Receptor Activator of NK-κB for Tumor Cells


02 Apr 2018

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Cancers figure among the leading causes of death worldwide, accounting for 8.2 million deaths in 2012 .Lung, liver, stomach, colorectal and breast cancers cause the most cancer deaths each year. It is expected that annual cancer cases will rise from 14 million in 2012 to 22 within the next two decades

Over the past several years, proteolytic cleavage and release of the ectodomain of membrane-bound proteins, also referred to as ectodomain shedding, has emerged as an important posttranslational regulatory mechanism for modifying the function of cell surface proteins. The cleavage of RANK should decrease its availability on osteoclasts and their precursors and simultaneously generate soluble decoy receptors that may inhibit the RANKL–RANK association and their by NFkB signaling[27]

It was previously demonstrated that tumor cells express RANK and activate RANKL-RANK pathway. The RANK/RANKL axis emerges as a key regulator of breast cancer initiation, progression and metastasis.In addition,RANKL can protect breast cancer cells from apoptosis in response to DNA damage, as well as control the self-renewal and anchorage-independent growth of tumor initiating cells [13].

In a recent study it’s demonstrated that NK-κB Signaling could be blocked by Enterokinase by cleavage of RANK (Receptor Activator of NK-κB), suggests its possible application in treating diseases like Osteoporosis, cancer and diseases associated with bone loss [14].

Enterospeptidase could specifically cleave RANK on the sequence NEEDK was demonstrated by a surrogate peptide blocking assay [28]


All animals need to digest exogenous macromolecules without destroying similar endogenous constituents. The regulation of digestive enzymes is, therefore, a fundamental requirement. Vertebrates have solved this problem, in part, by using a two-step enzymatic cascade to convert pancreatic zymogens to active enzymes in the lumen of the gut.

Enteropeptidase (synonym: enterokinase [E.C.]) is aglycoprotein enzymeof the digestive tract was discovered, by N. P. Schepovalnikow in 1899 in Russia (Walther 1900), as a element that is present in the duodenum and which can capable of activating pancreatic juice to digest fibrin. Enteropeptidase converts trypsinogen (a zymogen) convert into its active form trypsin by enteropeptidase, with selective cleavage of 6-Lys-|-Ile-7 bond which causes subsequent activation of digestive enzymes in pancreatic secretions.


Subsequent activation of trypsin,it cleaves and activates other zymogens in pancreatic secretions,including chymotrypsinogen, proelastase, procarboxypeptidases, and some prolipases [1]

In almost all vertebrate species, a short trypsinogen activation peptide is released that terminates with the sequence Asp-Asp-Asp-Asp-Lys(DDDK) (2). except for the similar sequences of trypsinogens from lungfish (IEEDK and LEDDK) and African clawed frog (FDDDK). The unique enteropeptidase substrate specificity has been exploited in protein engineering. The enteropepetidase recognizing sequence DDDDK↓I is often used in recombinant proteins that necessitate specific cleavage.Enteropeptidase's specificity for its recognizing sequence makes it an ideal tool in biochemical applications; following protein purification enteropeptidase can cleave a fusion protein containing a C-terminal tag (such as poly-His) linked by this sequence to obtain the target protein

1.1 Enteropeptidase

1.1.1 Enteropeptidase expression:

Enterokinase is believed to be Exclusively produced in the brush border by enterocytes and goblet cells of the duodenal mucosa, the enteropeptidase is ubiquitously distributed among vertebrates (Eggermont et al., 1971a,b; Rinderknecht, 1986; Mithoshi et al., 1990). It secreted in to the small intestine. It lacks in crypts but found substantialiy in villous enterocytes and maximal in the upper half of the villi, partucilarly on the brush border. enteropeptidaseis secreted from glands following the entry of swallowed food passing from the intestin. It resists destruction from the various enzymes in the small intestine but is destroyed by bacteria in the large intestine.

