In  breast cancer , multicellular tumor spheroids (MTS) are  3D cell cultures  which acquire differentiated cell-cell junctions and a defined microenvironment, differentially expressing a number of adhesion molecules such as  EPCAM ,  E-cadherin ,  integrins  and  syndecans  when compared to 2D  monocultures . [ 13 ]  NAALADL2 has been shown to be differentially expressed in MTS when compared to 2D cultures. [ 13 ]  These results support a role of NAALADL2 in cell-cell interactions and agree with evidence in prostate cancer which find NAALADL2 affects cell- ECM  interactions. [ 13 ] [ 7 ]


SNP's in  NAALADL2  have also been identified in cancer risk GWAS's for breast cancer and Lung cancer. [ 14 ] [ 15 ]


It has been shown that the gene encoding  NAALADL2  is located within a  fragile site , a genomic loci prone to breakage and subsequent repair. [ 16 ] [ 17 ]  In cancer, the fragile site located within  NAALADL2  has been recently shown to be the fifth most altered of all fragile sites. [ 18 ]  Therefore, it has been suggested that the copy-number gains in  NAALADL2  and gains in surrounding oncogenes such as  GATA2 ,  PIK3CB ,  ATR ,  SMC4 ,  TBL1XR1 ,  SOX2  and  MUC4  may likely arise due to breakage and attempted genomic repair in this region. [ 19 ]  Upon a break in this fragile site, through a process known as the fork stalling and template switching (FoSTeS), extra copies of the genes in the region surrounding the break may be duplicated. [ 19 ]  Extra copies (copy-number gains) of  NAALADL2  and the genes which surround it have been shown to increase the  mRNA expression  of these genes, leading to further dysregulation and activation of cancer-associated pathways involved in growth and proliferation. [ 10 ] [ 19 ]


NBEAL1 is a protein that in humans is encoded by the  NBEAL1  gene. [ 1 ]  It is found on chromosome 2q33.2 of  Homo sapiens .


Through the different domains of this protein, the function of NBEAL1 is predicted to be involved in the following cellular mechanisms: vesicle trafficking, membrane dynamics, receptor signaling, pre-mRNA processing, signal transduction and cytoskeleton assembly. [ 2 ] [ 3 ] [ 4 ]  NBEAL1 is also known as Amytorophic Lateral Sclerosis 2 Chromosomal Region, ALS2CR16 and ALS2CR17. [ 1 ]


The  mRNA  for this protein consists of 9058 base pairs in a linear sequence with the coding sequence begins at base pair number 334 and extends until base pair number 8418. [ 5 ]   The translated protein is a total 56  exons  that constitute a final length of 2694 amino acids. [ 6 ]  There are currently 9 known isoforms within humans. [ 3 ]


Neurobeachin-like1 contains five domains: DUF4704, DUF4800, PH_BEACH, Beach, and WD40 repeats. [ 6 ]


DUF4704 is a domain of unknown function. While the function of this domain is unknown, it is conserved within neurobeachin proteins in eukaryotes. [ 4 ]  It begins at amino acid 859 and spans until number 1115. [ 3 ]


DUF4800 is a domain of unknown function. It begins at amino acid 1580, spanning until 1833. [ 3 ]  While it is uncharacterized in function, it is found within eukaryotes. [ 7 ]


Spanning from amino acid 1886 until amino acid 1983, this domain is referred to as a Pleckstrin Homology domain in the BEACH domain. [ 8 ]  It has a PH because the fold of this domain is similar to the PH domain, but is not identical in the sequence of the canonical PH domains. The PH_BEACH domain is not able to bind phospholipids. [ 9 ]


The Beige and Chediak-Higashi (BEACH) domain is one of the most  significant domains within this protein. This domain is highly conserved roughly 280 amino acid domain, present in nine different human BEACH domains. [ 10 ]  It located after the PH_BEACH domain in the sequence. While not much is understood on the exact function of BDCP proteins within the BEACH domain, it is known that they serve many purposes within cellular mechanisms: vesicular transport, apoptosis, membrane dynamics and receptor signaling. [ 10 ]  This protein family is of great clinical importance currently because mutations in this domain have been identified in multiple human disorders. For example, neurobeachin-like1 is upregulated in glioma: as the pathological grade of the glioma increases, the expression of neurobeachin-like1 is decreased. [ 2 ]  In NBEAL1, this follows the PH_BEACH domain, beginning at amino acid 2005 and ending at amino acid 2284. [ 3 ]


NBEAL1 has one  WD40 domain  within NBEAL1. From amino acid 2409 to 2682 is the entire WD40 domain. Within the domain, from 2406 to 2439, there is a structural motif WD40 repeat. The WD40 domain is found in a number of eukaryotic proteins that have multiple functions. These include, but are not limited to, adaptor/regulatory modules in signal transduction, pre-mRNA processing, and cytoskeleton assembly. [ 3 ]


The secondary structure of NBEAL1 is predicted to be a combination of  alpha helices ,  beta sheets  and  random coils . [ 13 ]


I-TASSER was used to predict a 3D structure of NBEAL1. [ 14 ]  Since NBEAL1 is longer in amino acid length than allowed for input, it was split in half to predict the structure of the whole protein.


The following document illustrates the different post-translational modifications.


Using the EST abundance profile through Unigene, NBEAL1 expression was discovered based on both body sites and health states. [ 15 ]  NBEAL1 shows expression in the brain, embryonic tissue, eye, intestine, kidney, liver, lung, mammary glands, ovaries, pancreas, pharynx, placenta, prostate, skin, stomach, testis, thyroid, and trachea. Based on transcripts per million, expression is highest in the stomach at 62 transcripts per million, with pancreas and trachea being next with their transcripts per million being 37 and 38, respectively. The lowest transcripts per million in the brain, eye, placenta and testis, all at 4 per million. When looking at the breakdown by different health states, NBEAL1 is highly expressed in multiple tumors. [ 15 ]  Again, the abundance was highest in  gastrointestinal tumors , correlating to the high expression of NBEAL1 within the stomach. However, NBEAL1 expression is not seen in  pancreatic tumors , which may signify something about its function within the pancreas. The abundance also differs in developmental stages, the highest being the fetal stage with 21 transcripts per million and the adult at 14 transcripts per million.


The function of NBEAL1 is not yet well understood by the scientific community. However, given the function of the different domains and disease associations, it is predicted that the NBEAL1 protein may be involved in a variety of functions. As of now they include, but are not limited to, protein-protein interactions, vesicle trafficking, membrane dynamics, receptor signaling, apoptosis, adaptor/regulatory modules in signal transduction, pre-mRNA processing, and cytoskeleton assembly. [ 3 ] [ 2 ]


This protein has been associated with  NBEAL1  are  Amyotrophic Lateral Sclerosis , Juvenile and  Adenocarcinoma , [ 1 ]  although the function in these diseases has not yet been identified.


Neurobeachin-like1 is a highly conserved protein. It has orthologs found in many life forms, including but not limited to: reptiles, birds, amphibians, mammals, fish, and a few invertebrates. The following table presents some of the orthologs found using searches in BLAST [ 16 ]  and BLAT. [ 17 ]


According to GeneCards, NBEAL1 has a few paralogs:  NBEAL2 ,  WDFY3 , NBEA,  LRBA ,  Lysosomal trafficking regulator  (LYST), and WDFY3. [ 19 ]  The table below summarizes the paralogs of NBEAL1.


Neuroblastoma breakpoint family, member 1 , or  NBPF1 , is a protein that is encoded by the  gene   NBPF1  in humans. This protein is member of the  neuroblastoma breakpoint family  of proteins, a group of proteins that are thought to be involved in the development of the nervous system. [ 3 ]


The NBPF1 gene in humans is located on the minus strand of 1p36.3 in humans and is 51179 base pairs long including  exons  and  introns . It is located between the protein coding genes  NECAP2  and  CROCC . NBPF1 is one of the 26 known members of the Neuroblastoma Breakpoint Family genes and pseudogenes. The NBPF2 pseudogene and  NBPF3  gene are the most similar genes located close to NBPF1 and they reside on the chromosomal location 1p36.12. Most members of the NBPF gene family are located on chromosomal location 1q21.1-1q23.3 in humans, and these genes are more similar to each other in sequence than they are to NBPF1. [ 4 ]


The transcript for NBPF1 in humans is a 6183 base pair mRNA that is made of 28 exons. There are more than 14  alternative splicing  forms of NBPF1 predicted, but only seven of the splice forms have been observed. Out of all of the possible transcripts, only two are known to code proteins. One of these transcripts is 1139 amino acids long with 23 coding exons, while and the other is 1095 amino acids long and 23 coding exons. The noncoding transcripts are processed, but their function is unknown. [ 3 ] [ 5 ] [ 6 ]


NBPF1 is a 1214 amino acid long protein in humans that weighs 139 kD and has an  isoelectric point  of around 5. A feature about this protein's composition is that it is much richer than most other proteins in both glutamine and glutamic acid residues. Additionally, it contains amino acid repeats that are present in humans, other primates, and even armadillos. [ 7 ]  Another feature is that the NBPF1 protein contains residues that are predicted to have  post-translational modifications , including  glycation ,  N-linked glycosylation ,  O-GlcNAc  attachment,  O-linked glycosylation ,  Phosphorylation , and  Sumoylation . [ 8 ]   The two most important domain types in the NBPF1 protein are the  coiled coil  domains and  DUF1220  domains. NBPF1 contains three coiled coil domains and nine DUF1220 domains in humans. The coiled coil domains are 60-100 amino acids long, while the DUF1220 domains are approximately 65 base pairs long with high sequence similarity. [ 9 ]


Human NBPF1 has been shown to interact with  Ubiquitin C  via both  protein complex immunoprecipitation [ 10 ]  and  affinity chromatography . [ 11 ]   Two-hybrid screening  assays have shown that NBPF1 interacts with the  coiled coil  domains of  CBY1 , a repressor for the  Beta-catenin , a protein that is involved in  Wnt signaling  for  cell proliferation . [ 12 ]  Additionally, two hybrid screen have shown that NBPF1 interacted with bacterial proteins, such as an  Oxidoreductase  iron/ascorbate family protein from the bacterium  Francisella tularensis  and an uncharacterized protein from the bacterium  Bacillus anthracis . [ 13 ]


Although the function of the NBPF1 protein in unknown, its physical and chemical properties can give insight about its function. Like other NBPF proteins, the NBPF1 protein product contains a repeated domain called  DUF1220 , a  domain of unknown function  that is thought to be related to human brain complexity. [ 14 ]   First, NBPF1 is predicted to be a nuclear protein, as it contains positively charged  nuclear localization  signals. These nuclear localization signals in NBPF1 and a conserved DNA binding domain similar to the transcription factor  STAT3 /dna complex or  STAT1 /dna complex suggests that it could act as a  transcription factor [ 15 ]


Second, genes like NBPF1 with DUF1220 genes are expressed during human neurogenesis. The number of DUF1220 domains present in the human genome correlates with both brain size and the amount of neurons present in the brain. [ 16 ]  Additionally, higher copy numbers of the DUF1220 domain are associated with increasing  Autism  severity, which often results from an excess of neurons that do not under synaptic pruning. [ 17 ]


The NBPF1 protein is also found to be disrupted by a  chromosomal translocation  between chromosomes 1 and 17 with  in some cases of human  neuroblastoma . [ 18 ]  Additional studies show that NBPF1 is possibly a  tumor suppressor gene  because adding it to cell cultures lowers the incidence of  foci formation . Additionally, NBPF1 contains three coiled coil structures are commonly involved in  oligomerization  with other proteins. [ 19 ]  Based on the interactions listed above, NBPF1 is shown to interact with the coiled coil domain of CBY1, which represses Wnt signaling. [ 18 ]  Aberrant Wnt signaling in the brain is a common cause of tumor growth and drug resistance in neuroblastomas, [ 20 ]  further suggesting that NBPF1 could act as a tumor suppressor gene in the brain if it directly affects this pathway.


