Muhammad Naveed*ab,
Komal Imranb,
Ayesha Mushtaqb,
Abdul Samad Mumtazc,
Hussnain A. Janjuad and
Nauman Khalid*e
aDepartment of Biotechnology, Faculty of Life Sciences, University of Central Punjab, Lahore 5400, Pakistan. E-mail: naveed.quaidian@gmail.com; Tel: +92 301 5524624
bDepartment of Biotechnology and Biochemistry, University of Gujrat, Gujrat 50700, Pakistan
cDepartment of Plant Sciences, Quaid-i-Azam University, Islamabad 44500, Pakistan
dDepartment of Industrial Biotechnology, Att-Ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, H-12, Islamabad, Pakistan
eSchool of Food and Agricultural Sciences, University of Management and Technology, Lahore 54000, Pakistan. E-mail: nauman.khalid@umt.edu.pk; Fax: +92 42 3518478; Tel: +92 333 5278329
First published on 12th June 2018
Since the last decade, various genome sequencing projects have led to the accumulation of an enormous set of genomic data; however, numerous protein-coding genes still need to be functionally characterized. These gene products are called “hypothetical proteins”. The hypothetical protein pecanex-like protein 2 Homo sapiens (PCNXL2) is found to be mutated in colorectal carcinoma with microsatellite instability; therefore, annotation of the function of PCNXL2 in tumorigenesis is very important. In the present study, bioinformatics analysis of PCNXL2 was performed at the molecular level to assess its role in the progression of cancer for designing new anti-cancer drugs. The retrieved sequence of PCNXL2 was functionally and structurally characterized through the web tools Pfam, Batch CD (conserved domain) search, ExPASy, COACH and I-TASSER directed for pathway analysis and design to explore the intercellular interactions of PCNXL2 involved in cancer development. The present study has shown that PCNXL2 encodes multi-pass transmembrane proteins whose tumor suppressor function may involve regulating Notch signaling by transporting protons across the membrane to provide suitable membrane potential for γ secretase function, which may liberate the Notch intracellular domain NICD from the receptor to inside the cell. Furthermore, domain A of PCNXL2 may exhibit nuclear transport activity of NICD from the cytoplasm to the nucleus through interaction with a nuclear localization signal that may act as an activator for Notch signaling in the nucleus. Conclusively, the tumor suppressor role of PCNXL2 by regulation of the Notch signaling pathway and its functional and structural characteristics are important findings. However, further studies are required to validate the putative role of PCNXL2 as a cancer biomarker in cancer development.
Functional annotations of hypothetical proteins and docking sites provide an extensive outlook of essential biochemical pathways or the cellular processes responsible for the development and prognosis of any disease in the body.2 The associations between biologically relevant molecules such as proteins, nucleic acids, carbohydrates, and lipids play central roles in signal transduction; however, the relative orientation of the two interacting partners may affect the type of signal produced (e.g., agonism vs. antagonism).3 Therefore, docking is useful for predicting both the strength and type of a signal produced by hypothetical protein–ligand interactions to the appropriate target binding sites.4 Therefore, characterization of the binding behavior of hypothetical proteins will play an important role in the rational design of drugs with the elucidation of fundamental biochemical processes.
Pecanex homolog 2 (PCNXL2) is a protein-coding gene, and homologs of this gene are present from Drosophila to humans.5 In Drosophila, it is observed that loss of pcx (pecanex) may cause a neurogenic phenotype that may involve the N signaling pathway. pcx resides in the endoplasmic reticulum in Drosophila, and its disruption causes enlargement of the ER; this confirms its characteristic role in ER functioning.6 Previous studies reported that frameshift mutations have been found in FLJ11383 (PCNXL2) in patients suffering from colorectal carcinoma.7 Although the incidence of mutations is high, the role of these mutations in tumorigenesis is not yet clear. A study demonstrated that PCNXL2 encodes a multipass transmembrane protein with unknown functions in Drosophila that may be required during activation of the N signaling pathway. Actually, pecanex (pcx) functionally encodes a multi-pass transmembrane protein with unknown functions that is widely found from Drosophila to humans; due to its maternal neurogenic effects, it may be involved in activation and regulation of N signaling pathways.8 A study reported in Drosophila described pcx as a component of the Notch signaling pathway that is required upstream during activation of the pathway.6 Because pcx residues are present in the endoplasmic reticulum and its disruption can cause ER enlargement, pcx may lead to lateral inhibition of neuroblasts. No known predicted secondary structures of PCNXL2 have been identified to date in humans. Therefore, the detailed structure and function of this protein must be determined to understand the signaling transduction pathway. In humans, the Notch signaling pathway has diverse consequences due to its cross-talk with other pathways or cell-specific regulatory molecules; its disruption can lead to pulmonary arterial hypertension, leukemia and several cancers by inhibiting its tumor suppressor role in a cellular context.9 Notch signaling involves direct cell to cell communication through interaction of the expressed ligand with the Notch receptor of a neighboring cell. Ligand–receptor interaction causes Notch activation by cleavage of the receptor through activated γ secretase to release the activated Notch intracellular domain, NICD, in the cytoplasm; this is followed by translocation to the nucleus, where it may associate with the DNA binding protein CSL and regulate the transcription of other tumor suppressor genes.10 During cellular maturation, the extracellular domain of N is cleaved by furin protease, ADAM10/ADAM17 and γ secretase to liberate the intracellular domain for cell–cell Notch mediating signaling. N requires several posttranslational modifications, such as O-glycosylation, for optimal disulfide bond formation in the extracellular domain of N for activation of N signaling.11
pcx is a component of the N signaling pathway; however, the exact molecular function of the pcx encoded multi-pass transmembrane protein is not known. The present study was conducted to characterize PCNXL2 functionally and structurally to understand its gene regulatory network; molecular docking was performed to provide insight into its exact molecular process in the N signaling pathway, which may be related to its tumor suppressor role and its putative role as a cancer biomarker for patient prognosis and therapeutic intervention.