It has been purified from several sources including porcine (Barrati et al., 1973), bovine (Anderson et al.; Liepnieks and Light, 1979), human (Kitamoto et al., 1995), murine (Yang et al., 1998) and rat intestine (Yahagi et al., 1996). In all cases the protease seems to be expressed as a single-chain precursor, which must be cleaved to achieve the native disulfide-linked heterodimer, in the case of human enteropeptidase consisting of an 86 kDa heavy chain and a 28 kDa light chain. Most of the structural elements are highly conserved, especially between human, bovine and porcine enteropeptidase, which share more than

80% identity in their amino acid sequences. The heavy chain, which contains various domains including membranespanning hydrophobic membrane anchors, several receptorlike motifs and up to 10 intramolecular disulfide bridges, is responsible for specific macromolecular substrate recognition (Lu et al., 1997; Mikhailova et al., 2007). The light chain is connected to the heavy chain via one disulfide e bridge and contains the classical catalytic triad (His57, Asp102 and Ser195 in chymotrypsin numbering) with up to four intramolecular disulfide bridges.

The small and catalytically active light chain offers especially high potential for biotechnological applications, and several attempts to understand and improve the functionality of this protease have been made (Lu et al., 1999; Liew et al., 2007; Shahravan et al., 2008).

et al., 2004), Lu et al. (1997) have determined the crystal structure of a bovine light chain complex with a trypsinogen activation peptide analogue at a resolution of 2.3 A ° . Human enteropeptidase has been recombinantly expressed in E.coli by Gasparian et al. (2003), although this resulted in insoluble aggregates and no crystal structure has yet been reported. The subsequent refolding via 6 M guanidinium chloride resulted in a total refolding yield of 2% after two cycles of renaturation.

Enteropeptidase is serine protease it’s a heterodimer of a multidomain heavy chain and a catalytic light chain linked by a disulfide bond . Enteropeptidase anchored to intestinal brush border of duodenal enterocytes by a transmem brane segment in the a 82–140 kDa heavy chain and a 35–62 kDa light chain which consist the catalytic subunit. Replacement of the transmembrane domain by a cleavable signal peptide does not impair trypsinogen activation, indicating that membrane association is not required for substrate recognition (Lu et al., 1997)..[3]Enteropeptidase is a part of the chymotrypsin-group of serine proteases, and is structurally resemble to these proteins.[4]

1.1.2. Enteropeptidase gene ontology:

In humans, PRSS7 gene (also known as ENTK) encodes enteropeptidase enzyme on chromosome 21q21. The gene spans ~90-kb in length and has 25 exons .Enteropeptidase mRNA is expressed majorly in the duodenum and, at poor levels, in the proximal section of jejunum. The human enteropeptidase cDNA open reading frame of encodes a 1019 amino acids Type II transmembrane protein with a calculated mass of 113kDa and with particularly 17 potential N-linked glycosylation sites. Few frameshift and nonsense mutations in this gene lead to a rare recessive condition characterized by severe failure to thrive in affected newborns, because of enteropeptidase deficiency.[6] Conversely, duodenopancreatic reflux of proteolytically active enteropeptidase may cause acute and chronic pancreatitis.

1.1.4 Structure

Bovine enteropeptidase is synthesized as a single-chain precursor of 1035 amino acid residues (5) that appears to require proteolytic activation, suggesting that enteropeptidase may not be the “first” protease of the digestive hydrolase cascade. Active enteropeptidase has been cleaved after Arg-800 to produce a disulfide-linked heterodimer with an amino-terminal '120-kDa heavy chain and a '47-kDa light chain; 40% of the actual mass of these polypeptides is due to glycosylation (6, 7).