NBPF1 seems to be involved in proliferation during human neurogenesis, and growth suppression during adulthood. Showing that the DUF1220 and coiled coil domains may be important during different life stages. The DUF1220 domains are important in neurogenesis, while the coiled coil domains involved with the binding of cell proliferation inhibitors. Although there are no known differences between Autism and neuroblastoma rates are known, a study has shown the existence of  comorbid   microcephaly  and neuroblastoma conditions, although more research is needed to show this. [ 21 ]  Based on these predictions, the lack of NBPF1 during could prevent fetal neurogenesis and postnatal tumor suppression in the brain, although this connection is not well understood.


NBPF1 is ubiquitously expressed in all tissues in humans, but shows the highest levels of expression in the bone marrow, skeletal muscle, brain, and spinal cord. It is expressed at slightly lower levels in other tissues such as the pancreas, kidney, and lung. [ 22 ]  In the brain, NBPF1 expression is the highest in the frontal, temporal, and parietal lobes, and it lowest in the ventricles and cerebellum. [ 23 ]  Based on protein composition, NBPF1 in humans and its  orthologs  in related species is most likely to be localized in the nucleus. [ 24 ]


Predicted Localization of NBPF1 and its Orthologs [ 24 ]


Expression studies have shown changes in NBPF1 under different experimental conditions  in vitro . First, the depletion of nervous system  transcription factor   SOX11  causes a slight increase in NBPF1 expression. [ 25 ]  Additionally, the inactivation of  Far upstream element-binding protein 1  causes a decrease in NBPF1, while the inactivation of Far Upstream Binding Elements 2 and 3 causes an increase in NBPF1 expression. [ 26 ]  Far upstream binding elements are involved in transcriptional regulation using gene enhancers, each having different binding sites. [ 27 ]  The overexpression of  CLDN1 , a protein that forms  tight junctions  such as those of the  blood–brain barrier , causes a sharp decline in NBPF1 expression [ 28 ]


Although NBPF1 itself only exists in primates, a wide variety of NBPF like protein  orthologs  exist in other mammals such as cattle, felines, and cetaceans. In non-primate mammals, the gene sequences of NBPF-like genes have little similarity to the primate NBPF genes. These genes appear to be entirely absent in model mammals such as mice and rats. The large amount of NBPF genes in the human genome is most likely due to recent duplications because all of the NBPF genes are so similar and repetitive that they easily recombine with each other, causing duplications. Variation in the number of repetitive sequences in the NBPF genes also varies even within humans. [ 29 ]
DUF1220 domains also vary greatly from humans in other species in their NBPF proteins. The further away a species is from humans, the fewer DUF1220 domains the species has. Humans have on average 272 DUF1220 domains in their NBPF genes, while chimpanzees have 125, macaques have 35, and dolphins only have 4. [ 30 ]


Selected Orthologs of NBPF1 [ 31 ] [ 32 ]


The  paralogs  for NBPF1 are other members of the NBPF protein family. The highest similarity between these paralogs further shows evidence of gene duplication during human evolution.


Selected Paralogs of NBPF1


NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16kDa  is a  protein  that in humans is encoded by the NDUFAB1  gene . [ 4 ]  The NDUFAB1 protein is a subunit of  NADH dehydrogenase (ubiquinone) , which is located in the  mitochondrial inner membrane  and is the largest of the five complexes of the  electron transport chain . [ 5 ]


The NDUFAB1 gene is located on the p arm of  chromosome 16  at position 12.2 and it spans 15,327 base pairs. [ 4 ]  The NDUFAB1 gene produces a 17.4 kDa protein composed of 156  amino acids . [ 6 ] [ 7 ]  NDUFAB1 is a subunit of the enzyme  NADH dehydrogenase (ubiquinone) , the largest of the respiratory complexes. The structure is L-shaped with a long,  hydrophobic   transmembrane  domain and a  hydrophilic  domain for the peripheral arm that includes all the known redox centers and the NADH binding site. [ 5 ]  NDUFAB1 is one of about 31 hydrophobic subunits that form the transmembrane region of Complex I. It has been noted that the  N-terminal  hydrophobic domain has the potential to be folded into an  alpha helix  spanning the inner  mitochondrial membrane  with a  C-terminal  hydrophilic domain interacting with globular subunits of Complex I. The highly  conserved  two-domain structure suggests that this feature is critical for the protein function and that the hydrophobic domain acts as an anchor for the  NADH dehydrogenase (ubiquinone)  complex at the inner mitochondrial membrane. [ 4 ]


The human NDUFAB1 gene codes for a subunit of  Complex I  of the  respiratory chain , which transfers electrons from  NADH  to  ubiquinone . [ 4 ]  However, NDUFAB1 is an accessory subunit of the complex that is believed not to be involved in catalysis. [ 8 ]  Initially,  NADH  binds to Complex I and transfers two electrons to the  isoalloxazine ring  of the  flavin mononucleotide  (FMN) prosthetic arm to form FMNH 2 . The electrons are transferred through a series of  iron-sulfur (Fe-S) clusters  in the prosthetic arm and finally to coenzyme Q10 (CoQ), which is reduced to  ubiquinol  (CoQH 2 ). The flow of electrons changes the redox state of the protein, resulting in a conformational change and p K  shift of the ionizable side chain, which pumps four hydrogen ions out of the mitochondrial matrix. [ 5 ]


In a  genome-wide association study  (GWAS)  meta-analysis  conducted across European and African American populations, the  NDUFAB1  gene and two other genes ( MFAP3L  and  PALB2 ) were identified as genetic loci significantly associated with  anxiety disorders  (ADs). Since the  comorbidity  of ADs arises from their shared genetic basis, these candidate  genetic loci  may become therapeutic targets for AD treatments. [ 9 ]  Moreover, a study to identify  small molecule drug  targets for  Tetralogy of Fallot  (TOF), a  congenital  malformation of the heart, found the  NDUFAB1  gene to be a major hub gene of differentially expressed genes in TOF. [ 10 ]


NM_004547 NM_001168331


NP_001161803 NP_004538


NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 4, 15kDa  is a  protein  that in humans is encoded by the NDUFB4  gene . [ 4 ]  The NDUFB4 protein is a subunit of  NADH dehydrogenase (ubiquinone) , which is located in the  mitochondrial inner membrane  and is the largest of the five complexes of the  electron transport chain . [ 5 ]


The NDUFB4 gene, located on the q arm of  chromosome 3  in position 13.33, is 6,130 base pairs long. The NDUFB4 protein weighs 15 kDa and is composed of 129 amino acids. [ 6 ] [ 7 ]  NDUFB4 is a subunit of the enzyme  NADH dehydrogenase (ubiquinone) , the largest of the respiratory complexes. The structure is L-shaped with a long,  hydrophobic   transmembrane  domain and a  hydrophilic  domain for the peripheral arm that includes all the known redox centers and the NADH binding site. [ 5 ]  NDUFB4 is one of about 31 hydrophobic subunits that form the transmembrane region of Complex I and is of the non-catalytic subunits of the complex. It has been noted that the  N-terminal  hydrophobic domain has the potential to be folded into an  alpha helix  spanning the inner  mitochondrial membrane  with a  C-terminal  hydrophilic domain interacting with globular subunits of Complex I. The highly  conserved  two-domain structure suggests that this feature is critical for the protein function and that the hydrophobic domain acts as an anchor for the  NADH dehydrogenase (ubiquinone)  complex at the inner mitochondrial membrane. [ 4 ]


The human NDUFB4 gene codes for a subunit of  Complex I  of the  respiratory chain , which transfers electrons from  NADH  to  ubiquinone . [ 4 ]  However, NDUFB4 is an accessory subunit of the complex that is believed not to be involved in catalysis. [ 8 ]  Mammalian complex I is composed of 45 different subunits. It locates at the  mitochondrial inner membrane . This protein complex has NADH dehydrogenase activity and oxidoreductase activity. It transfers electrons from NADH to the  respiratory chain . The immediate electron acceptor for the enzyme is believed to be  ubiquinone . [ 4 ]  Initially,  NADH  binds to Complex I and transfers two electrons to the  isoalloxazine ring  of the  flavin mononucleotide  (FMN) prosthetic arm to form FMNH 2 . The electrons are transferred through a series of  iron-sulfur (Fe-S) clusters  in the prosthetic arm and finally to coenzyme Q10 (CoQ), which is reduced to  ubiquinol  (CoQH 2 ). The flow of electrons changes the redox state of the protein, resulting in a conformational change and p K  shift of the ionizable side chain, which pumps four hydrogen ions out of the mitochondrial matrix. [ 5 ]


NM_001142393 NM_001271033 NM_006403 NM_182966


NM_001111324 NM_017464


NP_001135865 NP_001257962 NP_006394 NP_892011


NP_001104794 NP_059492


Neural precursor cell expressed developmentally down-regulated protein 9  ( NEDD-9 ) is a  protein  that in humans is encoded by the  NEDD9   gene . [ 5 ]  NEDD-9 is also known as  enhancer of filamentation 1  ( EF1 ),  CRK-associated substrate-related protein  ( CAS-L ), and  Cas scaffolding protein family member 2  ( CASS2 ). An important  paralog  of this gene is  BCAR1 .


In 1992, Kumar,  et al.,  first described a sequence tag corresponding to the NEDD9 3′ untranslated region based on the  cloning  of a group of genes predominantly expressed in the brain of embryonic, but not adult mice, a group of genes designated neural precursor cell expressed, developmentally down-regulated. [ 6 ]  In 1996, two groups independently described the complete sequence of the NEDD9 gene, and provided initial functional analysis of NEDD9 protein. Law  et al.  overexpressed a human cDNA library in  S. cerevisiae , and screened for genes that simultaneously affected cell cycle and cell polarity controls, inducing a filamentous yeast budding phenotype, and thus identified the HEF1 protein (Human Enhancer of Filamentation 1). [ 7 ]  This study identified HEF1/NEDD9 as an interactive partner for  focal adhesion kinase  (FAK), connecting it to  integrin signaling . Separately, Minegishi  et al.  cloned the gene encoding a protein hyperphosphorylated following ligation of  β1-integrins  in  T cells  and hypothesized to play a role in the process of T cell  costimulation , designating this gene  Cas-L  (Crk-associated substrate-related protein, Lymphocyte type). [ 8 ]


The genomic coordinates of the NEDD9 gene are 6:11,183,530-11,382,580 in the GRCh37 assembly, or 6:11,183,298-11,382,348 in the GRCh38 assembly. The gene is on the minus strand. The cytogenetic location is 6p25-p24, based on the nomenclature developed by the  Human Genome Organization  (HUGO) gene nomenclature committee ( HGNC ). NEDD9 is the HGNC approved symbol. Official IDs are 7733 (HGNC), 4739 (Entrez Gene), and ENSG00000111859 (Ensembl).  CAS-L, CASL, HEF1, dJ49G10.2, dJ761I2.1, CAS2, CASS2 are alias symbols. The NEDD9 gene is conserved in Rhesus monkeys, dogs, cows, mice, rats, chickens,  zebrafish , and frogs.  In vertebrates, it is a member of a 4-gene family, with the other paralogous genes known as  BCAR1  (p130Cas), EFS (Sin), and CASS4 (HEPL)


The NEDD9 promoter has 2 transcriptional start sites. The transcript variants NM_006403.3 and NM_001142393.1 encode proteins that have distinct  N-termini  (MKYK and MWTR, respectively). In mouse, the two alternative first  exons  are MKYK and MWAR. Their function is not known. NM_001142393 initiates translation at an upstream location compared to NM_006403.3, but both transcripts have 7 exons. Shorter transcripts with missing exons or an alternative 3' terminal exon have been detected in various studies; however, their role in the cell is unclear.


