Difference between revisions of "Part:BBa K3848000"

Revision as of 20:22, 20 October 2021

TRAIL-Smac Fusion Protein with HlyA signal peptide

Fusion protein which can specifically induce apoptosis and kills Cancer cells. This fusion protein has N terminal 7 amino acid residues of Smac/DIABLO (small-mitochondria associated activator of Caspase) and the soluble portion of TRAIL (TNF related Apoptosis Inducing Ligand).

The smac-n7 is given a Cell Penetrating Peptide (CPP) consisting of 8 Arginine residues for efficient membrane penetration into mammalian cells .A protease cleavage site recognized by Matrix Metalloproteinases (MMP-3 and MMP-9) and Urokinase (uPa) will cleave the Smac portion once the FP reaches cancer cells .It is also linked with HlyA signal peptide (C terminal signal peptide) that allows secretion by Escherichia coli . A 6X-His Tag is given for efficient purification of the protein.

Figure 1:Schematic of TRAIL-Smac Fusion Protein with HlyA signal peptide.

Usage and Biology

TRAIL is a tumor specific apoptosis inducing ligand that binds to its receptors DR-4 and DR-5 which are overexpressed in cancer cells, and activate the extrinsic pathway of apoptosis through Caspase activation. Smac/DIABLO is a protein released from Mitochondria when apoptotic signals arrive. The Smac inhibits XIAP (X-linked Inhibitor of Apoptosis Protein) and promotes caspase- mediated apoptosis via the intrinsic pathway of apoptosis [2].

Figure 2:Mechanism of Apoptosis after our chassis organism secretes out the TRAIL-Smac FP at the tumor site -Extrinsic pathway (TRAIL) & Intrinsic pathway (Smac/DIABLO) of Apoptosis.

The fusion construct consists of a soluble portion of TRAIL (95-281) and N Terminal 7 amino acids of Smac which are the crucial region of both the proteins for inducing apoptosis in cancer cells. It has been observed that some cancer cells gain resistance against TRAIL by overexpressing IAP proteins like XIAP , that block the Caspase pathway of apoptosis. But as Smac/DIABLO can inhibit XIAP , combining both TRAIL and Smac in this fusion construct we have a potent cytotoxic drug that can efficiently induce apoptosis and kill the cancer cells .

Once the fusion protein reaches cancer cells, it will be cleaved by Metalloproteinases (MMP-3 & MMP-9) or Urokinase (uPA), which are proteasese overexpressed in most cancer cells. The TRAIL portion binds to the DR-4 , DR-5 receptors, while the Smac-n7 along with the octa-arginine CPP , internalize into the cancer cells.

Figure 3:Schematic of fusion peptide cleavage by proteases,TRAIL binding & Smac n7+CPP internalization. Picture courtesy: Maciej Masłyk et.al [1]

This FP was tested against a panel of cancer cells (including lung, colorectal, pancreatic, liver, kidney and uterine) and showed a potent cytotoxic effect, and it was well tolerated by animals and significantly reduced the rate of the tumor growth in colon and lung adenocarcinoma animal models [1]

References

[1] Jerzy S. Pieczykolan , Konrad Kubiński , Maciej Masłyk et.al; “AD-O53.2—a novel recombinant fusion protein combining the activities of TRAIL/Apo2L and Smac/Diablo, overcomes resistance of human cancer cells to TRAIL/Apo2L”; Springer, 04 September 2014; doi :10.1007/s10637-014-0153-y

[2] Tamer E Fandy1, Sharmila Shankar2 ,Rakesh K Srivastava et al; “Smac/DIABLO enhances the therapeutic potential of chemotherapeutic drugs and irradiation, and sensitizes TRAIL-resistant breast cancer cells”; Molecular Cancer; 2008 ; doi: 10.1186/1476-4598-7-60

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 655
Illegal BamHI site found at 661
Illegal XhoI site found at 197
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 775
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 796

@@ Line 30: / Line 30: @@
-Once the fusion protein reaches cancer cells, it will be cleaved by Metalloproteinases (MMP-3 & MMP-9)or Urokinase (uPA), which are proteasese overexpressed in most cancer cells. The TRAIL portion binds to the DR-4 , DR-5 receptors, while the Smac-n7 along with the octa-arginine CPP , internalize into the cancer cells.
+Once the fusion protein reaches cancer cells, it will be cleaved by Metalloproteinases (MMP-3 & MMP-9) or Urokinase (uPA), which are proteasese overexpressed in most cancer cells. The TRAIL portion binds to the DR-4 , DR-5 receptors, while the Smac-n7 along with the octa-arginine CPP , internalize into the cancer cells.
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