity like DNA-dependent RNA polymerase. Gene Ontology denoted it to have ATP binding, nucleotide binding, and transferase activity, while STRING revealed an absence of its protein interaction with others. Importantly, RNA polymerase controls the gene transcription and improves the efficiency in adaptability in different sort of stress. From these results, it can be surmised that these genes can modulate certain enzyme and their expression, thus, assisting in acclimatization and adaption of the fungus to a particular environment. It also plays a role in telomerase and RNA silencing which may be a target for designing various therapeutics agents.

Gasi_Mp30 through BLASTp was found to have similarity with GIY-YIG endonuclease, whereas HHpred homology detected it to be like Uvr abc system protein C. ProDom analyzed it as metal-binding iron heme domain of the oxidase transmembrane contained on the mitochondrion membrane, whereas BLOCK indicated it to have likeness to N-terminal of the c subunit of excinuclease ABC by the conserved sequence analysis. InterProScan, SUPERFAMILY predicted the protein families, domains and functional sites as GIY-YIG endonuclease. CATH and PANTHER analyzed sequences to have a domain similar to Uvr ABC system protein C while SVMProt deciphered it as zinc-binding protein family. CDART and SMART predicted the domain, further confirming the HP to be similar to GIY_YIG, whereas ProtoNet 6.1 showed its similarity with nuclease-associated modular DNA-binding 1. Gene Ontology used the term in cellular component associated with mitochondria whereas STRING database showed interaction with COX2 cytochrome c oxidase subunit 2.

Gasi_Mp34 protein was also seen to have similarity, as detected by BLASTp with, GIY-YIG endonuclease whereas HHpred by homology detected it be similar to Uvr abc system protein C. ProDom analyzed it as endonuclease intron-encoded hydrolase. In this case also BLOCK detected it to have a likeness to N-terminal of the c subunit of excinuclease ABC by the conserved sequence analysis. InterProScan, SUPERFAMILY predicted protein families, domains and functional sites as GIY-YIG endonuclease. GIY-YIG motif was confirmed by Motif whereas SVMProt deciphered Gasi_Mp34 protein as all DNA-binding. SMART predicted the domain and upheld the conclusion of it being GIY_YIG to be true. ProtoNet 6.1 hierarchically classified it as an intron-encoded nuclease. Gasi_Mp34 was observed to have no positive interaction with others proteins.

Hypothetical protein Gasi_Mp23 was seen to have similarity with GIY Cytb i2 grp ID protein as projected by BLASTp and further confirmed it as GIY-YIG endonuclease by HHpred. ProDom analyzed it as mitochondrial endonuclease GIY-YIG, whereas BLOCK indicated Intron-encoded nuclease two domains. InterProScan and SUPERFAMILY predicted it as GIY-Journal YIG endonuclease. Motif predicted NUMOD1 domain and SMART found it to possess introns encoded nuclease motif while ProtoNet 6.1 has Cytochrome b/b6, N-terminal. Gene ontology found it to have catalytic activity whereas STRING defined and characterized the CYT1: C-terminal fragment of CaP19.3527 indicating it to be likely cytochrome C1.

Other hypothetical proteins, Gasi_Mp09, and Gasi_Mp42, through different parameters (Table 2) were annotated function as GIY-YIG endonuclease. It is interesting to note that the GIY-YIG nuclease domain superfamily has been implicated in the cellular process including DNA repair and recombination, transfer of mobile genetic elements, and restriction of incoming foreign DNA. It serves as a scaffold for metal ion required for catalysis of phosphodiester bond cleavage. Moreover, the nucleases of the GIY-YIG have been seen to be involved in many cellular processes including DNA repair and recombination, transfer of mobile genetic elements, and restriction of incoming foreign DNA. The GIY-YIG domain also forms a compact structural domain serving as a scaffold for the coordination of a divalent metal ion required for catalysis of the phosphodiester bond cleavage.