The 5' region of the NEDD9 promoter is regulated by  all-trans retinoic acid  (ATRA), and contains a retinoic acid response element (RARE) that is specifically bound by a  retinoid X receptor  (RXR)/retinoic acid receptor (RAR)  heterodimer . [ 9 ] [ 10 ] [ 11 ]  NEDD9 is also induced by the environmental pollutant  dioxin , based on regulation through the  aryl hydrocarbon receptor  (AhR). [ 12 ]  One study has found NEDD9 repressed by  estrogen , based on binding of the SAFB1 co-repressor. [ 13 ]  NEDD9 is induced by  Wnt signaling  in  colon cancer , based on binding to T-cell factor (TCF) factors in the promoter region. [ 14 ]  NEDD9 is induced by  hypoxia  and loss of  VHL , based on binding of hypoxia-induced factor ( HIF )  transcription factors  to the NEDD9 promoter. [ 15 ] [ 16 ] [ 17 ]  Prostaglandin E2 induces NEDD9 transcription. [ 18 ]   The Fox transcription factor Forkhead box C1 ( FoxC1 ) [ 19 ]   and  PAX5  transcription factor  [ 20 ]  have been reported to induce NEDD9 transcription.  TGF-beta  induces NEDD9 transcription. [ 21 ]   Based on inspection of sequence, the NEDD9 promoter also has potential binding sites for a number of additional transcription factors, including  STAT5A  and  NF-kappa B .


In the  3'UTR  of NEDD9 is a match to positions 2-8 of mature  miR-145 . NEDD9-binding regions in the miR-145 locus would allow the direct binding of the NEDD9 3'UTR to the genomic region of miR-145, and some studies suggests this miR regulates NEDD9 in  glioblastoma [ 22 ]  prostate cancer, [ 23 ]  and renal cell carcinoma cells. [ 24 ]  A non-coding RNA, named B2, extending from 10 kb upstream of NEDD9 exon 1 to exon 4 has been described, but the functional role for this  ncRNA  is not yet clear. [ 25 ]  NEDD9 is highly expressed in the embryonal brain, [ 26 ]  and in numerous tissues in the embryo and adult organism.  Elevated expression is associated with cancer, as discussed below.


NEDD9 is a member of the  CAS  (Crk-associated substrate) protein family, which has 4 members in vertebrates.  The other  paralogous  genes are known  BCAR1  (p130Cas), [ 27 ]   EFS  (Sin), [ 28 ] [ 29 ]  and  CASS4  (HEPL). [ 30 ]  There is no detectable NEDD9-related gene in bacteria, yeast, or  C. elegans .   A single family member exists in  D. Melanogaster , termed DCas. [ 31 ] [ 32 ]


In humans, NEDD9 is 834 amino acids long.  NEDD9 is a noncatalytic scaffolding protein that contains docking sites for proteins involved in multiple  signal transduction  pathways, regulating magnitude and duration of cell signaling cascades  [ 33 ] [ 34 ] [ 35 ] [ 36 ]   The overall structure of NEDD9 is represented graphically in Figure 1.


These domains include:


In terms of  post-translational modifications , NEDD9 is subject to significant  phosphorylation  based on growth conditions. In most actively growing adherent cells, NEDD9 migrates as a doublet of 115 and 105 kDa. Serine/threonine hyper-phosphorylated p115 NEDD9 is more common in  G2 / M phase  cells, [ 50 ]  suggesting these modifications are associated with increased localization to  centrosome  and  mitotic spindle . One study indicated the conversion of p115 into p105 is activated by cell detachment through cytoskeletal regulation of phosphatase  PP2A , [ 51 ]  although other work has found conflicting results. [ 52 ]


NEDD9 is present throughout  cell cycle , but most abundant in G2/M phase cells. [ 50 ]   NEDD9 is subject to both  caspase  cleavage and  proteasomal  degradation. [ 34 ] [ 35 ]  In conditions of cell detachment, and particularly in early stages of anoikis or apoptosis, NEDD9 is rapidly cleaved by caspases 3 and/or 7 at a DLVD site  (residue 363), and at a DDYD site (residue 630)  [ 53 ]   to form N-terminal 55 KDa and C-terminal 28 KDa fragments forms. This cleavage is prevented by focal adhesion formation, which suggests NEDD9 as a sensor of altered adhesion states. [ 50 ] [ 54 ]  Overexpression of p28 in cells causes cellular rounding and detachment, and induces apoptosis, [ 54 ]  probably because of a dominant-negative effect on survival-promoting signaling complexes at focal adhesions.  Together this data suggests that production of different NEDD9 posttranslational modifications is regulated by cell de/attachment, which, in turn, allows regulation of NEDD9 turnover and participation in distinct cellular processes.


P115 is the primary target for proteasomal degradation of NEDD9. [ 51 ]  Proteasomal degradation of NEDD9 is triggered by a number of stimuli, including induction of TGF-beta signaling. [ 55 ]  An effector of the TGFbeta receptor, Smad3, may interact directly with APC subunit APC10 and thus recruit the APC complex. CDH1 subunit of the APC complex recognizes NEDD9 and regulates ubiquitination and subsequent degradation of NEDD9. [ 56 ]   NEDD9 is also degraded by the proteasome at the end of mitosis, following completion of activities with Aurora-A that support mitotic progression. [ 50 ]


In interphase cells, the majority of NEDD9 localizes to focal adhesions.  However, some of the protein is also cytoplasmic, and small pools localize to the centrosome  [ 43 ]  and the basal body of cilia. [ 57 ]  At mitotic entry NEDD9 moves along mitotic spindle, eventually localizing at midbody at cytokinesis. [ 43 ]


NEDD9 is an intermediate in a number of important signaling pathways relevant to the cellular processes of proliferation, survival, migration, and others (see figure to the right). [ 33 ] [ 34 ] [ 35 ]


Integrin signaling, which control cell movement, spreading and adhesion to extracellular matrix (ECM), and survival, is the best established signaling pathway for NEDD9. Integrins are transmembrane proteins that nucleate focal adhesions, structures that provide bi-directional signaling between ECM and actin cytoskeleton.  NEDD9 stabilizes formation and regulates turnover of focal adhesions, influencing cell motility and the invasion and metastasis of cancer cells. [ 58 ]  In response to integrin activation, FAK or the related kinase RAFTK recruits NEDD9 into a focal adhesion site, binds it via the N-terminal SH3 domain and phosphorylates the NEDD9 Src-binding site. This allows SRC or SRC family kinase to bind NEDD9 via its SH2 domain. Phosphorylation of the NEDD9 substrate domain by Src and other kinases results in the creation of binding sites for Crk and other adaptors that associate with SH2 binding motifs. NEDD9 Crk complexes activate Rho and Ras family GTPases via the recruitment of their nucleotide exchange factors (GEFs), such as DOCK1, DOCK3  [ 36 ]  DOCK180 and C3G. [ 59 ]


These GTPases regulate cell motility, proliferation and also contribute to tumor progression and invasion. In many cell types, NEDD9 overexpression increases spreading and crescent morphology (an indicator of high motility). [ 54 ]  However, in fibroblasts, some work has found that absence of NEDD9 leads to more rapid focal adhesion turnover, which led to increase of migration in NEDD9-/- compared to wild type. [ 58 ]


In cancer cells, NEDD9 can drive mesenchymal-type movement by activating RAC1 GTPase and WAVE in complex with its GEF DOCK3, which in turn cause inhibition of GTPase Rho and amoeboid movement. [ 60 ]   Invasion is accompanied by proteolysis of the ECM through activation of MMP14, MMP2 and MMP9 metalloproteinases. [ 61 ]


NEDD9 is involved in chemokine-induced T cell migration and T cell receptor (TCR)–mediated integrin activation. In lymphocytes, integrin or TCR signaling induces NEDD9 phosphorylation by tyrosine kinases Fyn and Lck (SRC family kinases), which is essential for T cell migration. [ 62 ]  In addition, in response to chemokine signals, Abl family kinases promote GTPase RAP1 activation by phosphorylating of NEDD9; [ 63 ]  NEDD9 associates with the transducer protein Chat-H/SHEP1/NSP3, a member of the NSP protein family, further supporting RAP1 activation, cell migration, and adhesion. [ 64 ]   In B cells, NEDD9 association with NSP3 enhances integrin-mediated NEDD9 serine/threonine hyperphosphorylation following B cell receptor (BCR) ligation, promoting B  lymphocyte adhesion, motility and homing into marginal zones of spleen  [ 65 ]
Estrogen Receptor. The NEDD9 interactors p130/CAS and the NSP protein NSP2/BCAR3 are implicated in antiestrogen resistance  [ 66 ] [ 67 ]  and breast cancer progression  [ 68 ]  Some data suggests a role for NEDD9 in the cellular response to estrogen, including the progression to anti-estrogen resistance, breast cancer progression and invasion  [ 69 ] [ 70 ] [ 71 ]


RTKs (EGFR). NEDD9 also contributes to the transduction of signals downstream receptor tyrosine kinases (RTKs). A role for NEDD9 in signaling crosstalk between epidermal growth factor receptor (EGFR) and integrins was established in non-small lung cancer (NSLC). It was shown that inhibition of EGFR reduces the tyrosine phosphorylation of NEDD9. [ 72 ]   Nedd9 interacts directly with the EGFR effector protein Shc, positioning it to affect downstream signaling relevant to EGFR; mice lacking Nedd9 have depressed activity of the EGFR effectors ERK and AKT. [ 73 ]  NSP proteins are also multidomain scaffolds, which bind activated RTKs in response to extracellular stimuli and recruit both NEDD9 and BCAR1 to assist in integrating signaling between RTKs and integrins. NEDD9 is also activated by PDGF  [ 74 ]  and other RTKs, although more study is required.


TGF-beta is a regulator of tissue remodeling and epithelial-mesenchymal transition (EMT) in development, and promotes metastasis in cancer.  A number of studies have identified NEDD9 as a downstream effector in the TGF-beta signaling pathway, essential for promoting EMT. [ 21 ] [ 55 ] [ 75 ] [ 76 ] [ 77 ]  In MCF-7 cells, NEDD9 negatively regulates expression of the epithelial protein E-cadherin, preventing association of E-cadherin with cell membrane and activating SRC-kinase. [ 78 ]  Activated SRC provides internalization and lysosomal degradation of E-cadherin. [ 78 ]  Consistent with these findings is a study demonstrating downregulation of epithelial markers (E-cadherin, occludin, β-catenin) and concurrent upregulation of mesenchymal markers (N-cadherin, vimentin, fibronectin) in response to NEDD9 overexpression in MCF-10 cells. [ 79 ]


NEDD9 binds directly to the Aurora-A mitotic kinase at the centrosome, and promotes its activity, allowing cells to enter mitosis. [ 43 ] [ 80 ]   Degradation of NEDD9 at the end of mitosis contributes to timely Aurora-A degradation. [ 43 ] [ 80 ] [ 81 ]   Cells overexpressing NEDD9 exhibit deficient cytokinesis resulting in the accumulation of multipolar mitotic spindles and abnormal numbers of centrosomes. On the other hand, cells with depleted NEDD9 have prematurely separated centrosomes and are deficient in microtubule organizing activity during mitosis, leading to an abundance of monopolar or asymmetric spindles, [ 43 ]  preventing cells from entering mitosis. NEDD9 also regulates Aurora-A activation at the basal body of cilia as cells resorb cilia during early G1. [ 57 ]   Cilia are small organelles that protrude from the surface of adherent cells that are the obligate site of action for proteins such as Hedgehog, and the polycystins: by influencing ciliary stability, NEDD9 is positioned to affect these signaling systems. Interaction of NEDD9 with Aurora A kinase may also play a role in tumor invasion. NEDD9 binds to and regulates acetylation of cortactin (CTTN) in an Aurora A kinase (AURKA)/HDAC6–dependent manner. The knockdown of NEDD9 or AURKA results in an increase in the amount of acetylated CTTN and a decrease in the binding of CTTN to F-actin. Overexpression of the deacetylation mimicking (9KR) mutant of CTTN is sufficient to restore actin dynamics at the leading edge and migration proficiency of the tumor cells. Inhibition of AURKA and HDAC6 activity by alisertib and tubastatin A in xenograft models of breast cancer has led to a decrease in the number of pulmonary metastases. [ 82 ]