The deduced amino acid sequences suggested that from a single-chain precursor, active two-chain enteropepetidase is derived. A potential sigal-anchor (SA) sequence near the amino terminus mediates membrane association of enter peptidase in intestine. The amino-terminal heavy chain consist the domains that are homologous to sections of the low density lipoprotein receptor(LDLR), two repeats found in complement serine proteases C1r and C1s, a MAM domain (so named for similar motifs first identified in the metalloprotease meprin, the Xenopus laevis neuronal recognition proteinA5, and protein-tyrosine phosphatase Mu), and a macrophage scavenger receptor cysteine-rich repeat ( MSCR). The light chain is a typical chymotrypsin-like serine protease. The activation cleavage site between the heavy and light chains has the sequence Val-Ser-Pro-Lys2Ile, which might be recognized by trypsin or other trypsin-like proteases. The carboxyl-terminal catalytic light chain is homologous to the class of trypsin-like serine proteases. Therefore, enteropeptidase is a mosaic protein with a complex evolutionary history. The enteropeptidase light chain amino acid sequence surrounding the amino terminus is ITPK-IVGG (human) or VSPK-IVGG (bovine), supporting that unidentified trypsin-like protease that cleaves Lys-fle bond to activate single-chain enteropeptidase.Therefore, enterokinase may not be the "first" enzyme of the cascade of intestinal digestive hydrolases. Enteropeptidase specificity for the DDDDK-I sequence of trpsinogen may be described by complementary basic-amino acid residues grouped in potential S2-S5 subsites.

1.1.3 enterokinase deficiency

Therefore, enterokinase has been recognized to play a key role in regulating intestinal protein digestion. Certainly, patients with primary enterokinase deficiency, a genetic disorder with little or no enterokinase activity in the duodenum, have been reported to suffer from malabsorption and malnutrition, predominantly in infancy, and need to take drugs containing pancreatic enzyme mixture for recovery [2]. Because of its physiological importance, there have been a number of studies on the purification and characterization of enterokinase from various species [3-9]



Entero kinase as Biotechnology tool

Protein purification is frequently aided by use of protein tags: therefore, fusion proteins or chimeric proteins produced by recombinant DNA technology are utilized in the forefront of protein science research for applications as various as vaccine development, biochemical purification, immunodetection, functional genomics, analysis of protein trafficking, protein therapies, and analyses of protein– nucleic acid or protein–protein interactions (Beckwith 2000). In structural biology, where milligram amounts of homogeneous protein sample are generally required, the most common usefulness of chimeras participates in the separation of the fusion protein from the cell lysate by affinity chromatography. The most common affinity tags include E. coli thioredoxin (TRX; LaVallie et al. 2000),the hexa-histidine (His-tag; Bornhorst and Falke 2000), Schistosoma japonicum glutathione-S-transferase (GST; Smith 2000), Escherichia coli maltose-binding protein (MBP; Sachdev and Chirgwin 2000)and avidin/streptavidin Strep tags (Skerra and Schmidt 2000). A number of other tags have also been developed (Stevens 2000). To produce crystals of a protein of interest to study its structure such as X-ray diffraction studies, bulky-affinity tags, such as MBP or GST, are generally removed using site-specific proteases in the engineered linker region, followed by purification to isolate the protein of interest from the affinity tag fusion protein and the used protease . However, certain problems may be faced during the cleavage step, including failure to recover active or structurally intact protein, high price of proteases (e.g., factor Xa and enterokinase),low yield, precipitation of the target protein or tedious optimization of cleavage conditions.

Recent estimates indicate using a His-tag, maybe one-third to one-half of all proteins of prokaryotes cannot be overexpressed in bacteria as a soluble form (Edwards et al. 2000; Stevens 2000). This number is may higher for eukaryotic proteins, indicated by three latest high throughput studies (Braun et al. 2002; Hammarstrom et al. 2002; Shih et al. 2002), specifically bigger multi domain proteins. In E. coli, if the problem of insoluble expression of the His-tagged protein is faced, one or more of the following options are usually explored: varying culture growth conditions, chaperones co-expression, altering cell lines, or changing to a different affinity tag such as, TRX, MBP, GST or NusA

Apart from affinity purification, the large-affinity tags offer numerous advantages. In a recent report, compared to the His-tag expression, TRX and MBP improved the solubility and expression of a set of 32, less than 20 kD small human of proteins in E. coli (Hammarstrom et al. 2002). For the sets of 32 larger human proteins (17–158kD; Braun et al. 2002) and 40 proteins of eukaryotes (9–100 kD; Shih et al. 2002), the large-affinity tags MBP (40 kD), NusA (54 kD), and GST (26 kD) were shown to be helpful in enhabcing the yield of soluble protein.