A Batch CD search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) was also performed to highlight conserved domains in PCNXL2 for further confirmation of our results. This program uses the NCBI web interface to explore the conserved domain database within protein-coding sequences using RPSBLAST, a variant of PSI-BLAST, and Position-Specific Scoring Matrices (PSSMs).14 ScanProsite (https://prosite.expasy.org/scanprosite/) allows searching of domains, functional sites and protein families by comparing amino acid patterns in the PCNXL2 protein.15 These predictions assisted in the characterization of the biochemical function of our hypothetical protein.
CYSPRED (https://gpcr.biocomp.unibo.it/cgi/predictors/cyspred/pred_cyspredcgi.cgi) determines whether the cysteine residues in your query protein form disulfide bridges/bonds. CYSPRED is a neural network-based predictor that is trained to distinguish the bonding states of cysteine in proteins, starting from the residue chain in its non-binding state, with high efficiency.24
DIANA (https://clavius.bc.edu/%7Eclotelab/DiANNA/) was also used because it helps predict the disulfide connectivity of an input protein sequence. Correct prediction of the disulfide bonds in a hypothetical protein is of crucial interest for understanding the protein function and is key for tertiary prediction methods.25 The tertiary structure of a hypothetical protein will be helpful in identifying docking sites, moving one step closer towards designing drugs that target diseases caused by mutations in the PCNXL2 hypothetical gene.
One additional tool, CYSCON (https://www.csbio.sjtu.edu.cn/bioinf/Cyscon/), was used for disulfide bond prediction to enhance and confirm the knowledge we obtained from the abovementioned software. CYSCON first identified the most confident disulfide bonds within the hypothetical protein; then, the prediction focused on the remaining cysteine residues based on Support Vector Regression (SVR) training.26
ProtParam tool | EMBOSS Pepstats | ||
---|---|---|---|
Sequence ID | gi|134254443|r | Charge | 9.5 |
Family (Pfam) | Pecanex_C superfamily | Improbability of expression in inclusion bodies | 0.747 |
Domain (ScanProsite) | DNAJ_1 | Average residue weight | 111.033 |
Alignment | 1619–1844 | A280 extinction coefficients 1 mg ml−1 | 1.039 |
HMM length | 229 | A280 molar extinction coefficients | 246635 |
Bit score | 369.2 | Tiny (A + C + G + S + T) | 662 |
E value | 6.3 × 10−111 | Small (A + B + C + D + G + N + P + S + T + V) | 1084 |
Number of AA | 2137 | Aliphatic (A + I + L + V) | 608 |
MW | 237276.9 | Aromatic (F + H + W + Y) | 238 |
pI | 6.29 | Non-polar (A + C + F + G + I + L + M + P + V + W + Y) | 1108 |
EC | 246635 | Polar (D + E + H + K + N + Q + R + S + T + Z) | 1029 |
II | 48.98 | Charged (B + D + E + H + K + R + Z) | 487 |
AI | 87.45 | Basic (H + K + R) | 265 |
GRAVY | −0.204 | Acidic (B + D + E + Z) | 222 |
ScanProsite predicted that DNAJ_1 PS00636 (Nt-dnaJ domain signature) belongs to the J protein family in PCNXL2, ranging from 1270 to 1289 amino acids. DNAJ_1 regulates the activity of hsp70, which is a molecular chaperone responsible for the translocation of polypeptides across organelle membranes, folding of nascent proteins, targeting proteins for degradation and inferring responses to stress.37
EMBOSS Pepstats shows statistics of the physiochemical properties of PCNXL2, such as the number and molar percent of each type of amino acid; by applying DayhoffStat, the number and molar percent of each physiochemical class of amino acid, the extinction coefficient at 1 mg ml−1 (A280) and the molar extinction coefficient (A280) can be obtained. The EMBOSS Pepstats results showed that PCNXL2 exhibits 9.5 charges and consists of up to 608 aliphatic, 238 aromatic, 1108 nonpolar and 1029 polar amino acids (Table 1). The aliphatic amino acids compose the core of the protein, while polar and charged amino acids are present on the surface of the protein and interact with solvent molecules. The positive and negative amino acids form salt bridges and maintain the three-dimensional structure of the protein. The molar percentage of each amino acid reflects the ability of the protein to form secondary structures. The amino acids M, A, L, E, K, G, Y, S, H, R, and Q prefer to adopt helical conformations, while T, W, Y, F, I, and V prefer to adopt β conformations. DayhoffStat predicted that most of the amino acids in PCNXL2 (A, R, N, Q, E, K, H, P, S, T, W, and Y) have negative scores; this characterizes PCNXL2 as hydrophilic.