Transgenic mice with homozygous depletion of NEDD9 are vital and fertile, but have immunological abnormalities that result in pre-malignant conditions later in life, defects are initially subtle, but increase in later life; B cell homing to the spleen and lymphocyte trafficking are deficient. [ 73 ] [ 83 ]


The NEDD9 rs760678 SNP located in an intronic region, has been studied for a possible association with late onset  Alzheimer's  disease (LOAD). [ 84 ] [ 85 ] [ 86 ] [ 87 ] [ 88 ]   However, in 2012, Wang  et al.,  performed a meta-analysis and concluded that more studies are required for solid conclusions. [ 87 ]  This SNP and relevant signaling is discussed more fully in. [ 89 ]


Altered (typically elevated) expression of NEDD9 is strongly associated with cancer. NEDD9 is rarely if ever mutated, but frequently show altered expression or phosphorylation (associated with increased activity) in pathological conditions including immune cell dysfunction and cancer.  NEDD9 overexpression is documented to occur and in some cases linked the process of tumorigenesis of many different malignances.  Besides examples in breast cancer discussed above, these malignancies include colon, [ 14 ] [ 15 ] [ 18 ] [ 90 ]  pancreatic, [ 91 ]  head and neck, [ 92 ]  ovarian, [ 93 ]  gastric, [ 94 ]  lung, [ 95 ]  genitourinary (including prostate), [ 23 ] [ 96 ]  liver, [ 19 ]  and kidney cancer, [ 17 ] [ 24 ]  gastrointestinal stromal tumors, [ 97 ]  glioblastoma, [ 22 ] [ 74 ] [ 98 ]  and neuroblastoma. [ 9 ] [ 10 ] [ 58 ]


Nedd9 expression may be important for recovery from stroke.  Nedd9 is upregulated in the neurons of the cerebral cortex and hippocampus after transient global ischemia in rats. Induced Nedd9 is tyrosine phosphorylated, bound to FAK in dendrite and soma of neurons, and promotes neurite outgrowth, contributing into recovery of neurologic function after cerebral ischemia. [ 99 ]  Nedd9 has recently been implicated in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). NEDD9 expression is elevated in human autosomal dominant polycystic kidney disease (ADPKD) and in mouse ADPKD models, and ADPKD-prone mice lacking NEDD9 developed a more severe form of ADPKD than those with normal NEDD9. [ 100 ]


Because of its roles in cancer, several studies have considered the potential value of NEDD9 as a therapeutic target or therapeutic guide. Because of lack of a kinase domain, or any defined catalytic domain, and because it is entirely intracellular, NEDD9 is a difficult molecule to target.
Because NEDD9 serves as a scaffolding molecule for other signaling proteins that play significant roles in cancer development, the effects of NEDD9 overexpression in supporting metastasis could in theory be mitigated by inhibition of its downstream targets. In one study, deletion of Nedd9 in MMTV-neu mammary tumors increased their sensitivity to inhbitiors of FAK and SRC. [ 101 ]  NEDD9 depletion sensitizes breast tumor cell lines to the Aurora A inhibitor alisertib. [ 81 ]   Consideration of NEDD9 as a biomarker for therapeutic response is a promising research direction.


NEDD9 has been shown to  interact  with:


The  NR4A1 gene  is a  transcription factor  important in the development of  cells  that secrete the  hormone   insulin-like 3  (INSL3). In general, the NR4A gene family regulates  cell growth  and  differentiation .


In humans, INSL3 aids in the regulation of  testicular descent  during  fetal development . In an adult INSL3 helps in keeping  germ cells  alive, in both males and females.


In the male  reproductive system  the NR4A1 gene is a promoter of insulin-like 3 in  Leydig cells . Leydig cells are located in the  testis  and known for the function as a support cell in  spermatogenesis . Along with other things they secrete  testosterone .


In females, INSL3 is also involved in the reproductive system. It is secreted by  thecal  and  luteal  cells in the  ovary  and thus is important in the maturation of developing  oocytes .  In the rabbit model, disorders observed involving NR4A1 expression include  testicular dysgenesis , and  cryptorchidism .


Immunoprecipitation  assays show that INSL3 does bind to NR4A1; however, much is still not known about INSL3 regulation and the direct involvement of the NR4A1 gene.


Luc J. Martin and Jacques J. Tremblay
Nuclear Receptors in Leydig Cell Gene Expression and Function
Biol Reprod July 2010 83 (1) 3–14; published ahead of print April 7, 2010, doi:10.1095/biolreprod.110.083824


Nicholas M. Robert, Luc J. Martin, and Jacques J. Tremblay
The Orphan Nuclear Receptor NR4A1 Regulates Insulin-Like 3 Gene Transcription in Leydig Cells
Biol Reprod February 2006 74 (2) 322–330; published ahead of print October 19, 2005, doi:10.1095/biolreprod.105.044560


NM_001099287 NM_001172292


NP_001092757 NP_001165763


Nipa‐Like Domain‐Containing 4 , also known as  NIPAL4  or  Ichthyin , is a  gene  that is predicted to  code  for a  transmembrane protein  with nine  transmembrane domains . [ 5 ]   NIPAL4  codes for the protein  magnesium   transporter  NIPA4 , which acts as a  Mg 2+  transporter.


NIPAL4  is mainly expressed in the skin, specifically in the  granular layer  of the  epidermis . [ 6 ]


NIPAL4  codes for a magnesium transporter that can also transport other  divalent cations  such as Ba 2+ , Mn 2+ , Sr 2+  and Co 2+ , though to a much less extent than Mg 2+ . [ 5 ]  There is also evidence that  NIPAL4  is involved in the synthesis of  very long chain fatty acids  involved in the epidermal  lipid metabolism . [ 7 ]  Disruptions to this pathway results in impaired skin function, causing the symptoms of  ARCI . [ 8 ]


Mutations in this gene account for 16% of  autosomal recessive congenital ichthyosis  (ARCI) cases, making it the 2nd most common gene involved with this disease. [ 9 ]  Since its first identification in 2004, 18 disease‐causing mutations have been reported in  NIPAL4 . [ 8 ]


Nix  is a  pro-apoptotic   gene  that is regulated by  Histotoxic hypoxia .  It  expresses  a  signaling protein  related to the  BH3-only family .  This protein induces  autophagy , an intracellular function by which  cytoplasmic  components are delivered to the  lysosome  to be broken down and used elsewhere or  excreted  from the  cell . [ 1 ]  This protein is important in development because it allows cells to have a consistent store of cellular components. [ 2 ]  It also holds an important role in the  differentiation  and maturation of  erythrocytes  and  lymphocytes  by the process of  mitophagy  with the help of its regulator  BNIP3 . [ 3 ]   Using a  gene knockout  technique in mice, scientists have been able to attribute this pruning of  mitochondria  and induction of cellular  necrosis  to the expression of the Nix gene. [ 1 ]  The Nix protein may be associated with certain kinds of cancer formation. In mouse models, loss of Nix resulted in a delayed onset of tumors for pancreatic cancer, and was additionally associated with reduced mitophagy and increased oxidative metabolism. Nix therefore may be a tumor promoter for pancreatic cancer. [ 4 ]


Not only does it hold a role in the differentiation of these immune and oxygen-carrying cells, but it also affects the development and maintenance of heart tissue.  It has been found to be a cause of pathologic  hypertrophy  and  cardiomyocyte  apoptosis involved in  congenital heart disease . [ 5 ]  The effects of Nix are amplified in the neonatal heart compared to the adult heart. Overexpression of Nix in the fetal mouse has been found to cause severe growth retardation and massive cardiomyocyte apoptosis often followed by lethality.  These early interactions between the fetal heart and Nix expression are thought to have a role in the development of adult heart disease. [ 3 ]


NM_005601 NM_001363693


NP_005592 NP_001350622


Natural killer cell granule protein 7  is a  protein  that in humans is encoded by the NKG7  gene .
 [ 5 ]


1MH9 ,  1Q91 ,  1Q92 ,  1Z4I ,  1Z4J ,  1Z4K ,  1Z4L ,  1Z4M ,  1Z4P ,  1Z4Q ,  2JAU ,  2JAW ,  4L6A ,  4L6C ,  4MUM ,  4MWO ,  4NFL ,  4YIK


5',3'-nucleotidase, mitochondrial , also known as 5'(3')-deoxyribonucleotidase, mitochondrial (mdN) or deoxy-5'-nucleotidase 2 (dNT-2), is an  enzyme  that in humans is encoded by the  NT5M   gene . This gene encodes a  5'   nucleotidase  that  localizes  to the  mitochondrial matrix . This enzyme de phosphorylates  the 5'- and 2'(3')-phosphates of  uracil  and  thymine  deoxyribonucleotides. The gene is located within the  Smith–Magenis syndrome  region on chromosome 17. [ 5 ] [ 6 ]


The  cDNA  of mdN encodes a 25.9-kDa polypeptide, and the crystal structure of this enzymes reveals a 196-long  amino acid  sequence in the mature protein. [ 7 ] [ 8 ]  The first 32 amino acids, which contain the mitochondrial  targeting sequence , are removed during the processing of the premature protein for its import into the mitochondrial matrix. The enzyme is likely a  dimer  protein formed by the interaction of alpha and beta loops between the cores of the two  monomers . Each monomer is composed of a large and small domain connected by two loops. The large domain forms an alpha/beta  Rossmann fold  as well as 2 helix loops in the fold, whereas the small domain forms a truncated four-helix bundle which is inserted between a  beta strand  and  alpha-helix  in the large domain. The active site is found in a cleft between the two domains and binds a  magnesium   ion  that is coordinated by three exogenous  ligands , a  phosphate  ion, and two  water  molecules in an octahedral shape. [ 8 ]


mdN is one of seven 5' nucleotidases identified in humans, all of which differ in tissue specificity, subcellular location,  primary structure  and  substrate  specificity. [ 8 ] [ 9 ]  Of the seven, the  cytosolic  counterpart of mdN,  cdN , is the most closely related to mdN. Their genes,  NT5M  and  NT5C , share the same  exon / intron  organization, and their  amino acid  sequences are 52% identical. [ 7 ] [ 8 ]  In addition, mdN structurally resembles members of the HAD family despite no significant sequence similarity. [ 8 ]


This enzyme functions in dephosphorylating nucleoside triphosphates, especially the 5′- and 2′(3′)-phosphates of uracil and thymine  deoxyribonucleotides  (dUMPs and dTMPs). [ 7 ] [ 8 ] [ 10 ]  Due to this function, mdN regulates the size of pyrimidine deoxyribonucleotide pools within mitochondria, in conjunction with the mitochondrial  thymidine   kinase , as part of the thymidine (dTTP)/dTMP substrate cycle. Since excess dTTP leads to aberrant  mitochondrial DNA   replication , the regulatory role of mdN serves to maintain dTTP levels to ensure proper mitochondrial DNA replication. [ 9 ] [ 10 ]


Similar to other mitochondrial enzymes, mdN  mRNA  is found in  heart ,  brain , and  muscle , and to a lesser degree in  kidney  and  pancreas , while it is absent from  placenta ,  liver , and  lung . [ 7 ]  Though the enzyme is ubiquitous, mdN activity has only been detected in brain and heart tissue. [ 7 ] [ 8 ]


Since the  NT5M  gene is located in the Smith–Magenis syndrome region of chromosome 17,  mutations  in this gene could contribute to the disease. Moreover, its location may indicate that the disease involves a mitochondrial component. [ 7 ]
The protein mdN is essential to counteract dTTP accumulation, as excess dTTP has been linked to mitochondrial  genetic disease . [ 10 ]  In addition, this enzyme's dephosphorylation function could be applied to  anticancer  and  antiviral  treatments which use  nucleoside   analogs . [ 8 ] [ 9 ]  These treatments rely on the  kinase  activation of the analogs, which then are incorporated into the DNA of the  tumor  cell or  virus  to act as DNA chain terminators. [ 9 ]  mdN can be used to maintain the concentrations of nucleoside analogs at low levels to avoid mitochondrial toxicity. Thus, only analogs whose 5' phosphates are rapidly and specifically degraded by mdN should be used. [ 8 ] [ 9 ]


mdN binds and dephosphorylates uracil and thymine deoxyribonucleotides. [ 7 ] [ 8 ]


A nuclear gene is a gene that has its DNA nucleotide sequence physically situated within the cell nucleus of a eukaryotic organism. This term is employed to differentiate nuclear genes, which are located in the  cell nucleus , from genes that are found in mitochondria or  chloroplasts . The vast majority of genes in eukaryotes are nuclear.