One of the significant features of enteropeptidase is its exclusive substrate specificity, which recognizes Lys at P1 and a group of four Asp amino acid at P2-P5. Within this recognition sequence, a Lys or Arg residue at P1 and Lys residues at P2 and P3 seems to be highly essential for efficient cleavage (87). The structural factors for enteropeptidase substrate specificity have been contained in its catalytic light chain. There is a cluster of four conserved basic residues, R/KRRK at sites 96–99, which were assumed to interact with the acidic P2-P5 residues in the trypsinogen activation spot (74). Lys99 residue was found to have extensive contacts with the P2 and P4 Asp residues indicated in crystal structure of enteropeptidase light chain of bovine, (22). Lys99 amino acid is conserved in enteropeptidase from various species. Substitution of Lys99 with Alanine by site directed mutagenesis blocked enteropeptidase from activating trypsinogen. In compare, Lys96, Arg97 substitution, and Arg98 residues on activity of bovine enteropeptidase had less significant effects (22). The exclusive enteropeptidase specificity for its substrate has been exploited in protein engineering. The DDDDK↓I sequence is ofen used in recombinant proteins where specific cleavage is required.

Enteropeptidase has a high potential as a fusion protein cleavage reagent, because of high specificity for the amino-acid sequence (Asp) 4Lys,.

An important benefit of enteropeptidase is that no crucial specificity residues are positioned on the C-terminal side of the scissile bond in its recognition site .Accordingly, when an affinity tag is linked to N-terminus the protein of interest,in maximum cases enteropeptidase is able to produce a digestion product with a native N-terminus.

A study inspecting the significance of the P1–P5 positions concluded that the P1 lysine was the most important specificity element, followed by the aspartate amino acids in the P2, P3, P5 and P4 positions, respectively, with the latter position donating very little to specificity

In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function

Activating Proteses

The N-terminal pro-sequence of proteases which must be cleaved prior to activation can be mutated to enable activation with enteropeptidase.[7]

Target for Obesity Treatment

Congenital enteropeptidase deficiency now days attracted attention as a novel target for obesity Treatment, among the identified genetic diseases related with starvation human phenotype,

Obesity is a complex metabolic disorder, in which various environmental factors and several genes are involved [1], [2]. Previous research targeting to develop drugs for treatment of obesity and type II diabetes has targeted genes that are linked with a fat human phenotype. Certainly, substantial work has been dedicated to participates developing drugs against these so called “obesity genes,” all of which are, directly or indirectly, in energy controlling; e.g., control of appetite, energy generation; carbohydrate and protein metabolisms, satiety, or thermogenesis, fatty acid, etc. [3], [4]. However, in humans, obesity is hardly attributable to the role of a single gene (wild or mutated). Additionally, the high redundancy of genes participates in energy management makes it unlikely that obesity will ever be controlled by affecting just one gene

The foregoing proposes that EP activity may aid as selective and competent target for metabolic disorders treatment. While complete of blocking enteropeptidase would cause the unwanted side effects observed in patients affected by CEP, partial inhibition should reduce the efficacy of energy absorption through the gastrointestinal (GI) tract. A 15–20% decrease in the daily absorption of deriving energy from both fatty acids and proteins should have a major influence on long-term weight controlling, and it should be an extra effective weight-control measure than a treatment centered only on pancreatic lipase inhibitors such as Alli or Xenical. It should be noted that these drugs endorse the buildup of undigested lipids in the intestinal tract, causing in leaky stool and diarrhea. An additional benefit of partial enteropeptidase inhibition is that the combination of undigested proteins and lipids would be more reliable than just fat, debatably fading or suppressing the above unpleasant effects.


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