Another hypothetical protein, Gasi_Mp32, was predicted as reverse transcriptase enzyme, a key enzyme in antiviral drugs and insulin production. Initially, BLASTp predicted it as RNA-directed DNA polymerase, and HHpred detected to be telomerase reverse transcriptase. ProDom database consisting of an automatic compilation of homologous domains found RNA-directed transcriptase which was further confirmed by BLOCK as telomerase. InterProScan identified it as reverse transcriptase based on a prediction of protein families, domains and functional sites of the Gasi_Mp32. SUPERFAMILY verified its evolutionary domain as reverse transcriptase which was also verified by PANTHER database. Motif annotated the protein as reverse transcriptase by Pfam. SVMProt predicted its functional family based on the primary structure and having the capability for the classification of distantly related proteins and homologous proteins of different function and predicts it as zinc binding. CDART, SMART and ProtoNet 6.1 predicted and confirmed this hypothetical protein to be reverse transcriptase.

Homing endonuclease

In addition to the above discussed hypothetical proteins, others including Gasi_Mp21, Gasi_Mp11, Gasi_Mp33, Gasi_Mp31, Gasi_Mp40, Gasi_Mp24, Gasi_Mp41, Gasi_Mp06, Gasi_Mp05, Gasi_Mp27, Gasi_Mp44, Gasi_Mp25, Gasi_Mp35, Gasi_Mp10, Gasi_Mp22, Gasi_Mp37, Gasi_Mp39, Gasi_Mp26, Gasi_Mp36 and Gasi_Mp38 were analyzed with respect to their molecular weight, theoretical pI, aliphatic index and hydropathcity (Table 2). The information thus obtained taken together indicated them to be behaving as LAGLIDADG endonuclease. BLASTp, HHpred, ProDom, BLOCK and various bioinformatics tools with high confidence predicted its function as LAGLIDADG. Homing endonucleases, encoded by open reading frame in self-splicing introns and having an independently folded domain of self-splicing introns known as inteins facilitates self-propagation [43]. It endorses the homing of their respective genetic elements into allelic intronless and inteinless sites and thus playing a vital role in recombination. LAGLIDADG motif plays a crucial role in protein folding, dimerization or interdomain packing and catalysis [42]. Homing endonuclease plays a pivotal role in genome analysis, gene manipulation, cloning, recombination events, double-stranded repair, and transposition as rare cutting endonucleases to uphold chromosomal integrity and viability. LAGLIDADG plays a crucial role in protein folding, dimerization or interdomain packing and catalysis. Importantly, endonuclease plays a pivotal role in DNA repair, and little deviation in normal functioning may lead to the genesis of anomalies.

(Table 6) List of HPs with gene and UNIPROT ID, SVM Prot with annotated function of HPs in Ganoderma lucidum

Conclusion

Prediction and annotation of function to hypothetical proteins forms an indispensable part of bioinformatics and proteomics. Annotating functions to the uncharacterized proteins can assist in fathoming the various mechanisms fundamental in the adaptation of this fungus to a various stress condition. Literature has time and again declared the importance of protein which is unexplored, thus, motivating us to annotate functions to the 33 hypothetical proteins, being carried out for the first time. This endeavor reached it fruition by the assistance of various in silico tools, helping in understanding various decisive parameters along with the characteristics that shape the protein function. Some determining features of the proteins are also discussed. Among the 33 hypothetical proteins sequenced and characterized, all were predicted precisely with high confidence, except Galu_Mp10 and Galu_Mp16, which was mainly due to the absence of insufficient data. The information, thus obtained, reinstates the versatility and multifaceted nature of the fungal proteins. Delving deeper in knowing the revealed functions may help in understanding the regulation of various signaling pathways modulating cell cycle, providing a more lucid view for medical interventions. Lastly, but not all the least, the exploring the functional nature of these proteins may provide a platform for charting out effective therapies and designing drugs. In-depth predictions and functional annotation of the HPs, if and when, carried out, would assist in understanding the nuances of proteomics better.

Acknowledgement

Authors thanks Central University of Punjab, Bathinda for providing the necessary facilities to carry out the present work.

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