Mitochondria and  plastids  evolved from free-living  prokaryotes  into current cytoplasmic organelles through  endosymbiotic  evolution. [ 1 ]  Mitochondria are thought to be necessary for eukaryotic life to exist. They are known as the cell's powerhouses because they provide the majority of the energy or ATP required by the cell. The mitochondrial genome ( mtDNA ) is replicated separately from the host genome. Human mtDNA codes for 13 proteins, most of which are involved in  oxidative phosphorylation  (OXPHOS). The  nuclear genome  encodes the remaining mitochondrial proteins, which are then transported into the mitochondria. [ 2 ]  The genomes of these organelles have become far smaller than those of their free-living predecessors. This is mostly due to the widespread transfer of genes from prokaryote progenitors to the nuclear genome, followed by their elimination from organelle genomes. In  evolutionary  timescales, the continuous entry of organelle DNA into the nucleus has provided novel nuclear genes. [ 1 ]  Furthermore, Mitochondria depend on nuclear genes for essential protein production as they cannot generate all necessary proteins independently. [ 3 ]


Though separated from one another within the  cell , nuclear genes and those of mitochondria and chloroplasts can affect each other in a number of ways. Nuclear genes play major roles in the expression of chloroplast genes and mitochondrial genes. [ 4 ]  Additionally, gene products of mitochondria can themselves affect the expression of genes within the cell nucleus. [ 5 ]  This can be done through  metabolites  as well as through certain  peptides  trans-locating from the mitochondria to the nucleus, where they can then affect gene expression. [ 6 ] [ 7 ] [ 8 ]


Eukaryotic genomes have distinct higher-order  chromatin  structures that are closely packaged functional relates to gene expression. Chromatin compresses the genome to fit into the cell nucleus, while still ensuring that the gene can be accessed when needed, such as during  gene transcription ,  replication , and  DNA repair . [ 9 ]   The entirety of genome function is based on the underlying relationship between nuclear organization and the mechanisms involved in genome organization, in which there are a number of complex mechanisms and biochemical pathways which can affect the expression of individual genes within the genome. [ 9 ]  The remaining mitochondrial proteins, metabolic enzymes, DNA and  RNA polymerases ,  ribosomal proteins , and mtDNA regulatory factors are all encoded by nuclear genes. Because nuclear genes constitute the genetic foundation of all eukaryotic organisms, anything that might change their  genetic expression  has a direct impact on the organism's cellular  genotypes  and phenotypes. [ 2 ]  The nucleus also contains a number of distinct subnuclear foci known as  nuclear bodies , which are dynamically controlled structures that help numerous nuclear processes run more efficiently. [ 9 ]  Active genes, for instance, might migrate from chromosomal regions and concentrate into subnuclear foci known as  transcription factories . [ 9 ]


The majority of proteins in a cell are the product of messenger RNA  transcribed  from nuclear genes, including most of the proteins of the organelles, which are produced in the  cytoplasm  like all nuclear gene products and then transported to the organelle. Genes in the nucleus are arranged in a linear fashion upon chromosomes, which serve as the scaffold for replication and the regulation of gene expression. As such, they are usually under strict copy-number control, and replicate a single time per cell cycle. [ 10 ]  Nuclear cells such as platelets do not possess  nuclear DNA  and therefore must have alternative sources for the RNA that they need to generate proteins. With the nuclear genome's 3.3 billion DNA  base pairs  in humans, one good example of a nuclear gene is  MDH1  or the  malate dehydrogenase 1  gene. In various metabolic pathways, including the citric acid cycle, MDH1 is a protein-coding gene that encodes an enzyme that catalyzes the NAD/ NADH -dependent, reversible oxidation of malate to oxaloacetate. This gene codes for the cytosolic isozyme, which is involved in the  malate-aspartate shuttle , which allows malate to cross past the mitochondrial membrane and be converted to oxaloacetate to perform further cellular functions. [ 11 ]  This gene among many exhibits its huge purposeful role in the entirety of an organism’s physiologic function. Although non-nuclear genes may exist in its functional nature, the role of nuclear genes in response and in coordination with non-nuclear genes is fundamental.


Many nuclear-derived transcription factors have played a role in respiratory chain expression. These factors may have also contributed to the regulation of mitochondrial functions.  Nuclear respiratory factor  (NRF-1) fuses to respiratory encoding genes proteins, to the rate-limiting enzyme in  biosynthesis , and to elements of replication and transcription of mitochondrial DNA, or  mtDNA . The second nuclear respiratory factor (NRF-2) is necessary for the production of cytochrome c oxidase subunit IV (COXIV) and Vb (COXVb) to be maximized. [ 4 ]


The studying of gene sequences for the purpose of speciation and determining genetic similarity is just one of the many uses of modern day genetics, and the role that both types of genes have in that process is important. Though both nuclear genes and those within endosymbiotic organelles provide the genetic makeup of an organism, there are distinct features that can be better observed when looking at one compared to the other. Mitochondrial DNA is useful in the study of speciation as it tends to be the first to evolve in the development of a new species, which is different from nuclear genes' chromosomes that can be examined and analyzed individually, each giving its own potential answer as to the speciation of a relatively recently evolved organism. [ 12 ]


Low-copy nuclear genes in plants are valuable for improving phylogenetic reconstructions, especially when universal markers like  Chloroplast DNA , or cpDNA and Nuclear ribosomal DNA, or  nrDNA  fall short. Challenges in using these genes include limited universal markers and the complexity of gene families. Nonetheless, they are essential for resolving close species relationships and understanding plant phylogenetic studies. While using low-copy nuclear genes requires additional lab work, advances in sequencing and cloning techniques have made it more accessible. Fast-evolving introns in these genes can offer crucial phylogenetic insights near species boundaries. This approach, along with the analysis of developmentally important genes, enhances the study of plant diversity and evolution. [ 13 ]


As nuclear genes are the genetic basis of all eukaryotic organisms, anything that can affect their expression therefore directly affects characteristics about that organism on a cellular level. The interactions between the genes of endosymbiotic organelles like mitochondria and chloroplasts are just a few of the many factors that can act on the nuclear genome.


OCC-1  (overexpressed in colon carcinoma-1) is a  protein , which in humans is encoded by the  gene  C12orf75. [ 1 ]  The gene is approximately 40,882 bp long and encodes 63  amino acids . OCC-1 is  ubiquitously  expressed throughout the human body. OCC-1 has shown to be overexpressed in various colon  carcinomas . [ 2 ]
Novel splice variant of this gene was also detected in various human cancer types; in addition to encoding a novel smaller protein (51 amino acids), OCC-1 gene produces a non-protein coding RNA splice variant lncRNA (called OCC-D variant).


Location and size :
C12orf75 is found along the plus strand of  chromosome 12  (12q23.3). [ 3 ]  The gene is 40,882 bp long with the genomic sequence beginning at 105,330,636 bp and ends at 105,371,518 bp. C12orf75 contains 6  exons  and is flanked by KCCAT198 (renal clear cell carcinoma-associated transcript 198). [ 4 ]


C12orf75 encodes several transcripts of  mRNA  that the longest one is 1386 bp. [ 5 ]  The mature mRNA of this splice variant contains six exons.


Primary structure:
OCC-1 protein is 63 amino acids long  [ 6 ]  and has a  molecular weight  of 6.4 kdal. [ 7 ]  OCC-1 contains an “opioid growth factor receptor repeat” ( OGFr )  [ 8 ]  motif from residue 8 to 27. OCC-1 is an acidic protein with an  isoelectric point  of OCC-1 is 6.6. [ 9 ]


Secondary structure:
The secondary structure of OCC-1 is a combination of multiple coils, a few  α-helices , and few  β-sheets . The Phyre 2 program 52% α-helices, 6% β-sheets, and 70% disordered. [ 10 ]  The predicted region β-sheet from residues 31 to 38 coincide with the α-helices and β-sheets regions predicted by other programs. OCC-1 is a soluble protein; according to the SOUSI program therefore there are no  transmembrane domains . [ 11 ]


Tertiary structure:
Predicted folding by iTASSER is shown. [ 12 ]


Post translational modifications:
OCC-1 is predicted to undergo the post-translational modifications of  O-glycosylation ,  phosphorylation , and  myristoylation . [ 13 ]


Subcellular location:
The k-NN tool places OCC-1 in the nucleus of the cell with 65.2% certainty, 13% in the  mitochondria , 8.7% in the vesicles of the secretory system, 4.3%  cytoplasm , and 4.3%  vacuole .


Paralogs:
OCC-1 has no known  paralogs .


Orthologs:
OCC-1 has been found in  mammalia ,  reptilia ,  amphibian ,  aves , and  actinopterygii . [ 14 ]  The gene is not found in  plants ,  protists ,  fungi ,  archaea , or  bacteria . The most distant ortholog is the Oryzias latipers or the  Japanese rice fish , which diverged from the human gene approximately 436.5 million years ago. [ 15 ]


Phylogeny:
The  phylogenetic  tree to the right shows the evolution of OCC-1 among humans and the orthologs from the various taxa that contain OCC-1. The results of this phylogenetic tree follow in accordance with the predicted evolutionary history of animals on Earth.


Expression level:
OCC-1 has moderate to high expression throughout the body, therefore OCC-1 is ubiquitously expressed in humans; notably high expression in the  kidney ,  skeletal muscle  and  pancreas  and low expression in the  heart . OCC-1 has shown to be overexpressed in various colon carcinomas. [ 16 ]
In regards to homologous expression,  in situ hybridization  data revealed that OCC-1 is expressed in the primary  visual cortex  of the  macque , in an activity dependent manner. [ 17 ]


Disease state expression:
Profiles from NCBI UniGene show the expression of OCC-1 in adrenal tumors,  chondrosarcoma , gastrointestinal tumors, kidney tumors,  leukemia ,  liver tumors ,  prostate cancer , soft tissue/muscle tissue tumors, and  uterine tumors . [ 18 ]


Promoter:
The promoter of OCC-1 is GXP_4407929 and 601 bp in length. [ 19 ]  The promoter can be found on the plus strand and begins at 105234790 bp and ends at 105235390 bp.


OCC-1 is shown to interact with HRG4,  ELAVL1 , c- REL , and  IRS4 . ELAVL1 functions to stabilized mRNA for gene expression. C-REL is involved in  lymphoid  and cell growth/survival, with a specific presence in T cell malignancies and cancer. [ 20 ]  HRG4 plays a role in signal transduction and trafficking sensory neurons and is located primarily in the  retina , [ 21 ]  which relates to the discovery of expression in the brain of the macque through in situ hybridization data. IRS4 functions as interface between growth factor receptors consisting of tyrosine kinase activity, such as  insulin  receptors. [ 22 ]  IRS4 also is involved with the IGF1R  mitogenic  signaling pathway.


OCC-1 has shown to be overexpressed in multiple colon carcinomas. [ citation needed ]


NM_001293800 NM_029865


NP_001280729 NP_084141


OCEL1 , also called Occludin//ELL Domain Containing 1, is a  protein  encoding  gene  located at  chromosome 19p 13.11 in the  human genome . [ 5 ]  Other aliases for the gene include FLJ22709, FWP009, and S863-9. The function of OCEL1 has not yet been identified.


