PI3K Pathway

Phosphoinositide 3-Kinase enzyme

It is a signal transduction system that connects oncogenes and multiple receptor classes to many cellular functions such as cell survival, proliferation and differentiation. The key enzyme family in this pathway is PI3Ks (Phosphoinositide 3-Kinases) which transducer signals from various growth factors and cytokines into intracellular messages by generating phospholipids. These phospholipids activate the serine- threonine protein kinase AKT (also known as protein kinase B (PKB)) and other downstream effector pathways.

PIP2 (PtdIns (4,5) P₂) à PIP3 (PtdsIns (3,4,5) P₃)

The enzyme family is classified into three classes based on the structural characteristics and substrate specificity:

Class I enzyme: à These enzymes are activated directly by the cell surface receptors. They are further classified into two sub classes. i.e. class IA and class IB.

Class IA enzyme à they are heterodimers consisting of a P110 catalytic subunit and p85 regulatory subunit.

            p85 regulatory subunit mediates receptor binding, activation and localization of enzyme.

This subunit directly interacts with tyrosine phosphate motif of activated receptor (eg: platelet growth factor receptor) or to adapter proteins associated with receptor (Eg: IRS1)

Activated P110 catalytic subunit generates phosphoinositide 3,4,5 triphosphate which further activates multiple downstream signalling pathway.

Class IB enzyme à It’s also a heterodimer with p110γ catalytic subunit and p101 regulatory subunit. Apart from this they also contain some adapter proteins such as p84, p87.

P110γ subunit is activated by GPCRs through interaction of regulatory subunit with Gβγ subunit of trimeric G proteins. P110γ is expressed in leukocytes but also found in heart, pancreases, liver, and skeletal muscles.

Class II enzyme: à

It consists of only a single catalytic subunit. Phosphatidylinositol 4-phosphate (PtdsIns4P) is used as the substrate.

It is found in three isoforms: a) PI3KC2α b) PI3KC2β c) PI3KC2γ

And these can be activated by receptor tyrosine kinases, cytokine receptors, and integrins.

But still their specific role in cellular function remains unclear.

Class III enzyme: à

 It consists of a single catalytic subunit VPS34 (Homologue of the yeast vacuolar protein sorting associated protein 34). It’s also known as PIK3C3 which only producesPtdIns3P, which is an important regulator of membrane trafficking.

The subunit function as a nutrient regulated lipid kinase that mediates signalling throught mTOR (mammalian target of rapamycin).

The negative regulator of this PI3K pathway is a tumor suppressor protein called PTEN (Phosphotase and Tensin homologue). PIP3 (PtdsIns (3,4,5) P₃) is the key second messenger that drives several downstream signalling cascades that regulate cellular processes. The cellular levels of PIP3 are tightly regulated by the opposing activity of PTEN. PTEN functionally antagonizes PI3K activity through its intrinsic lipid phosphatase activity that reduces the cellular pool of PIP3 by converting PIP3 back to PtdIns (4,5) P₂.

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How Selenocysteine is incorporated into polypeptide chain during translation?

1. No free pool of Selenocysteine exists in the cell.


2. Its high reactivity would incur damage to cells. Instead, cells store selenium in the less reactive selenide form (H₂Se).


3. Selenocysteine synthesis occurs on a specialized tRNA, which also functions to incorporate it into nascent polypeptides.


4. The primary and secondary structure of Selenocysteine tRNA, tRNA(Sec), differ from those of standard tRNAs in several respects, most notably in having an 8-base (bacteria) or 10-base (eukaryotes) pair acceptor stem, a long variable region arm, and substitutions at several well-conserved base positions.


5. The Selenocysteine tRNAs are initially charged with serine by seryl-tRNA ligase, but the resulting Ser-tRNA(Sec) is not used for translation because it is not recognized by the normal translation factor (EF-Tu in bacteria, eEF1A in eukaryotes). Rather, the tRNA-bound seryl residue is converted to a selenocysteine-residue by the pyridoxal phosphate-containing enzyme selenocysteine synthase.


6. Finally, the resulting Sec-tRNA(Sec) is specifically bound to an alternative translational elongation factor (SelB or mSelB (a.k.a. eEFSec)), which delivers it in a targeted manner to the ribosomes translating mRNAs for selenoproteins.


7. The specificity of this delivery mechanism is brought about by the presence of an extra protein domain (in bacteria, SelB) or an extra subunit (SBP2 for eukaryotic mSelB/eEFSec) which bind to the corresponding RNA secondary structures formed by the SECIS elements in selenoprotein mRNAs.

