HBVx could be a drug target for HBV induced hepatocellular carcinoma.

Hepatitis B Virus X Protein: A Key Regulator of the Virus Life Cycle

Hepatitis B virus (HBV) infection is the main risk factor for hepatocellular carcinoma (HCC) worldwide. Epidemiologic studies have shown that individuals who are chronic HBV carriers have a greater than 100-fold increase in the risk of developing liver cancer. The HBV genome is a partially double-stranded, circular DNA containing four overlapping genes: S/preS, C/preC, P, and X. The X gene encodes a 17-kd HBV X protein (HBx), which is a multifunctional transactivator of both viral and cellular genes. It is widely accepted that HBx plays crucial roles in the pathogenesis of HBV-induced HCC. HBV belongs to the family hepadnaviridae. It is a small, enveloped DNA virus that replicates via reverse transcription of an RNA intermediate. HBV virions, also called Dane particles, are spherical lipid-containing structures with a diameter of ~42 nm. The inner shell of the virus consists of an icosahedral capsid, which is assembled from 180 or 240 subunits of the core protein. The capsid is covered by a lipid bilayer membrane densely packed with the three envelope proteins, large (L), middle (M), and predominantly small (S) protein, and is acquired by budding into the endoplasmic reticulum. They are translated from individual start codons but share the open reading frame and the same C-terminal amino acids, called the S domain. As a consequence, the M protein shares the S and has an extra N-terminal domain called preS2, and the L protein encompasses the S and two extra domains: preS2 and preS1. Capsids contain a single copy of the HBV genome consisting of a 3.2-kb partially double-stranded relaxed circular (rc) DNA molecule. The viral polymerase serves as a protein primer and remains covalently linked to the 5’ end of the complete strand, also called viral (–) strand DNA of the rcDNA after reverse transcription. Besides virions, HBV infection leads to secretion of huge amounts of subviral particles, which consist of empty viral envelopes with filamentous or spherical shapes containing mainly S and little L protein. Subviral particles are the most abundant HBV structures released into the blood stream, are commonly defined as hepatitis B surface (HBs) antigen and are thought to facilitate virus spread and persistence in the host by adsorbing virus-neutralizing antibodies and tolerizing T cell responses. In addition to polymerase and the structural proteins, the HBV genome also encodes for two non-structural proteins, which have less well-defined functions.

Life cycle of HBV

HBV infection is restricted to hepatocytes. HBV entry into these cells is thought to be a multistep process. Virions are first trapped at the surface of the cell by heparan sulfate proteoglycans and then bind to a receptor allowing uptake into the cells via an endocytosis process. So far, this cellular receptor has not been identified. Proteolytic cleavage of the surface protein occurs within the endosomal compartment, probably resulting in a conformational change that exposes some translocation motifs at the surface of the viral particle allowing fusion of viral and cellular membranes and release of the capsid into the cytosol. The naked capsid is then directed towards the nucleus, and the HBV genome is translocated to the nucleus. In the nucleus, the rcDNA genome is converted by cellular enzymes into a covalently closed circular DNA (cccDNA), the episomal persistance form of the virus serving as transcription template. The 3.5 kb RNA species serves as pregenomic RNA (pgRNA) and as messenger RNAs for the synthesis of polymerase and core proteins as well as HBeAg. The 2.1 and 2.4 kb subgenomic RNAs encode for the three viral envelope proteins, a small 0.7 kb RNA for the HBx. The pgRNA is exported in an unspliced form, encapsidated together with the viral polymerase and used as a template for reverse transcription. The capsid spontaneously self-assembles from core dimers present in the cytoplasm due to the nucleic acid-binding domain of the core protein. Specific packaging of pgRNA into the capsid is mediated by binding of the primer region of the viral polymerase to the stem-loop in the 5’ region of pgRNA. The pgRNA is then reverse transcribed by the reverse transcriptase domain of the polymerase within the capsid in the cytoplasm of the infected cell. Upon minus and then plus strand DNA synthesis the capsid matures and can be enveloped or reimported into the nucleus to fill up a cccDNA pool.