No. | N-terminal | Transmembrane region | C-terminal | Type | Length |
---|---|---|---|---|---|
1 | 63 | TNSCHLYLWLFLLLLPLALHLAF | 85 | PRIMARY | 23 |
2 | 92 | VFFYCSAVTIFFTIIKLVSYRLH | 114 | PRIMARY | 23 |
3 | 881 | IVLVSLLGFLTLSQGFCKDMWVL | 903 | PRIMARY | 23 |
4 | 936 | TYSRPIYFCVLCGLILLLDTGA | 957 | PRIMARY | 22 |
5 | 982 | RDYLIVFLYCFPAISLLGLFPQI | 1004 | PRIMARY | 23 |
6 | 1008 | CTYLLEQIDMLFFGGSAVSGITS | 1030 | SECONDARY | 23 |
7 | 1034 | SVARSVLAAALLHAVCFSAVKEP | 1056 | PRIMARY | 23 |
8 | 1059 | MQHIPALFSAFCGLLVALSYHLS | 1081 | SECONDARY | 23 |
9 | 1129 | WDLIVCAVVAVLSFAVSASTVFL | 1151 | PRIMARY | 23 |
10 | 1157 | LSIVLFALAGAVGFVTHYVLPQL | 1179 | PRIMARY | 23 |
11 | 1224 | KYILYPALILNALTIDAFLISN | 1245 | PRIMARY | 22 |
12 | 1249 | LGTHWDIFLMIIAGMKLLRTSFC | 1271 | SECONDARY | 23 |
13 | 1273 | PVYQFINLSFTVIFFHFDYKDIS | 1295 | SECONDARY | 23 |
14 | 1301 | DFFMVSILFSKLGDLLHKLQFVL | 1323 | SECONDARY | 23 |
15 | 1333 | WGSSFHVFAQLFAIPHSAMLFFQ | 1355 | SECONDARY | 23 |
16 | 1360 | SIFSTPLSPFLGSVIFITSYVRP | 1382 | SECONDARY | 23 |
Other built-in software in the SOSUI server, such as SOSUI (Batch), confirmed that PCNXL2 is a 100% membrane protein. SOSUIsignal predicted that PCNXL2 has no signal peptide, which shows that PCNXL2 is not involved in the secretory pathways of the cell. SOSUIgramN predicted that the inner membrane is the subcellular localization site of PCNXL2. SOSUImp1 showed that PCNXL2 is a multi-spanning membrane protein with potential to perform functions such as signal transduction, immunological reaction and cell adhesion (Table 3).