There are more than 20 human proteins that contain Selenocysteine.

where does Selenocysteine comes from?

Selenocysteine is not coded for directly in the genetic code. Instead, it is encoded in a special way by a UGA codon, which is normally a stop codon. When cells are grown in the absence of selenium, translation of selenoproteins terminates at the UGA codon, resulting in a truncated, nonfunctional enzyme.

The UGA codon is made to encode Selenocysteine by the presence of a SECIS element (Selenocysteine Insertion Sequence) in the mRNA. The SECIS element is defined by characteristic nucleotide sequences and secondary structure base-pairing patterns. In bacteria, the SECIS element is located immediately following the UGA codon within the reading frame for the selenoproteins. In archaea and in eukaryotes, the SECIS element is in the 3' untranslated region (3' UTR) of the mRNA, and can direct multiple UGA codons to encode Selenocysteine residues.

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Inhibitors of DNA Replication in Eukaryotic cells

DOXORUBICIN

The exact mechanism of action of doxorubicin is complex and still somewhat unclear, though it is thought to interact with DNA by intercalation. Doxorubicin is known to interact with DNA by intercalation and inhibition of macromolecular biosynthesis. This inhibits the progression of the enzyme topoisomerase II, which relaxes supercoils in DNA for transcription. Doxorubicin stabilizes the topoisomerase II complex after it has broken the DNA chain for replication, preventing the DNA double helix from being resealed and thereby stopping the process of replication.

The planar aromatic chromophore portion of the molecule intercalates between two base pairs of the DNA, while the six-membered daunosamine sugar sits in the minor groove and interacts with flanking base pairs immediately adjacent to the intercalation site, as evidenced by several crystal structures.

Intercalation occurs when ligands of an appropriate size and chemical nature fit themselves in between base pairs of DNA.

ETOPOSIDE

Etoposide phosphate (brand names: Eposin, Etopophos, Vepesid, VP-16) is an anti-cancer agent. It inhibits the enzyme topoisomerase II, which unwinds DNA, and by doing so causes DNA strands to break.

Etoposide forms a ternary complex with DNA and the topoisomerase II enzyme, preventing re-ligation of the DNA strands. This causes errors in DNA synthesis and promotes apoptosis of the cancer cell.

CAMPTOTHECIN

Camptothecin (CPT)cytotoxic quinoline alkaloid which inhibits the DNA enzyme topoisomerase I. CPT binds to the topo I and DNA complex (the covalent complex) resulting in a ternary complex, and thereby stabilizing it. This prevents DNA re-ligation and therefore causes DNA damage which results in apoptosis. CPT binds both to the enzyme and DNA with hydrogen bonds.

Toxicity of CPT is primarily a result of conversion of single-strand breaks into double-strand breaks during the S-phase when the replication fork collides with the cleavage complexes formed by DNA and CPT

RIFAMPICIN

Rifampicin inhibits DNA-dependent RNA polymerase in bacterial cells by binding its beta-subunit, thus preventing transcription to RNA and subsequent translation to proteins. Its lipophilic nature makes it a good candidate to treat the meningitis form of tuberculosis, which requires distribution to the central nervous system and penetration through the blood-brain barrier.

Rifampicin acts directly on messenger RNA synthesis. It inhibits only prokaryotic DNA-primed RNA polymerase, especially those that are Gram-stain-positive and Mycobacterium tuberculosis. Much of this acid-fast positive bacteria's membrane is mycolic acid complexed with peptidoglycan, which allows easy movement of the drug into the cell. Evidence shows that in vitro DNA treated with concentrations 5000 times higher than normal dosage remained unaffected; in vivo eukaryotic specimens' RNA and DNA polymerases suffered few problems as well. Rifampicin interacts with the β subunit of RNA polymerase when it is in an α2β trimer. This halts mRNA transcription, therefore preventing translation of polypeptides. It should be made clear, however, that it cannot stop the elongation of mRNA once binding to the template-strand of DNA has been initiated. The Rifampicin-RNA polymerase complex is extremely stable and yet experiments have shown that this is not due to any form of covalent linkage. It is hypothesized that hydrogen bonds and π-π bond interactions between naphthoquinone and the aromatic amino acids are the major stabilizers, though this requires the oxidation of naphthohydroquinone which is found most commonly in Rifampicin. It is this last hypothesis that explains the explosion of multi-drug-resistant bacteria: mutations in therpoB gene that replace phenylalanine, tryptophan, and tyrosine with non-aromatic amino acids result in poor bonding between Rifampicin and the RNA polymerase.

Well due to blocking in RNA transcription, DNA initiation is also not possible since the primer synthesis is also blocked by the drug.