General features and functions of HBVx

HBx is translated from a small subgenomic RNA controlled by the HBx promoter. Alternatively, HBx may be produced form a very long RNA (3.9 kb) containing all the HBV open reading frames (ORF). The ORF was originally designated X because of the lack of homology with known sequences. HBx is a protein composed of 154 amino acid residues with a molecular mass of around 17.5 kDa.

HBx selectively promotes degradation of Smc5/6 via an E3-ubiquitin ligase pathwaw by hijacking the DDB1–E3 ligase to target Smc5/6 for degradation. Smc5/6 is a complex that directly binds DNA and is required for chromosome dynamics and stability. Smc5/6 play a role in homologous recombination as well as in resolving replication-induced DNA supercoiling. In addition, a recent study demonstrated that Smc5/6 binds and topologically entraps plasmid DNA in an ATP-dependent manner. Smc5/6 binds episomes (including cccDNA) and blocks episome transcription.

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 Tang H, Oishi N, Kaneko S, Murakami S. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci 2006;97: 977–83.

Motavaf M, Safari S, Saffari JM, Alavian SM. Hepatitis B virus-induced hepatocellular carcinoma: the role of the virus x protein. Acta Virol 2013;57:389–96

Bertoletti, A. & Gehring, A. J. (2006). The immune response during hepatitis B virus infection. J Gen Virol 87, 1439-1449.

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Chen, M. T., Billaud, J. N., Sallberg, M., Guidotti, L. G., Chisari, F. V., Jones, J., Hughes, J. & Milich, D. R. (2004). A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc Natl Acad Sci U S A 101, 14913-14918.

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BIOMET RESOURCES: Eukaryotic Translation Initiation Factor 3b is bot...

BIOMET RESOURCES: Eukaryotic Translation Initiation Factor 3b is bot...: Eukaryotic Translation Initiation Factor 3b is both a Promising Prognostic Biomarker and a Potential Therapeutic Target for Patients with...

FLAVONOIDS AND THEIR HEALTH BENEFITS

FLAVONOIDS AND THEIR HEALTH BENEFITS

 Flavonoids are low molecular weight bioactive polyphenols which play a vital role in photosynthesising cells. They are a large family of over 5,000 hydroxylated polyphenolic compounds that carry out important functions in plants. The original "flavonoid" research apparently began in 1936, when Hungarian scientist Albert Szent-Gyorgi was uncovering a synergy between pure vitamin C and as yet unidentified co-factors from the peels of lemons, which he first called "citrin," and, later, "vitamin P". Flavonoids are secondary metabolites characterised by flavan nucleus and C6-C8-C6 carbon-skeleton. These are group of structurally related compounds with a chromane-type skeleton having phenyl substituent in C2-C3 position. The basic structural feature of flavonoid is 2-phenyl-benzo-γ-pyrane nucleus consisting of two benzene rings (A and B) linked through a heterocyclic pyran ring (C) as shown in fig (I). Flavonoids are one of the largest groups of secondary metabolites and widely distributed in leaves, seeds, bark and flowers of plants with more than 4000 different structures which are classified according to their chemical structures as follows; flavones, flavonols, flavanones, dihydroflavonols, isoflavones, anthocyanins, catechins and calchones. Flavonoids which are part of human diet are thought to have positive effects on human health such as reducing risk of cardiovascular diseases and cancer. Most of the beneficial effects of flavonoids are attributed to their antioxidant and chelating abilities. Flavonoids are structurally related compounds with a chromane-type skeleton with a phenyl substituent in the C2 or C3 position . They are consisting of phenylpropane (C6-C3) unit derived from shikimic acid pathway and C6 unit derived from polyketide pathway biosynthetically.