SOSUI (Batch) | SOSUI signal | SOSUIgramN | SOSUImp1 | ||
---|---|---|---|---|---|
Nature | Percentage | Signal peptide | Nature | Subcellular localization site | Spanning membrane protein |
Membrane protein | 100% | No | Soluble | IM (inner membrane) | Multi |
Fig. 2 Predicted model for the whole sequence of PCNXL2. (RaptorX results: current status of the model of PCNXL2 is complete). |
Actin-like 6-A (ACTK6A) is a member of the SWI/SNF like BRG1/brm associated factor (BAF) chromatin remodeling complex that encodes BAF53a, the 53 kDa subunit of the BAF complex in mammals.38 ACTL6A is involved in diverse cellular processes, such as nuclear migration, chromatin remodeling and transcriptional regulation. Recently, a study by Bao et al.39 demonstrated that SOX2 is a potential target of ACTL6A and ACTL6A ectopic expression; enhanced SOX2 expression up-regulates Notch 1 and triggers the Notch signaling pathway in the cell. Hence, ACTL6A can enhance Notch activity in cells, exhibiting a tumor suppressor function by targeting reduced proliferation and apoptosis of cancerous cells. Another study by Rowland and Peeper40 suggests that ACTL6A regulates the SWI/SNF chromatin remodeling complex to suppress differentiation in the epidermis by preventing SWI/SNF from binding to promoters of KLF4 and other differentiation genes. KLF4 is a transcription regulator that dictates diverse biological processes, including pluripotency, cell differentiation, tumorigenesis and proliferation.40
TATA box binding associated factor RNA polymerase 1 B (TAF1B) is a component of the RNA polymerase 1 core factor complex; it plays active roles in multiple steps during transcription initiation, such as pre-initiation complex (PIC) assembly and post polymerase recruitment events. TAF1B, the second largest component of TATA-binding protein (TBP), binds to ribosomal DNA by recruiting POL1 to the SL1/TIF-IB complex through its interaction with RRN3.41 A study demonstrated that MARCK2, PCNXL2 and TAF1B are putative target genes in colorectal carcinoma with microsatellite instability.7 PCNXL2 (74%) and TAF1B (82%) were found to be mutated in MSI-H colorectal carcinomas. This study reported that frameshift mutations of the three abovementioned target genes are related to functional inactivation by generating truncated proteins and may be related to tumorigenesis. Cell cycling and protein synthesis are key regulatory tasks for cancerous cells. TAF1B is involved in the synthesis of proteins and mutations and has been found in many cancers; therefore, we can conclude that mutations result in truncated proteins which are unable to perform their functional roles in the homeostasis of cells. Furthermore, these mutations may inactivate tumor suppressor genes and DNA repairing genes in cells by providing a suitable cancerous microenvironment which initiates tumorigenesis.
Fibulin-5 (FBLN5) is a member of the fibulin family that is widely expressed as an extracellular matrix protein (ECM) and is involved in cell-to-matrix or cell-to-cell communication, elastogenesis, cell adhesion and cell mobility.42 Fibulin-5 is a 66 kDa glycoprotein that is localized in elastic fiber and is involved in regulating elastic fiber assembly. It has a unique RGD domain which can bind to integrins on cell surface receptors. Integrins regulate intracellular signaling through extracellular signal-regulated kinase (ERK), and focal adhesion kinase (FAK) mediates cell adhesion to the extracellular matrix.43 The Notch signaling pathway performs its tumor suppressor role by inhibiting Wnt/β catenin signaling through increased p21 expression, which may act as a negative modulator of Wnt-4. Fubilin-5 also acts as a metastasis suppressor by inhibiting the Wnt/β catenin pathway through inhibition of ERK via its unique RGD domain, leading to activation of GSK3β. GSK3β downregulates β-catenin by proteasomal degradation, preventing its translocation to the nucleus; this leads to suppression of the oncoproteins metalloproteinase-7 (MMP-7) and c-Myc.44
CYSPRED | DIANA | CYSCON | ||||
---|---|---|---|---|---|---|
Cysteine | Prediction | Reliability | Sequence ID | Bonded cysteine | SSBOND | Cysteine residues |
CYS 144 | Bonding state | 8 | gi|134254443|r | 34–1239, 64–337, 144–1402, 290–880, 312–614, 315–874, 331–797, 445–467, 519–1634, 652–715, 736–1472, 865–1473, 912–1587, 915–2086, 959–1418, 976–1475, 1017–1038, 1064–1781, 1102–2101, 1189–1873, 1454–2114, 1474–1924, 1486–1821, 1597–1704, 1607–1728 | SSBOND#1 | 34–2114 |
CYS 290 | Bonding state | 1 | SSBOND#2 | 64–2086 | ||
CYS 312 | Bonding state | 2 | SSBOND#3 | 144–2101 | ||
CYS 315 | Bonding state | 8 | SSBOND#4 | 290–1924 | ||
CYS 331 | Bonding state | 8 | SSBOND#5 | 312–1821 | ||
CYS 337 | Bonding state | 2 | SSBOND#6 | 315–1873 | ||
CYS 467 | Bonding state | 8 | SSBOND#7 | 331–1781 | ||
CYS 519 | Bonding state | 1 | SSBOND#8 | 337–1728 | ||
CYS 715 | Bonding state | 1 | SSBOND#9 | 445–1704 | ||
CYS 736 | Bonding state | 1 | SSBOND#10 | 467–1634 | ||
CYS 1418 | Bonding state | 8 | SSBOND#11 | 614–652 | ||
CYS 1472 | Bonding state | 8 | SSBOND#12 | 715–736 | ||
CYS 1473 | Bonding state | 8 | SSBOND#13 | 1017–1038 | ||
CYS 1474 | Bonding state | 4 | SSBOND#14 | 1402–1418 | ||
CYS 1475 | Bonding state | 4 | SSBOND#15 | 1587–1597 | ||
CYS 1607 | Bonding state | 4 | ||||
CYS 2086 | Bonding state | 2 |
CYSCON, a disulfide bond predicting software program, predicted SSBonds for the cysteine residues of PCNXL2 (Table 4). These results display a new path to improve the ab initio structure modeling of cysteine-rich proteins such as PCNXL2.