APHIDICOLIN

Aphidicolin is defined as a tetracyclic diterpene antibiotic with antiviral and antimitotical properties. Aphidicolin is a reversible inhibitor of eukaryotic nuclear DNA replication. It blocks the cell cycle at early S phase. It is a specific inhibitor of DNA polymerase A, D in eukaryotic cells and in some viruses and an apoptosis inducer in HeLa cells.

NOVOBIOCIN

The molecular basis of action of novobiocin, and other related drugs clorobiocin and coumermycin A1 has been examined. Aminocoumarins are very potent inhibitors of bacterial DNA gyrase and work by targeting the GyrB subunit of the enzyme involved in energy transduction. Novobiocin as well as the other aminocoumarin antibiotics act as competitive inhibitors of the ATPase reaction catalysed by GyrB. The potency of novobiocin is considerably higher than that of the fluoroquinolones that also target DNA gyrase, but at a different site on the enzyme. The GyrA subunit is involved in the DNA nicking and ligation activity.

CIPROFLOXACIN

Ciprofloxacin is a broad-spectrum antibiotic active against both Gram-positive and Gram-negative bacteria. It functions by inhibiting DNA gyrase, a type II topoisomerase, and topoisomerase IV, enzymes necessary to separate bacterial DNA, thereby inhibiting cell division.

1. Actinomycin -binding between adjacent G-C bases in DNA (intercalation
2. chloramphenicol- inhibits peptidyltransferase of the 70S ribosome
3. erythromycin - binds to the 50S particle and arrests synthesis of the 70S ribosome
4. neomycin- binds to the 30S ribosomal subunits and inhibits binding of a tRNA
5. puromycin- premature chain termination
6. Rifamycin- inhibits RNA synthesis by binding to the β subunit of the RNA polymerase holoenzyme
7. streptomycin as erythromycin
8. tetracyclin -inhibits binding of tRNA to the 30S ribosomal subunit in eukaryotes
9. α-amanitin - inhibits polymerase II
10. chloramphenicol - inhibits peptidyltransferase of the mitochondrial ribosome
11. cycloheximide -inhibits peptidyltransferase
12. diptheria toxin - inhibits factor 2 and translocation  

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Telomere and Its enzyme

In eukaryotes, the linear chromosomes ends with a specific nucleoprotein structure called telomere.

In human, telomere consists of thousands of repeats of six base pair motif (5’-TTAGGG-3’) ending in a G rich 3’ overhang 30-300 nt long, associated with a complex of six proteins named shelterin.

The structure prevents chromosome ends from being recognized as DNA double strand breaks and therefore being processed by the DNA repair machinery.

End replication problem is the inability of DNA polymerase to completely replicate the linear genomes, in which cells undergo telomere shortening at every replication round.

The loss of terminal DNA is counteracted by the activity of telomerase enzyme, which adds de novo telomeric repeats to the G rich 30- overhang.

Telomerase is active only in germ line and in stem cells, but not in somatic cells where telomeres shorten till they reach a critical length that activates a DNA damage response (DDR) leading to replicative senescence or to apoptosis.

Thus, limiting the number of cell division this constitutes an important barrier against cancer proliferation, because it reduces the risk of accumulating harmful mutations that could lead to malignant transformation.   

Telomere erosion could result in high chromosomal instability and facilitates the generation of tumor promoting mutations, if cells escape p53 and Rb-dependent DNA damage checkpoints.

Telomeres are extremely dynamic structure and their organisation switch between a protected and a deprotected state throughout the cell cycle and cell differentiation, inorder to accomplish their multiple tasks.

During cell cycle, telomere structure has to change from a closed conformation concealing chromosome ends from repairing enzymes, to an open one in S phase inorder to allow controlled access to DNA replication factors.

In the telomerase negative cells, after reaching the critical short length of telomere, the uncap region will trigger the ATM/ATR signalling cascade, that eventually leads to a p53 dependent cell cycle arrest or apoptosis.

Capping of telomere requires the binding of specific proteins that recognize telomeric DNA and shields single stranded G overhangs by hiding them into specific structures.

T loops (Telomeric loop) are lasso like structures were the 3’ overhang folds back and invade the upstream telomeric region, generating a displacement loop (D-loop).

G – quadruplex are four stranded DNA structure, derived from folding of single stranded DNA containing runs of three to four consecutive guanines to form stacked tetrad  of Gs, stabilized by Hoogsteen hydrogen bonding and cation coordination.

Both these structures need RTEL1 helicase inorder to be resolved and allow an efficient telomere replication.

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