Chemical structure of Flavonoids

 

Figure 1: diphenylpropane, a basic structure of flavonoids

 

Chemically flavonoids are based upon a fifteen-carbon skeleton consisting of two benzene rings (A and B as shown in Figure 1) linked via a heterocyclic pyrane ring (C). They can be divided into a variety of classes such as flavones (e.g., flavone, apigenin, and luteolin), flavonols (e.g.,

quercetin, kaempferol, myricetin, and fisetin), flavanones (e.g., flavanone, hesperetin, and naringenin), and others. Their general structures are shown in Table 1. The various classes of flavonoidsdiffer in the level of oxidationandpattern of substitution of the C ring, while individual compounds within a class differ in the pattern of substitution of the A and B rings.

Flavonoids occur as aglycones, glycosides, and methylated derivatives. The basic flavonoid structure is aglycone (Figure 1). Six-member ring condensed with the benzene ring is either a 𝛼-pyrone (flavonols and flavanones) or its dihydroderivative (flavonols and flavanones). The position of the benzenoid substituent divides the flavonoid class into flavonoids (2-position) and isoflavonoids (3-position). Flavonols differ from flavanones by hydroxyl group at the 3- position and a C2–C3 double bond. Flavonoids are often hydroxylatedinpositions 3, 5, 7, 2, 3󸀠, 4󸀠, and5󸀠.Methyl ethers and acetyl esters of the alcohol group are known to occur in nature. When glycosides are formed, the glycosidic linkage is normally located in positions 3 or 7 and the carbohydrate can be L-rhamnose, D-glucose, glucorhamnose, galactose, or arabinose.

 

Classes of flavonoids

Flavonoids are classified into eight classes based on differences in their arrangement of hydroxyl, methoxy and glycosidic side groups and in the conjuction between A and B rings. A variation in C ring provides division of subclasses. According to their molecular structure, they are divided into eight classes, namely: Flavone, Flavonones, Flavonol, Isoflavone , Anthocyanidin, Catechin, Dihydroflavonol and Chalcone.

Metabolism of flavonoids

The absorption of the dietary flavonoids liberated from the food by chewing will depend on its physicochemical properties such as molecular size, configuration, lipophilicity, solubility, and pKa. The flavonoid can be absorbed from the small intestine or has to go to the colon before absorption. It may depend upon structure of flavonoid, that is, whether it is glycoside or aglycone. Most flavonoids, except for the subclass of catechins, are present in plants bound to sugars as b-glycosides. Aglycans can be easily absorbed by the small intestine, while flavonoid glycosides have to be converted into aglycan form. The hydrophilic flavonoid glucoside such as quercetin are transported across the small intestine by the intestinal

Na+-dependent glucose cotransporter (SGLT1). An alternative mechanism suggests that flavonoid glucosides are hydrolyzed by lactase phloridzin hydrolase (LPH), a 𝛽-glucosidase on the outside of the brush bordermembrane of the small intestine. Subsequently, the liberated aglycone can be absorbed across the small intestine. The substrate specificity of this LPH enzyme varies significantly in a broad range of glycosides (glucosides, galactosides, arabinosides, xylosides, and rhamnosides) of flavonoids. The glycosides which are not substrates for these enzymes are transported toward the colon where bacteria have ability to hydrolyze flavonoid glycosides, but simultaneously they will also degrade the liberated flavonoid aglycones . Since absorption capacity of the colon is far less than that of the small intestine, only trivial absorption of these glycosides is to be expected. After absorption, the flavonoids are conjugated in the liver by glucuronidation, sulfation, or methylation or metabolized to smaller phenolic compounds. Due to these conjugation reactions, no free flavonoid aglycones can be found in plasma or urine, except for catechins. Depending on the food source bioavailability of certain flavonoids differs markedly; for example, the absorption of quercetinfrom onions is fourfold greater than that from apple or tea. The flavonoids secreted with bile in intestine and those that cannot be absorbed fromthe small intestine are degraded in the colon by intestinal microflora which also break down the flavonoid ring structure (Figure 3). Oligomeric flavonoids may be hydrolyzed tomonomers and dimers under influence of acidic conditions in the stomach. Larger molecules reach the colon where they are degraded by bacteria. The sugar moiety of flavonoid glycosides is an important determinant of their bioavailability. Dimerization has been shown to reduce bioavailability. Among all the subclasses of flavonoids, isoflavones exhibit the highest bioavailability. After ingestion of green tea, flavonoid content is absorbed rapidly as shown by their elevated levels in plasma and urine. They enter the systemic circulation soon after ingestion and cause a significant increase in plasma antioxidant status.