The second section contains information about the domain number. The best template is 3m1iC; it has a probability P-value of 7.8 × 10−7, which is less than the threshold and is considered to be a good model. The current model comprises 2131 residues with uGDT (136) and GDT (6), further confirming the viability of the model (Table 5).
Summary of predicted model | |
---|---|
Domains | 2 |
Best template | 3m1iC |
Probability value ‘P-value’ | 7.8 × 10−7 |
Overall uGDT | 136 |
GDT | 6 |
Modeled residue count | 1144 |
Disordered position count | 913 |
Secondary structure details | 31% H (helix), 8% E (beta sheet), 60% C (loop) |
The most important and targeted section of our study is the 3rd section, ‘detailed prediction results’, which contains relevant functional and structural information about the two predicted domains. The best model was selected on the basis of P-value using the templates 3m1iC, 3gjxA, 4gmxC, 4fgvA, and 4c0oA for the first domain and 4he8: I for the second domain; all the templates have different functions in the cell. To select the best-predicted domain, various parameters were used, such as rank, P-value, uGDT (GDT), uSeqId (SeqId), and Score. The first domain (domain A) has rank: 0, P-value: 7.8 × 10−7, uGDT (GDT): 98 (9), uSeqId (SeqId): 93 (9), and Score: 237; it contains 1054 amino acids, with a sequence from 821 to 1875. In contrast, the second domain has rank: 1, P-value: 4.55 × 10−3, uGDT (GDT): 38 (43), uSeqId (SeqId): 10 (11), and Score: 53; it contains 89 amino acids, from 1 to 89. All values for the first domain A are close to the threshold, with P-values less than 0.005; the uGDT must be greater than 50 and the Score must be in the range of 0 to the domain sequence length. This domain was considered to be the best domain compared to a second domain, whose values varied considerably from this threshold value (Fig. 4).
I-TASSER was applied to obtain comprehensive structural and functional understanding of the RaptorX-predicted domain. Domain A consists up of 1054 amino acids, from amino acids 821 to 1875 in the PCNXL2 protein. Pfam revealed the presence of a pcx domain from amino acids 1619 to 1844 in PCNXL2; thus, the pcx domain can be considered to be part of domain A. Therefore, the sequences of domain A and pcx were submitted separately in I-TASSER for structure and function predictions.
The secondary structure of domain A showed that it is enriched with long stretches of α helixes (M, A, L, E, K, G, Y, S, H, R, Q) and loops with a confidence score of 9, followed by a few short stretches of β sheets (T, W, Y, F, I, V), suggesting that the core region in domain A is an α helix secondary structure element. The remaining amino acids (P, G, S, D, and N) disrupt the secondary structure and give rise to loops or turns called secondary structure breakers. The remaining amino acids (P, G, S, D, and N) disrupt the secondary structure and give rise to loops or turns called secondary structure breakers. Prediction of the solvent accessibility discovered buried and exposed regions of domain A with values ranging from 0 (buried residue) to 9 (exposed residue). Most of the residues in the domain have 0 values, showing their involvement in the core region of the protein; residues with higher values are hydrophilic and can potentially be involved in hydration processes. The predicted normalized B factor estimates the thermal mobility of residues in a protein. The residues at the N and C termini have positive normalized B factors, indicating that these regions are relatively stable; meanwhile, the α and β regions have values that are either negative or close to 0, revealing that these regions are structurally stable. The arrangements of particular amino acids in a primary structure determine the secondary and tertiary globular structures of a protein by sequestration of hydrophobic amino acids in the core and enrichment of hydrophilic amino acids on the surface. The top five models were generated by I-TASSER. The model with the highest confidence score (C score; −1.18), TM score (0.57 ± 0.15) and RMSD (11.9 ± 4.4 Å) was considered to be the best-predicted model for domain A (Fig. 5). After selection, the best-predicted model was aligned against all known protein structures in the PDB database to find proteins that are structurally close to our target protein. Ten protein structures close to the target were predicted. One PDB structure (3w3tA) with a C score close to 0.9 was observed and was selected for further functional study of the target protein. The model (2vdcA) with the highest EC score (0.122) and TM score (0.338) was selected; it was found to be a glutamate synthase with an EC number of 1.4.1.13. Finally, gene ontology (GO) was performed to determine the function of domain A. This section comprises