Though the gene’s function is currently unknown, it is part of a family of genes that are related to  occludin , which is an enzyme localized at tight junctions of epithelial and endothelial cells, and are also related to eleven-nineteen lysine-rich leukemia (ELL), which is an elongation factor that can increase the rate of  RNA polymerase II  transcription. [ 6 ] [ 7 ]   There are five paralogs of OCEL1 in this gene family:  ELL , ELL2,  ELL3 ,  MARVELD2 , and  OCLN . [ 8 ]  OCEL1 and each of its five paralogs all contain the Occludin/ELL Domain (pfam07303), suggesting their functions may be related to protein interactions. [ 6 ] [ 7 ]


The protein encoded by OCEL1 is called Occludin/ELL Domain Containing Protein 1. [ 9 ]  The protein is 264 amino acids in length. It is made up of 6 distinct exons and contains the ELL/Occludin Domain. OCEL1 has 15 different splice variations. The molecular weight of OCEL1 is approximately 28-29 kDa. [ 10 ] [ 11 ]  The predicted  isoelectric point  for the protein is 10. [ 11 ]  There are 3 isoforms for the OCEL1 protein: isoform X1, isoform X2, and isoform X3. [ 9 ]  Only isoforms X1 and X2 contain the ELL/Occludin Domain. The OCEL1 protein contains a proline-rich domain spanning from amino acid 28-106; the protein has two standard deviations more of proline than average. [ 12 ] [ 13 ]


The secondary structure of OCEL1 is predicted to contain several alpha helices but no beta pleated sheets. [ 14 ] [ 15 ]  There are no predicted sulfide bridges or transmembrane domains in the protein. [ 16 ] [ 17 ]  The tertiary structure as predicted by iTASSER shows multiple alpha helices coiling with each other.


The OCEL1 protein is localized in the Golgi apparatus, but can also be found at low levels in the nucleus. [ 18 ]


OCEL1 is expressed at detectable levels in all human tissues with low tissue specificity. [ 18 ]  Expression in the kidney is low relative to other tissues during human fetal development, but is relatively higher in adults. [ 19 ] [ 20 ]


Expression of OCEL1 may be regulated by several  miRNA  binding sites predicted to be in the  3' UTR  of the gene: [ 21 ]


In addition to its five paralogs in the human genome, the OCEL1 gene has 234 known orthologs in other species. [ 9 ] [ 8 ]  Its orthologs are found in jawed vertebrates, including birds, alligators, turtles, lizards, mammals, amphibians, coelacanths, bony fish, and cartilaginous fish. Some orthologs contain both the ELL/Occludin Domain and the MARVELD2 Domain, suggesting the OCEL1 protein and MARVELD2 protein are closely related. [ 18 ]


The OCEL1 protein has been found to  interact  with two proteins via protein kinase assays;  SRPK1 , known as SRSF protein kinase I, and  SRPK2 , known as SRSF protein kinase II, which are both localized to the nucleus of the cell. [ 28 ]  SRSF protein kinase I and II both primarily function to phosphorylate proteins at serine residues and also phosphorylates serine residue splicing factors. [ 29 ] [ 30 ]


The OCEL1 gene may be related to some cancers. Low expression of OCEL1 is associated with poor prognosis in patients with human non-small cell lung cancer. [ 31 ]  OCEL1, when grouped with three other genes, is thought to be an effective predictor for the paclitaxel response in humans with HER2-negative breast cancer. [ 32 ]


Olfactory receptor family 4 subfamily F member 5  is a  protein  that in humans is encoded by the OR4F5  gene .
 [ 5 ]


Olfactory receptors interact with odorant molecules in the nose, to initiate a neuronal response that triggers the perception of a smell. The olfactory receptor proteins are members of a large family of G-protein-coupled receptors (GPCR) arising from single coding-exon genes. Olfactory receptors share a 7-transmembrane domain structure with many neurotransmitter and hormone receptors and are responsible for the recognition and G protein-mediated transduction of odorant signals. The olfactory receptor gene family is the largest in the genome. The nomenclature assigned to the olfactory receptor genes and proteins for this organism is independent of other organisms.


OR4F5 is the first protein coding gene on human chromosome 1.


An  orphon  is a  gene  located outside the main chromosomal  locus , i.e., it may be dispersed to an unconnected genomic location. [ 1 ] [ 2 ]


Orphons have been found in both protein-coding and non-protein-coding gene families, which suggests that most gene transcription processes do not constitute a restriction on the development of orphons. Extensive  polymorphism  in this feature between individuals of the same species was shown. The gene class was first discovered in yeast, sea urchins, and fruitflies, [ 1 ]  and has since been reported from the genome of many other  eukaryote  groups including molluscs, [ 3 ]  amphibians, [ 4 ]  and mammals including humans. [ 5 ]


NM_001261422 NM_001261423 NM_006812


NM_001171026 NM_177614


NP_001248351 NP_001248352 NP_006803


NP_001164497 NP_808282


Protein OS-9  is a  protein  that in humans is encoded by the  OS9   gene . [ 5 ] [ 6 ] [ 7 ] [ 8 ] [ 9 ]


This gene encodes a protein that is highly expressed in osteosarcomas. This protein binds to the hypoxia-inducible factor 1 (HIF-1), a key regulator of the hypoxic response and angiogenesis, and promotes the degradation of one of its subunits. Alternate transcriptional splice variants, encoding different isoforms, have been characterized. [ 9 ]


An  overlapping gene  (or  OLG ) [ 1 ] [ 2 ]  is a  gene  whose expressible  nucleotide sequence  partially overlaps with the expressible nucleotide sequence of another gene. [ 3 ]  In this way, a nucleotide sequence may make a contribution to the function of one or more  gene products . Overlapping genes are present in and a fundamental feature of both  cellular  and  viral   genomes . [ 2 ]  The current definition of an overlapping gene varies significantly between eukaryotes, prokaryotes, and viruses. [ 2 ]  In  prokaryotes  and  viruses  overlap must be between  coding sequences  but not  mRNA  transcripts, and is defined when these coding sequences share a nucleotide on either the same or opposite strands. In  eukaryotes , gene overlap is almost always defined as mRNA transcript overlap. Specifically, a gene overlap in eukaryotes is defined when at least one nucleotide is shared between the boundaries of the primary mRNA transcripts of two or more genes, such that a DNA base  mutation  at any point of the overlapping region would affect the transcripts of all genes involved. This definition includes 5′ and 3′  untranslated regions  (UTRs) along with  introns .


Overprinting  refers to a type of overlap in which all or part of the sequence of one gene is read in an alternate  reading frame  from another gene at the same  locus . [ 4 ]  The alternative open reading frames (ORF) are thought to be created by critical  nucleotide substitutions  within an expressible pre-existing gene, which can be  induced  to express a novel  protein  while still preserving the function of the original gene. [ 5 ]  Overprinting has been hypothesized as a mechanism for  de novo  emergence of new genes  from existing sequences, either older genes or previously  non-coding  regions of the genome. [ 6 ]  It is believed that most overlapping genes, or genes whose expressible nucleotide sequences partially overlap with each other, evolved in part due to this mechanism, suggesting that each overlap is composed of one ancestral gene and one novel gene. [ 7 ]  Subsequently, overprinting is also believed to be a source of novel proteins, as de novo proteins coded by these novel genes usually lack remote  homologs  in databases. [ 8 ]  Overprinted genes are particularly common features of the  genomic  organization of viruses, likely to greatly increase the number of potential expressible genes from a small set of viral genetic information. [ 9 ]  It is likely that overprinting is responsible for the generation of numerous novel proteins by viruses over the course of their  evolutionary history .


Genes may overlap in a variety of ways and can be classified by their positions relative to each other. [ 3 ] [ 11 ] [ 12 ] [ 13 ] [ 14 ]


Overlapping genes can also be classified by  phases , which describe their relative  reading frames : [ 3 ] [ 11 ] [ 12 ] [ 13 ] [ 14 ]


Studies on overlapping genes suggest that their evolution can be summarized in two possible models. [ 4 ]  In one model, the two proteins encoded by their respective overlapping genes evolve under similar  selection pressures . The proteins and the overlap region are highly conserved when strong selection against  amino acid  change is favored. Overlapping genes are reasoned to evolve under strict constraints as a single nucleotide substitution is able to alter the structure and function of the two proteins simultaneously. A study on the  hepatitis B virus  (HBV), whose DNA genome contains numerous overlapping genes, showed the mean number of synonymous nucleotide substitutions per site in overlapping coding regions was significantly lower than that of non-overlapping regions. [ 15 ]  The same study showed that it was possible for some of these overlapping regions and their proteins to diverge significantly from the original when there's weak selection against amino acid change. The  spacer  domain of the  polymerase  and the pre-S1 region of a surface protein of HBV, for example, had a percentage of conserved amino acids of 30% and 40%, respectively. [ 15 ]  However, these overlap regions are known to be less important for  replication  compared to the overlap regions that were highly conserved among different HBV strains, which are absolutely essential for the process.


The second model suggests that the two proteins and their respective overlap genes evolve under opposite selection pressures: one frame experiences  positive selection  while the other is under  purifying selection . In  tombusviruses , the proteins  p19  and  p22  are encoded by overlapping genes that form a 549 nt coding region, and p19 is shown to be under positive selection while p22 is under purifying selection. [ 16 ]  Additional examples are mentioned in studies involving overlapping genes of the  Sendai virus , [ 17 ]   potato leafroll virus , [ 18 ]  and human  parvovirus B19 . [ 19 ]  This phenomenon of overlapping genes experiencing different selection pressures is suggested to be a consequence of a high  rate  of nucleotide substitution with different effects on the two frames; the substitutions may be majorly  non-synonymous  for one frame while mostly being  synonymous  for the other frame. [ 4 ]


Overlapping genes are particularly common in rapidly evolving genomes, such as those of  viruses ,  bacteria , and  mitochondria . They may originate in three ways: [ 20 ]


The use of the same nucleotide sequence to encode multiple genes may provide  evolutionary  advantage due to reduction in  genome  size and due to the opportunity for  transcriptional  and  translational   co-regulation  of the overlapping genes. [ 12 ] [ 21 ] [ 22 ] [ 23 ]  Gene overlaps introduce novel evolutionary constraints on the sequences of the overlap regions. [ 14 ] [ 24 ]


In 1977,  Pierre-Paul Grassé  proposed that one of the genes in the pair could have originated  de novo  by mutations to introduce novel ORFs in alternate reading frames; he described the mechanism as  overprinting . [ 25 ] : 231   It was later substantiated by  Susumu Ohno , who identified a candidate gene that may have arisen by this mechanism. [ 26 ]  Some de novo genes originating in this way may not remain overlapping, but  subfunctionalize  following  gene duplication , [ 6 ]  contributing to the prevalence of  orphan genes . Which member of an overlapping gene pair is younger can be identified  bioinformatically  either by a more restricted  phylogenetic  distribution, or by less optimized  codon usage . [ 9 ] [ 27 ] [ 28 ]  Younger members of the pair tend to have higher  intrinsic structural disorder  than older members, but the older members are also more disordered than other proteins, presumably as a way of alleviating the increased evolutionary constraints posed by overlap. [ 27 ]  Overlaps are more likely to originate in proteins that already have high disorder. [ 27 ]


Overlapping genes occur in all  domains of life , though with varying frequencies. They are especially common in  viral  genomes.