Health benefits of Flavonoids

Flavonoids act as antioxidants by suppressing reactive oxygen specie (ROS) formation either by inhibition of enzymes or by chelating trace elements involved in free radical generation. Flavonoids inhibits the enzymes involved in ROS generation, that is, microsomal monooxigenase, glutathione s-transferase, mitochondrial succinoxidase, NADH oxidase etc. they also inhibits lipid peroxidation. Other health benefits of flavonoids include, anti-inflammation, antibacterial, anticancer antiviral.

Eukaryotic Translation Initiation Factor 3b is both a Promising Prognostic Biomarker and a Potential Therapeutic Target for Patients with Clear Cell Renal Cell Carcinoma

Eukaryotic Translation Initiation Factor 3b is both a Promising Prognostic Biomarker and a Potential Therapeutic Target for Patients with Clear Cell Renal Cell Carcinoma

Eukaryotic initiation factor 3b (el3b) is an RNA-binding component of the eukaryotic translation initiation factor 3 (eif-3) complex, which is required for several steps in the initiation of protein synthesis. The elf-3 complex associates with the 40s ribosome and facilitates the recruitment of elf-1, elf-1A, elf-2:GTP:methionyl-tRNAi and elf-5 to form the 43s pre-initiation complex (43S PIC). The elf-3 complex is also required for disassembly and recycling of post-termination ribosomal complexes and subsequently prevents premature joining of the 40S and 60S ribosomal subunits prior to initiation. The elf-3 complex specifically targets and initiates translation of a subset of mRNAs involved in cell proliferation, including cell cycling, differentiation and apoptosis, and uses different modes of RNA stem-loop binding to exert either translational activation or repression. Studies showed that cell proliferation was significantly inhibited after eIF3b Knockdown. Additionally, cell colony numbers fell after eIF3b depletion. Furthermore, migration capacity was significantly impaired after eIF3b knockdown. EIF3b depletion reduced the number of cells traversing the membrane. Therefore, eIF3b depletion impaired both cell migration and invasion. Notably, eIF3b depletion caused the cells to become smaller and rounded, suggesting that both migration and adhesion were impaired compared with control cells. In a study conducted to explore the effects of eIF3b depletion on the cell cycle, the proportion of cells in S-phase was lower in eIF3b-depleted cells and the proportion in the G1 phase was higher  than that in the negative control. Western blotting showed that the G1/S arrest was caused by eIF3b depletion. During the G1/S transition, cyclins D and E combine with cyclin-dependent kinases (CDK) to form the cyclin/CDK complexes required for the transition. The complexes phosphorylate the retinoblastoma protein (Rb), releasing the E2F transcription factor that activates expression of G1/S progression genes. It was found that the levels of both cyclins D and E decreased after eIF3b knockdown. Cyclins D and E, Rb, and the inactivated form of Rb (p-Rb) were downregulated after eIF3b knockdown. In addition, the levels of p27Kip1 and p21 Cip1, inhibitors of the cyclin/CDK complexes, significantly increased after eIF3b knockdown. Interestingly, Western blotting of G2/M-related proteins indicated that the G2/M transition was also inhibited after eIF3b knockdown Cyclin A, which accumulates steadily during the G2 phase and is abruptly destroyed at mitosis, was upregulated after eIF3b depletion. Cyclin B is required for the G2/M transition; we found that the cyclin B level fell. Also, the levels of Myt1 and Wee1 increased after eIF3b depletion; these proteins inhibit cell entry into mitosis. The observed decrease in histone H3 phosphorylation indicated that chromosome condensation was reduced by eIF3b depletion; fewer cells were in the mitotic phase. The levels of apoptosis increased slightly after eIF3b depletion. Further analysis showed that, after eIF3b knockdown, the pro-apoptotic factors Bax, caspase-3, and the activated form thereof (cleaved caspase-3) were all upregulated and the