two parts; the first shows 10 homologous GO templates, while the second is a consensus prediction of GO terms, including molecular function, biological process, and cellular components. A true positive template was selected by analyzing the functional homology score (Fh score) between the target and template proteins and by estimating the confidence level. The first two templates, 1qgkA and 2z5kA, were selected with Fh scores of 0.08 and C scores of 0.09 and 0.08, respectively. The GO term predicted that the cellular component of domain A is the cytosol. A literature review of these proteins revealed that domain A exhibits nuclear transport activity; thus, it may transport macromolecules or substrates from the cytoplasm to the nucleus by interacting with nuclear localization signals.45 This domain may also play roles in biological processes by phosphodiester bond hydrolysis.46
The secondary structure of the pcx domain showed that it is enriched with long stretches of α helixes (M, A, L, E, K, G, Y, S, H, R, Q) with a confidence score of 9, followed by a very small number of short stretches of β sheets (T, W, Y, F, I, V), suggesting that the core region in the pcx domain is an α helix secondary structure element. Most of the residues in the pcx domain have scores of 9, showing that they are hydrophilic and may undergo hydration or act as active sites. In the pcx domain, the residues at the N and C termini were predicted with positive normalized B factors, indicating that these regions are relatively stable; meanwhile, the values in the α and β regions are either negative or close to 0, revealing that these regions are structurally more stable. The best-predicted model by I-TASSER for the pcx domain has a higher C score (−4.20), TM score (0.27 ± 0.08) and RMSD (15.9 ± 3.2 Å) (Fig. 6). After selection, the best-predicted model was aligned against all known protein structures in the PDB database, and ten protein structures close to the target were predicted. Three closer protein structures, 5cwcA, 5cwoA and 2zxeA, with TM scores greater than 5 were selected for further functional study of our target protein. The enzyme commission number and active site predicted 5 models for the target protein. The model (imhsA) was selected with the highest EC score of 0.065 and TM score of 0.503, illustrating that it is a proton transport ATPase with an EC number of 3.6.3.6. Gene ontology (GO) demonstrated the molecular function of the pcx domain using 10 homologous GO templates. True positive templates, 3ixzA and 2zxeA, were selected by analyzing their Fh scores (0.07 and 0.06, respectively). The GO term predicted that the cellular component of the pcx domain is an intrinsic component of the cell membrane which spans or embeds both leaflets around the membrane. A literature review of these proteins revealed that the pcx domain exhibits ATPase activity coupled to transmembrane movement of ions (protons) by establishing membrane potential for essential biological processes, such as the liberation of an activated protein from receptors. A domain can play roles in biological processes by performing particular functions in the cell.
COACH also predicted 10 models for ligand binding interactions of the pcx domain. The best model was considered to be 3fu1A, with a C score of 0.05 and a cluster size of 3; it binds to the zinc (Zn) ligand. The RAP ligand model can also bind with other ligands available in the “mult” link. The combined results of five algorithms, including COFACTOR, TM-SITE, S-SITE, FindSite and ConCavity, are illustrated in Fig. 8.
The miRNA-target expression profiles of the enlisted microRNAs were also studied in miRTarBase to evaluate which microRNA expression is high in a particular cancer based upon the Pearson coefficient (r) and probability value (P).49 In this expression profile, miRNA was checked against the PCNXL2 mRNA level in a particular cell by viewing and studying graphical charts. miRNAs which have positive R values in particular cancers are considered to have a positive linear relationship, justifying that targeted PCNXL2 expression is directly proportional to miRNA expression for a particular cell. Meanwhile, miRNAs which have negative R values are considered to have a negative linear relationship, justifying that targeted PCNXL2 expression is down-regulated with increased expression of the miRNA in a particular cancer. To accept the null hypothesis, the miRNA must have a P-value greater than 0.005, justifying that a high expression level of a particular microRNA causes cancer by inhibiting PCNXL2 expression in a particular cell type. The expression of selected miRNAs has been experimentally validated to have a negative linear relationship with PCNXL2 mRNA in different cancers and cells, as shown in Table 6.