The existence of overlapping genes was first identified in the virus  ΦX174 , whose  genome  was the first DNA genome ever sequenced by  Frederick Sanger  in 1977. [ 29 ]  Previous analysis of ΦX174, a small single-stranded DNA  bacteriophage  that infected the bacteria  Escherichia coli , suggested that the  proteins  produced during infection required  coding sequences  longer than the measured length of its genome. [ 31 ]  Analysis of the fully sequenced 5386 nucleotide genome showed that the virus possessed extensive overlap between coding regions, revealing that some genes (like genes D and E) were translated from the same DNA sequences but in different reading frames. [ 29 ] [ 31 ]  An  alternative start site  within the genome replication gene A of ΦX174 was shown to express a  truncated protein  with an identical coding sequence to the  C-terminus  of the original A protein but possessing a different function [ 32 ] [ 33 ]  It was concluded that other undiscovered sites of  polypeptide synthesis  could be hidden through the genome due to overlapping genes. An identified de novo gene of another overlapping  gene locus  was shown to express a novel protein that induces lysis of E. coli by inhibiting biosynthesis of its cell wall[56], suggesting that de novo protein creation through the process of overprinting can be a significant factor in the evolution of  pathogenicity  of viruses. [ 4 ]  Another example is the   ORF3d  gene in the  SARS-CoV 2  virus. [ 1 ] [ 34 ]  Overlapping genes are particularly common in  viral  genomes. [ 9 ]  Some studies attribute this observation to  selective pressure  toward small genome sizes mediated by the physical constraints of packaging the genome in a  viral capsid , particularly one of  icosahedral  geometry. [ 35 ]  However, other studies dispute this conclusion and argue that the distribution of overlaps in viral genomes is more likely to reflect overprinting as the evolutionary origin of overlapping viral genes. [ 36 ]  Overprinting is a common source of  de novo  genes in viruses. [ 28 ]


The proportion of viruses with overlapping coding sequences within their genomes varies. [ 2 ]  Double-stranded  RNA viruses  have fewer than a quarter that contains them while almost three-quarters of  retroviridae  and viruses with  single-stranded DNA genomes  contain overlapping coding sequences. [ 37 ]  Segmented viruses in particular, or viruses with their genome split into separate pieces and packaged either all in the same  capsid  or in separate capsids, are more likely to contain an overlapping sequence than non-segmented viruses. [ 37 ]  RNA viruses have fewer overlapping genes than DNA viruses which possess lower  mutation rates  and less restrictive genome sizes. [ 37 ] [ 38 ]  The lower mutation rate of DNA viruses facilitates greater genomic novelty and evolutionary exploration within a structurally constrained genome and may be the primary driver of the evolution of overlapping genes. [ 39 ] [ 40 ]


Studies of overprinted viral genes suggest that their protein products tend to be accessory proteins which are  not  essential  to viral proliferation, but contribute to  pathogenicity . Overprinted proteins often have unusual  amino acid  distributions and high levels of intrinsic  disorder . [ 41 ]  In some cases overprinted proteins do have well-defined, but novel, three-dimensional structures; [ 42 ]  one example is the  RNA silencing suppressor p19  found in  Tombusviruses , which has both a novel  protein fold  and a novel binding mode in recognizing  siRNAs . [ 28 ] [ 30 ] [ 43 ]


Estimates of gene overlap in  bacterial  genomes typically find that around one third of bacterial genes are overlapped, though usually only by a few base pairs. [ 12 ] [ 44 ] [ 45 ]  Most studies of overlap in bacterial genomes find evidence that overlap serves a function in  gene regulation , permitting the overlapped genes to be  transcriptionally  and  translationally  co-regulated. [ 12 ] [ 23 ]  In prokaryotic genomes, unidirectional overlaps are most common, possibly due to the tendency of adjacent prokaryotic genes to share orientation. [ 12 ] [ 14 ] [ 11 ]  Among unidirectional overlaps, long overlaps are more commonly read with a one-nucleotide offset in reading frame (i.e., phase 1) and short overlaps are more commonly read in phase 2. [ 45 ] [ 46 ]  Long overlaps of greater than 60  base pairs  are more common for convergent genes; however, putative long overlaps have very high rates of  misannotation . [ 47 ]  Robustly validated examples of long overlaps in bacterial genomes are rare; in the well-studied  model organism   Escherichia coli , only four gene pairs are well validated as having long, overprinted overlaps. [ 48 ]


Compared to prokaryotic genomes, eukaryotic genomes are often poorly annotated and thus identifying genuine overlaps is relatively challenging. [ 28 ]  However, examples of validated gene overlaps have been documented in a variety of eukaryotic organisms, including mammals such as mice and humans. [ 49 ] [ 50 ] [ 51 ] [ 52 ]  Eukaryotes differ from prokaryotes in distribution of overlap types: while unidirectional (i.e., same-strand) overlaps are most common in prokaryotes, opposite or antiparallel-strand overlaps are more common in eukaryotes. Among the opposite-strand overlaps, convergent orientation is most common. [ 50 ]  Most studies of eukaryotic gene overlap have found that overlapping genes are extensively subject to genomic reorganization even in closely related species, and thus the presence of an overlap is not always well-conserved. [ 51 ] [ 53 ]  Overlap with older or less taxonomically restricted genes is also a common feature of genes likely to have originated  de novo  in a given eukaryotic lineage. [ 51 ] [ 54 ] [ 55 ]


The precise functions of overlapping genes seems to vary across the domains of life but several experiments have shown that they are important for virus lifecycles through proper protein expression and stoichiometry  [ 56 ]  as well as playing a role in proper protein folding. [ 57 ]  A version of  bacteriophage   ΦX174  has also been created where all gene overlaps were removed  [ 58 ]  proving they were not necessary for replication.


The retention and evolution of overlapping genes within viruses may also be due to  capsid  size limitations. [ 59 ]  Dramatic viability loss was observed in viruses with genomes engineered to be longer than the wild-type genome. [ 60 ]  Increasing the single-stranded DNA genome length of  ΦX174  by >1% results in almost complete loss of  infectivity , believed to be the result of the strict physical constraints imposed by the finite capsid volume. [ 61 ]  Studies on  adeno-associated viruses  as  gene delivery   vectors  showed that viral packaging is constrained by genetic cargo size limits, requiring the use of multiple  vectors  to deliver large human genes such as CFTR81. [ 62 ] [ 63 ]  Therefore, it is suggested that overlapping genes evolved as a means to overcome these physical constraints, increasing genetic diversity by utilizing only the existing sequence rather than increasing genome length.


Standardized methods such as  genome annotation  may be inappropriate for the detection of overlapping genes as they are reliant on already curated genes while overlapping genes are generally overlooked contain atypical sequence composition. [ 2 ] [ 64 ] [ 65 ] [ 66 ]  Genome annotation standards are also often biased against feature overlaps, such as genes entirely contained within another gene. [ 67 ]  Furthermore, some bioinformatics pipelines such as the  RAST pipeline  markedly penalizes overlaps between predicted ORFs. [ 68 ]  However, rapid advancement of genome-scale protein and RNA measurement tools along with increasingly advanced prediction algorithms have revealed an avalanche of overlapping genes and ORFs within numerous genomes. [ 2 ]   Proteogenomic  methods have been essential in discovering numerous overlapping genes and include a combination of techniques such as  bottom-up proteomics ,  ribosome profiling ,  DNA sequencing , and  perturbation .  RNA sequencing  is also used to identify genomic regions containing overlapping transcripts. It has been utilized to identify 180,000 alternate ORFs within previously annotated coding regions found in humans. [ 69 ]  Newly discovered ORFs such as these are verified using a variety of  reverse genetics  techniques, such as  CRISPR-Cas9  and  catalytically dead Cas9 (dCas9) disruption . [ 70 ] [ 71 ] [ 72 ]  Attempts at proof-by-synthesis are also performed to show beyond doubt the absence of any undiscovered overlapping genes. [ 73 ]


NM_001114734 NM_001363585


Poly(A) binding protein, cytoplasmic 4-like  is a  protein  that in humans is encoded by the PABPC4L  gene .
 [ 5 ]


PfATP6 , also known as PfSERCA or PfATPase6, is a  calcium ATPase  gene encoded by the malaria parasite  Plasmodium falciparum . [ 1 ]  The protein is thought to be a  P-type ATPase  involved in  calcium  ion  transport .


Mutations in PfATP6 that had been identified in field isolates (such as S769N) and in laboratory clones (such as L263E) were shown to have decreased sensitivity to artemisinin but conversely were more susceptible to other compounds targeting SERCAs. [ 2 ]  In a yeast expression system looking at mutations L263E, A623E, S769N, and A623E/S769N it was shown that there was a fitness cost to these mutations compared to the wild-type. [ 3 ]


Research in 2003 indicated that PfATP6 is a target of artemisinin (a potent antimalarial drug). [ 4 ]  It was observed that single amino acid mutations in PfATP6 could abolish sensitivity to artemisinin compounds. [ 5 ]  Evidence came from a  Xenopus oocyte  system describing specific interactions between artemisinins and PfATP6 as well as E255L-mutated mammalian SERCA and from parasites in French Guiana with mutations in PfATP6 making them less susceptible to inhibition by artemether. [ 6 ]  An independent assessment using the  Xenopus oocyte  system reported in 2016 that while PfATP6 protein could be detected, activity was not observed. [ 7 ]  In the independent oocyte work, mammalian SERCA and its E255L-mutated version were active but both were insensitive to artemisinin, again in contrast to the original claims. The authors suggested that the original results might have been affected by low ATPase signals, few experimental repeats and large standard deviations. [ 8 ]  The lack of artemisinin inhibition of E255L mammalian SERCA matched results from highly purified extracts obtained after heterologous expression in yeast cells. [ 9 ] [ 10 ]


More recently, research has supported the role of PfATP6 in artemisinin therapy; direct interaction of artemisinin with PfATP6 was further noted in 2016 in an in vivo screen of the malaria parasite with a tagged drug molecule in two independent studies; 124 separate Pf proteins and >60 proteins that bound to this molecule were identified. [ 11 ] [ 12 ]  This work was substantiated in 2022 using a functional whole cell assay after yeast heterologous expression and in vitro identifying PfATP6 as both a binding partner and capable of functional inhibition by artemisinin compounds. [ 13 ]  The same system confirmed  that mutated mammalian  SERCA1 (E255L) is more susceptible to inhibition by artemisinins. Intraparasitic free calcium concentrations are increased after exposure to an artemisinin.  In 2022 it was observed in murine neutrophils that artemisinins were inhibiting migration of these cells. Further investigation pinpointed the mechanism of neutrophil functions to inhibition of the activity of a homologue of PfATP6, SERCA3. [ 14 ]  This research supports the case for artemisinin activity with malaria SERCAs.


PfATP6 mutations play no role in the reduced artemisinin susceptibility observed in southeast Asia. The consensus is that PfATP6 is a validated target for artemisinins and mammalian SERCA3, in neutrophils, has emerged as a newly identified target.


NM_022835 NM_001351693 NM_001351694


NM_001083912 NM_001290542 NM_138752


NP_073746 NP_001338622 NP_001338623


NP_001077381 NP_001277471 NP_620091


Pleckstrin homology domain containing, family G member 2  (PLEKHG2) is a  protein  that in humans is encoded by the  PLEKHG2   gene . It is sometimes written as ARHGEF42, FLJ00018.


The PLEKHG2 protein is a huge protein of about 1300 amino acids, 130 kDa and has a Dbl homology (DH) domain and a pleckstrin homology (PH) domain near the N terminus of its structure. The DH domain is a domain responsible for guanine nucleotide exchange activity that converts GDP on the Rho family Small GTPase (RhoGTPase) to GTP, and PLEKHG2 having this domain also acts as a Rho-specific guanine nucleotide exchange factor ( RhoGEF ).


Activation of RhoGTPase reconstitute the actin cytoskeleton and changes the cell morphology, so PLEKHG2 might be contributes to cell motility and neuronal network development of neurons via RhoGTPase and actin remodeling (see later).


Recombinant BXH2 and AKXD inbred mice mutated by retroviral transduction are known to develop myeloid leukemia, B cell and T cell leukemia at high frequency. [ 5 ]


In 2002, Himmel et al., used this model of acute myelogenous leukemia and showed that a novel Dbl family guanine nucleotide exchange factor gene is contained downstream of the retroviral uptake site called Evi24. They named this gene Clg. Hemmel and colleagues cloned Clg and showed homology with PLEKHG2 contained in human chromosome 19 chromosome 19q13.1 region. From these observations they pointed out association with acute myeloid leukemia. [ 6 ]


In a paper published by Hemmel et al., in 2002, they showed that a construct containing a DH-PH domain of Clg promotes guanine nucleotide exchange of Cdc42 but does not promote guanine nucleotide exchange of Rac1 or RhoA. In addition, DH-PH domains or full-length Clg were introduced into NIH3T3 cells and transformation occurred.