pro-survival factor Bcl-2 was downregulated. More importantly, cleavage of poly-ADP-ribosepolymerase (c-PARP), a marker of apoptosis, was also increased. Therefore, apoptosis increased after eIF3b knockdown. However, interestingly, knockdown reduced the level of cleaved caspase-12, which is required for endoplasmic reticulum-stress-induced apoptosis. The EMT is a key event in tumor invasion and metastasis, including RCC. It was found that the epithelial marker E-cadherin was upregulated and the levels of repressors thereof (Slug and Snail) were downregulated after eIF3b depletion. Additionally, the mesenchymal markers N-cadherin and vimentin were downregulated. Therefore, the EMT was inhibited by eIF3b depletion (Figure 5A). The β-catenin pathway is involved in regulation of the EMT. It was found that β-catenin expression was significantly downregulated after eIF3b depletion. The level of cyclin D1, a target of β-catenin, was also downregulated after eIF3b depletion. Together, our data showed that eIF3b depletion inactivated the β-catenin pathway and inhibited the EMT of ccRCC. The serine/threonine kinase Akt (also termed protein kinase B or PKB) has attracted a great deal of attention because Akt plays critical roles in the regulation of many cellular functions including metabolism, growth, proliferation, survival, transcription, and protein synthesis. Studies found that eIF3b depletion was not associated with any significant change in Akt levels; however, the level of the activated form, p-Akt, fell in parallel with the extent of eIF3b depletion, indicating that the Akt signaling pathway was involved in such depletion. The significant morphological changes in cells after eIF3b depletion suggested that the integrin pathway might be affected; integrin links the extracellular matrix to the intracellular cytoskeleton to facilitate focal adhesion. After eIF3b knockdown, studies showed that the levels of integrins α2 and α5 fell significantly, as did the level of the upstream phosphorylated Focal adhesion kinase (p-FAK) protein, suggesting that integrin/FAK/Akt signaling was inhibited Akt plays a critical role in cell growth by directly phosphorylating the mechanistic target of rapamycin (mTOR). Studies has shown that p-mTOR was downregulated after eIF3b knockdown, indicating that the Akt/mTOR pathway was impaired. Furthermore, the observed downregulation of HIF-1α, HIF-2α, and p-NF-κB after eIF3b depletion may indicate that the Akt/mTOR/HIF and Akt/mTOR/NF-κB pathways were also downregulated, compromising cell proliferation and inducing apoptosis. Akt promotes cell survival by inhibiting apoptosis via phosphorylation (inactivation) of several proteins, including Bcl-2 and Bax. Apart from the roles played in survival and apoptosis, the Akt pathway is also involved in cell cycle regulation, preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 and negatively regulating the actions of p27 Kip1  and p21 Waf1/Cip1. Research showed that GSk-3β and cyclin D1 were upregulated and p27 Kip1 and p21. Cip1 were downregulated after eIF3b depletion indicating that the Akt/GSK-3β pathway was involved in cell cycle regulation of A498 and CAKI-2 cells.

Clear cell renal cell carcinoma (CCRCC) is a renal cortical tumor typically characterized by malignant epithelial cells with clear cytoplasm and a compactalveolar (nested) or acinar growth pattern interspersed with intricate, arborizing vasculature. A variable proportion of cells with granulareosinophilic cytoplasm may be present. CCRCC is characterized genetically by alterations to chromosome 3p. CCRCC is proposed to arise from epithelial cellsof the proximal convoluted tubules of the nephron, within the renal cortex. Extension into the renal sinus is the most common pathway of spread for most histologic types of RCC because no connective tissue separates thecortical columns of Bertin from the abundant lymphatics and vasculature within the sinus fat. Elf-3b will therefore be a good target for pharmacological response against CCRCC.

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