MicroRNA | Colorectal carcinoma | Breast cancer | Pancreatic cancer | Prostate cancer | CNS germ cell tumor | Multiple myeloma | Differentiated embryonic stem cell tumor |
---|---|---|---|---|---|---|---|
hsa-miR-154-5p | ✓ | ✓ | ✓ | ■ | ✓ | ✓ | ✓ |
hsa-miR-382-5p | ✓ | ✓ | ■ | ✓ | ✓ | ■ | ✓ |
hsa-miR-335-3p | ✓ | ■ | ■ | ✓ | ■ | ■ | ■ |
hsa-miR-548d-3p | ✓ | ✓ | ■ | ■ | ✓ | ■ | ■ |
hsa-miR-512-3p | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ■ |
hsa-miR-520e | ✓ | ✓ | ✓ | ■ | ✓ | ✓ | ■ |
hsa-miR-520d-3p | ✓ | ■ | ■ | ✓ | ✓ | ✓ | ■ |
hsa-miR-520c-3p | ✓ | ✓ | ■ | ■ | ■ | ✓ | ■ |
hsa-miR-520b | ✓ | ✓ | ✓ | ■ | ■ | ■ | ■ |
hsa-miR-520a-3p | ■ | ✓ | ■ | ■ | ■ | ■ | ■ |
hsa-miR-373-3p | ✓ | ✓ | ✓ | ■ | ■ | ✓ | ■ |
hsa-miR-302c-3p | ■ | ✓ | ✓ | ■ | ■ | ✓ | ■ |
hsa-miR-302b-3p | ■ | ✓ | ■ | ■ | ■ | ✓ | ■ |
hsa-miR-302a-3p | ✓ | ✓ | ✓ | ■ | ■ | ✓ | ■ |
hsa-miR-93-5p | ✓ | ✓ | ■ | ■ | ✓ | ✓ | ■ |
hsa-miR-20b-5p | ✓ | ✓ | ✓ | ■ | ✓ | ■ | ■ |
hsa-miR-20a-5p | ■ | ✓ | ✓ | ■ | ■ | ■ | ■ |
hsa-miR-17-5p | ✓ | ■ | ■ | ■ | ■ | ■ | ■ |
hsa-miR-106b-5p | ✓ | ✓ | ■ | ■ | ■ | ■ | ■ |
hsa-miR-106a-5p | ■ | ■ | ■ | ■ | ✓ | ■ | ■ |
hsa-miR-1283 | ■ | ✓ | ✓ | ■ | ✓ | ✓ | ■ |
Proper folding of Notch (N) protein is essential for its activation; therefore, the formation of disulfide bonds in the extracellular domain of N is required for its proper folding by Ero1L. When an unfolded and misfolded protein appears in the ER, the organelle suffers stress known as “ER stress”. This stress can induce apoptosis. The cell can reduce its stress level through a response known as unfolded protein response (UPR), described by Yamakawa et al.8 The unfolded protein response (UPR) is a response that enables the body to cope with ER stress. In stressed cells, Ire-1 is an ER tethered endonuclease that acts as a sensor of ER stress and splices Xbp1 mRNA, which promotes protein folding and results in the transcriptional induction of genes such as Ero1L.50 ER-associated degradation (ERAD), aggresome formation and autophagic processes are also coping responses of the body to ER stress.
Therefore, the disruption of the N-signaling pathway in the absence of pcx function may be partially due to misfolding of N because pcx acts upstream of the activated form of N and probably functions in signal receiving in cells by providing stability to the N protein to aid its proper functioning. The structure of pcx suggests that it plays a role in the formation of disulfide bridges in the extracellular domain of N by providing cohesion and stability for its proper folding by enabling its expression in the cell. Pecanex-like protein 2 Homo sapiens (PCNXL2) releases a multi-pass transmembrane protein with 1054 amino acids comprised of domain A and the pcx domain from the inner membrane of the ER into the cytosol. Because PCNXL2 also contains a DNA_J domain, it facilitates the translocation of polypeptides across organelle membranes.51 Domain A is found to be present in the cytosol, while the pcx domain is an intrinsic component of the cell membrane. Notch signaling is activated by binding Notch ligands to Notch receptors on signal-receiving cells, which leads to activation of γ secretase for cleavage of the receptor to release and translocate the Notch intracellular domain NICD to the nucleus. For proper function of γ secretase, the active site must contain water to carry out hydrolysis in the hydrophobic environment of the membrane; however, it is not understood how water and proton exchange are effected. Here, we demonstrate that the pcx domain may exhibit ATPase activity coupled to transmembrane movement of ions (protons) by establishing membrane potential for essential biological processes such as the liberation of an activated protein (NICD) from receptors. The pcx domain may play its role by establishing localization of the activated domain in a cell. Furthermore, domain A exhibits nuclear transport activity of macromolecules or substrates (NICD) from the cytoplasm to the nucleus by interacting with a nuclear localization signal (Fig. 9). Domain A may also play a role in biological processes by phosphodiester bond hydrolysis. In the nucleus, NICD binds to transcriptional factor CSL to activate the expression of Notch signaling.52
There are diverse consequences of the Notch signaling pathway; among these, cyclin/CDK inhibitor p21WAF1/CLIP1 is an important target of Notch signaling. Increased Notch 1 activity leads to increased p21 expression, a key modulator of negative regulation of Wnt-4 expression of the Wnt/β catenin signaling pathway.53 The crosstalk between the p21WAF1/CLIP1, Notch and the Wnt/β catenin pathway is important for regulation of tumor development by preventing the proliferation of tumor cells through down-regulating the Wnt signaling pathway and expression of its oncogenes.54 Another target of Notch activation is induction of the NF-κB pathway, promoting cytokine production for the development of innate and adaptive immunity in cells for growth control and apoptosis.55 A negative feedback loop is present between Notch and p63, and p63 overexpression has been found in many cancers; p63 expression is suppressed by the activation of Notch through selective modulation of interferon-responsive genes. Like p63, the small GTPases Rho, Rac and CD42 have inverse relationships with Notch activation in the control of stem cell potential and tumor development.56 Notch CBF-dependent mechanisms achieve tumor suppression by downregulating ROCK1/2 and MRCKα kinases, which are key