Later, Ueda and his colleagues introduced the expression construct of full-length human PLEKHG2 into HEK 293 cells. In this cell the Gβγ subunit of the trimeric G protein were interacted with PLEKHG2 directly. Ueda and colleagues also showed that PLEKHG2 were activated by Gbg and PLEKHG2 activates Rac1, Cdc42 of RhoGTPase and contributes to cell morphological change. [ 7 ]


In 2013, Runne et al., showed that PLEKHG2 is elevated in several leukemia cell lines, including Jurkat T cells. In addition, they showed that GPCR signal-dependent activation of Rac and Cdc42 regulates the chemotaxis of lymphocytes via actin polymerization. From this observation PLEKHG2 was considered to an important regulator of cell motility. [ 8 ]


Furthermore, in recent years, it has become clear that PLEKHG2 undergoes regulation through modification such as phosphorylation and interaction with other proteins by various intracellular signals (see the section on interaction / protein modification). However, the function in vivo is still unclear.


In 2016, Edvardson et al., identified homozygosity for Arg204Trp mutation in the PLEKHG2 gene in the patients with dystonia or postnatal microcephaly. [ 9 ]


PLEKHG2 is known to interacted with the following proteins.


・Gβγ [ 7 ]


・β-actin [ 10 ]


・Four and a Half LIM domain1 (FHL1) [ 11 ] [ 12 ]


・Gαs [ 13 ]


It is known that PLEKHG2 undergoes modification such as phosphorylation by the following signals.


・SRC [ 14 ]


・EGFR [ 15 ]


Polycystic kidney disease 3 (autosomal dominant)  is a  protein  that in humans is encoded by the  PKD3   gene . [ 2 ]


Polycystic kidney disease  (ADPKD) is a life threatening hereditary disorder; it is characterized by the development of fluid-filled cyst formation and expansion of the kidney and other organs. [ 3 ]  It is an autosomal dominant disease, and it is the most common hereditary disorders with a rate of occurrence of approximately 1 in 1000. [ 4 ]


ADPKD is an autosomal dominant disease, classified into three types, with the chromosomal location of the responsible gene in parenthesis:  PKD1  (16 Chr),  PKD2  (4 Chr) PKD3 (11 Chr, this gene). Mutations in the three different genes  PKD1 ,  PKD2  and  PKD3  cause a very similar disorder of the autosomal dominant form of polycystic kidney disease (ADPKD). [ 5 ]  The PKD3 gene is located on chromosome number 11q12.3; the phenotype MIM number is 600666.


PKD3 it is the result of a mutation in the  GANAB  gene. The  GANAB  gene codes the catalytic alpha subunit of glucosidase II and noncatalytic beta subunit; the glycosyl hydrolase 31 families of proteins. The heterodimeric glucosidase II enzyme has an important function in protein folding and catalyzes the hydrolysis of glucose residues. In the endoplasmic reticulum, the quality control is done by cutting the residues of glucose from the immature glycoproteins. The rise in the expression of the protein leads to the lung tumor tissue. Also, any mutation in this gene causes autosomal dominant polycystic liver disease. [ 6 ]


PKD3 occurs in adults and it sometimes shows symptoms in children. It is known as 'polycystic kidneys'. Polycystic kidney disease (PKD3) is an autosomal dominant inheritance that leads to renal cysts. It is related to the liver cysts that sometimes causes organ dysfunction. It is usually detected in middle to late aged individuals developing severe cysts in kiddy and liver. However, the renal disease is mild and very few patients have hypertension, it does not happen regularly. Unlike the liver disease where it develops a wide spectrum of severity; some have no cyst, while others have severe liver involvement polycystic kidney disease. [ 3 ]


In an analysis of 20 patients from nine unrelated families develop polycystic kidney disease with heterozygous mutations in the  GANAB  gene.  Five of the mutations from the  GANAB  gene were predicted to result in a short protein (frameshift, nonsense, or splicing), and three missense mutations.  Seven of the families had a diagnosis of PKD, while the other two families had a diagnosis of polycystic liver disease (PCLD). Most of the patients developed during adulthood, except one showed symptoms at the age of 9. The recordings indicate that the renal disease was insignificant. Also, very few of them had high blood pressure. Renal imaging has also been done, which present variable numbers of multiple cysts (from less than 10 to more than 40).Those suffering from PCLD, some had no cyst while others develop severe disease, requiring surgery.   Therefore, this shows that GANAB-related PKD and PCLD are not necessarily separate diseases, but there was significant phenotypic overlap between the two, and that the PKD3 is responsible for this disease. [ 4 ]


There is no specific treatment for PKD3. However, a lot of research is going on to mimic the symptoms. Regular monitoring and following up on the complications can assist in maintaining the health and extend a person's life. Recent research shows that the treatment with protein kinase C (PKC) inhibitor may help to mimic the increase of PKD3, by RacV12 and aluminum fluoride. [1]


The  PPP6R3  gene (also termed  C11orf23, PP6R3, SAP190, SAPL, SAPLa , and  SAPS3 [ 1 ] ) is located at  band  13.2 on the  long (or "q") arm  of  chromosome 11  and is expressed in all tissues tested in humans. [ 2 ]  It  encodes  protein phosphatase 6 regulatory subunit 3 (PP6RS3). [ 3 ]  Also termed serine/threonine-protein phosphatase 6 regulatory subunit 3, PP6RS3 is the function-regulating subunit of  protein phosphatase 6  (PPP6C). [ 4 ]  PPP6C is a component of a signaling pathway that regulates various cell functions including  cell division ,  the repair of damaged DNA ,  inflammatory responses , [ 5 ]   messenger mRNA splicing , and the stability of chromosomes. [ 6 ]


Two cases of  nodular fasciitis  have  malignant  tumors in which the  neoplastic  cells expressed a  PPP6R3  gene merged to the  USP6  (i.e. ubiquitin carboxyl-terminal hydrolase 6) gene to form an  USP-PPP6R3   fusion gene . In both of these cases, the  USP-PPP6R3  fusion genes was considered to contribute to these tumors malignancy. [ 3 ] [ 6 ] [ 7 ]


GeneCard for PPP6R3


PR domain zinc finger protein 12  is a  protein  that in humans is encoded by the  PRDM12   gene .  This gene is normally switched on during the development of pain-sensing nerve cells. People with homozygous mutations of the PRDM12 gene experience  congenital insensitivity to pain  (CIP). [ 5 ] [ 6 ]  PRMD12 is a part of a larger domain that mediate histone methyltransferases. Enzymes target gene promoters in order to control gene expression. [ 7 ]


The human protein isoform is made up of 367 amino acids containing a PR domain (related to the  SET methyltransferase domain ), 3  zinc fingers , and a  C-terminal  polyalanine tract. [ 5 ]


PRDM12 influences the development of nerve cells that assist in perception and sensation of pain, which is an important evolutionary advantage. In humans, mutations in the PRDM12 gene can cause loss of pain perception brought on by defects in the development of sensory neurons. [ 8 ]  It also has a range of interactions with and affects on various proteins. In vertebrates, PRDM12 directly represses the  DBX1  and  NK X6  genes. This is thought to be accomplished by utilizing  G9a , a strong H3K9 methyltransferase. The indicated result of PRDM12's cross-repressive interaction with the DBX1 and NKX6 genes is that the PRDM12 partially acts as a promoter of V1 interneurons (which are essential to the locomotion of vertebrates). [ 9 ]  It is a member of the group of PR- domain-containing zinc-finger familyfingers, "which appear to function as negative regulators of oncogenesis and include the tumor-associated genes  MDS1-EVI1 ,  RIZ ,  BLIMP1 ,  MEL1  and  PFM1 .  PRDM12  therefore represents an attractive candidate tumour suppressor gene within the der(9) [derivative chromosome 9] CDR [commonly deleted region]." [ 10 ]  Several members of the PRDM family are found to be acting as a tumor suppressor or a factor driving oncogenic processes in human diseases, specifically and most notably in solid cancers and hematological malignancies. It is hoped that further study may reveal target genes of PRDM proteins so a greater understanding of the functions of the PRDM family can be achieved. [ 11 ]  In  Xenopus  embryos, PRDM12 expression "was partially co-localized with the lateral expression regions" of the  SIX1 ,  PAX3 , ISLET1, and  PAX6  genes, but not those of the  FOXD3  and  SIX3  genes. In cases where  BMP4  was overexpressed, embryos showed an increase in PRDM12 expression. Data indicated that the regulation of PRDM12 expression in  Xenopus  embryos was controlled by BMP and  Wnt signaling . [ 12 ]


PRDM12 codes for a protein which regulates the neurological path through which pain in perceived, known as PR domain zinc finger protein 12. [ 13 ]  The protein plays a vital role in the regulation of histone H3-K9 dimethylation. [ 12 ] [ 14 ]  PRDM12’s protein also directly affects the development of nerve-endings. The protein is synthesized at the same developmental point as the neurons which sense pain and the growth of the two is linked. [ 6 ]  The mutation of this gene results in a non-functioning protein, which in-turn causes a failure to develop the pain-sensing nerve endings and an organism without sensitivity to pain. [ 15 ]  This lack of pain-sensing nerve endings can cause severe harm to the individual, as they cannot sense when they are injured by something such as a hot stove-eye or broken bone. [ 6 ]  PRDM12’s protein has also been found to be a tumor suppressor for chronic myloid leukemia. [ 10 ]  The protein controls gene expression by modifying chromatin. [ 15 ]  PRDMs as a family tend to require enzyme help to modify histones, with some exceptions. [ 16 ]


In humans, mutations in the PRDM12 gene can cause loss of pain perception brought on by defects in the development of sensory neurons. [ 8 ]  There are a number of diseases and conditions that can result from mutations in the PRDM12 gene.


Congenital insensitivity to pain (CIP) is a characterized by an inability to feel pain. [ 17 ]  This is a rare condition that is present at birth due to a lack of, or malfunction of, nociceptors. [ 17 ]  There are three different genes that can be mutated to cause CIP. First, a mutation in the  SCN9A  makes it impossible for nociceptors to respond to harmful stimuli because it causes the gene to lose its function. [ 17 ]  Second, a mutation in the  NTRK1  causes a loss of function for the gene and leads to a failure in nociceptor development. [ 17 ]  Finally, researchers have identified 10 homozygous mutations on PRDM12 that appeared to be linked to this condition. [ 15 ]  Past research has shown that PRDM12 is involved in the modification of chromatin. [ 15 ]  Chromatin can turn genes off and on by attaching itself to chromosomes and acting as an epigenetic switch. [ 15 ]  Chromatin play a huge role in neuron development, so researcher hypothesized that mutations in the PRDM12 gene prevent nociceptors and nerve fibers from developing normally.  [ 15 ]  They then studied the nerve biopsies of patients with this condition and found that the patients affected by this condition are lacking pain sensing never fibers in their legs, or only have half the amount they should have. [ 15 ]


Another condition caused by mutations in the PRDM12 gene is hereditary sensory and autonomic neuropathy type VIII. [ citation needed ]  HSAN VIII is a very rare autosomal recessive inherited disorder that also begins at birth and is characterized by an inability to feel pain and an inability to sweat ( anhidrosis ). [ citation needed ]  Anhidrosis can cause frequent episodes of high body temperature of high fever. [ citation needed ]  Other signs of this condition can include early loss of teeth, server soft tissue injuries, dental caries and submucosal abscesses, hypomineralization of primary, and mandibular  osteomyelitis . [ 18 ]  Abnormal functioning of the sensory nerves is what causes the sensory loss in patients with this condition. [ citation needed ]