regulators of overexpressed Rho GTPase in tumors. Another consequence of Notch activation is the down-regulation of HPV oncogene expression; Notch 1 is under the direct effect of p53, so increased levels of p53 upregulate the expression of Notch in respective cells either through the inhibition of endogenous AP-1 activity or through other indirect mechanisms.57 P53 and its relatives are also key regulators of noxa expression; the P53 status of a cell regulates noxa expression. Noxa activation is followed by its interaction with Bcl-2 family members, freeing Bax/Bak and promoting apoptosis to mitochondria through caspase cascade activation.58 An additional target of Notch activation is the suppression of FoxO3a, which is a key transcriptional factor responsible for deciding the fate of a cell either by promoting tumor metastasis or increased cancer cell invasion. The canonical Notch/HERP pathway is a promising negative regulator of FoxO3a by binding of the HSE/HRE/Tle transcription repressor complex to the promoter of FoxO3a.59 Cross-talk between the Notch and transforming growth factor-β (TGF-β) signaling pathways plays critical roles in the control of cell fate by regulating Hes-1 expression, an important target of the Notch pathway. NICD and Smad3 interact directly in a ligand-dependent manner; Smad3 can be recruited to CSL-binding sites on DNA in the presence of CSL and NICD, indicating that Notch and TGF-β signaling are integrated through direct protein–protein interactions.60
We conclude that the pecanex protein is a tumor suppressor protein regulating Notch signaling to perform its tumor-suppressing function in developing cells, preventing misfolding of N proteins that can cause proliferation, cell survival, tumor invasion and expression of oncogenes.
Previously, frame shift mutations were found in FLJ11383 (PCNXL2) in patients suffering from colorectal carcinoma. Although the incidence of mutations was high, the role of these mutations in tumorigenesis was not clear.7 The current study suggests that pcx is independently involved in activation of the Notch signaling pathway by transporting protons across the membrane, thus establishing a membrane potential gradient to release embedded water to the active site of γ secretase channeling; this provides a suitable environment for hydrolysis to proceed, with liberation of NICD from the receptors. Therefore, it was not previously understood how water and proton exchange were effected to carry out hydrolysis in the hydrophobic environment of the membrane, which is the focus of the current research.62 Furthermore, domain A induced interaction with nuclear localization to transport NICD in the nucleus for the activation of tumor repressor genes in signal receiving cells.
However, the results suggest that the downregulation of PCNXL2 in signal receiving cells may inhibit the proper function of γ secretase. It may also partially cause truncated N protein synthesis with deregulation of the Notch signaling pathway, resulting in cancer development; this is consistent with our hypothesis that pcx regulates the N signaling pathway by providing a suitable membrane environment for the genesis of activated N protein in signal-receiving cells.
Other possible reasons include the induction of several downstream events by the activated N protein synthesis, including regulation of the Notch signaling pathway, overexpression of tumor suppressor genes and inhibition of Wnt signaling. Therefore, Notch signaling cross-talk with other pathways may enhance the complex sub branched mechanism of PCNXL2, with compensated up-regulation of Notch targeted tumor suppressor genes p21, p53, and p27; this can lead to down-regulation of oncogenes, proliferation, differentiation, and cell cycle and growth arrest.63
Previous literature studies also confirmed that improper or misfolded PCNXL2 protein-induced ER stress can lead to apoptosis.11 ER stress due to misfolded proteins can cause cell death and dysfunction, which can pave the road for other diseases such as diabetes, ischemia, neurodegeneration and reperfusion injury by disturbing cell homeostasis.64 Here, we suggest that PCNXL2 is involved in regulation of endoplasmic reticulum architecture by facilitating regulatory machinery. As is evident from this study, PCNXL2 constitutes a J protein family domain (DNA_J) that is a member of the hsp40 molecular chaperone family; this may correspond to stress by degradation of misfolded proteins. Therefore, it can be concluded that aberrant expression of PCNXL2 may lead to accumulation of misfolded proteins, and dysregulation of UPR can affect not only the stress responses of the ER but also the regulation of the architecture of ER.65
This work has shown that interference with the gene regulatory network can challenge the expression of tumor suppressor genes; especially, PCNXL2 can up-regulate the Notch signaling pathway, which determines cell fate by avoiding the deregulation of N signaling. The current results challenge the role of the pecanex protein as a cancer biomarker for patient prognosis and therapeutic intervention by resolving the tumor suppressor function of Notch in many cancers, such as prostate and colorectal cancer, and other biological processes. Therefore, in future experiments, it will be important to determine the link between human hereditary diseases and ER structural dysregulation and the cancer biomarker role of PCNXL2 in the absence of pcx function.
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