Difference between revisions of "Part:BBa K3170998"

Latest revision as of 04:23, 21 October 2019

casp3(with a signal peptide)

Cysteine-aspartic protease，plays a central role in the execution-phase of cell apoptosis,

• Apoptosis is the natural death of genetically controlled cells. It is now widely accepted that apoptosis is the result of a series of highly regulated caspase cascade events, and caspase-3 (also known as CPP32, apopain) is confirmed to be downstream of this cascade, which passes Degradation of the corresponding substrate in the cell causes cell death, and other downstream caspase members may be the true performers of the apoptotic event. The caspase-3 gene (CASP-3) was originally cloned in human Jurkat-T lymphocytes, which encodes a 32 kD cysteine protease CPP32. CPP32 and its p20 and p11 recombinants induce sf9 insect cell development. Apoptosis. Later, Nicholson et al reported that apoptin from CPP32 zymogen can cleave poly[ADP-ribose] polymerase (PARP) in mammalian cells, leading to apoptosis. The dysfunction consists of two large and small subunits of mass 17kD and 12kD, and is later named caspase-3. Caspase-3 has both the commonality of the caspase family and its own characteristics. Caspase-3 is synthesized in its zymogen form. The zymogen has a prodo-main at the N-terminus. Compared with the initial caspase such as caspase-8 and 9, the caspase-3 and other downstream caspase anterior regions are shorter. There is no death effec-tor domain (DED), which must be activated after the initial caspase is activated. There is a safety-catch regulatory tripeptide in the caspase-3 zymogen, which consists of "Asp-Asp-Asp", located in the deformable loop region of the junction of large and small subunits. The tripeptide residue is acidified and cleaved in the cytosol and plays an important role in the trigger activation of caspase-3, and thus it may be a key regulatory point in the entire apoptotic cascade. When caspase-3 is activated, it is cleaved at the Asp(p1)-x(p1') position to form large and small subunits (α and β), and the N-terminal prodomain is removed, and α and β are combined to form a heterodimer. (α β), the two active sites of the enzyme are located at the junction of the large and small subunits, and the heterodimer will further function by forming a quaternary structure (α β) 2 through the secondary bond. When the caspase zymogen is cleaved or the enzyme exerts its action, its recognition sequence requires a minimum of 4 amino acids from the N-terminus to the cleavage site, and the P1 site must be Asp(D). When caspase-3 exerts its action, it is cleaved after DExD (x represents any amino acid), as the substrate PARP is cleaved by caspase-3 at DEVD↑G. The internal cleavage site of caspase protease is consistent with the activation or activation of other caspase by the zymogen itself and amplification of the caspase cascade. Caspase-3 includes a QACXG motif containing a cysteine residue at the active site and a SHG motif containing a histidine residue, both of which are located in the large subunit, and the imidazole group of His is in Cys The polarization activation of the side chain plays an important role. Arginine residues (Arg341 and Arg179) are located at the interface between the large and small subunits, which form the S1 subsite and bind to the aspartic acid at the P1 site. The three-dimensional structure of caspse-3 reveals that the large and small subunits participate in the binding cavity (S4-S1) that constitutes its specificity. The S4 subsite is the most important specific determinant and consists of only small subunits.

Experimental Results

• Fig.F is a photo of DNA electrophoresis. Apoptosis caused by activation of casp3, which is randomly cleaved between nucleosomes and degraded into 200 bp or an integral multiple thereof. Since the integrity of the nuclear membrane is maintained during DNA extraction, there is almost no DNA in the extract of normal cells, and DNA in apoptotic bodies of apoptotic cells
• The extracted DNA molecules with a length difference of 200 bp or an integral multiple thereof exhibit a ladder-like strip.

• Different cells can produce different degrees of apoptosis under different conditions. Under the fluorescence microscope, the normal cells stained with Hochest33258 showed a uniform blue color, while the apoptotic cells showed dense dense stained particles with a cluster-like distribution.

• A: blank control, under high oxygen concentration, sw1990 cell status is normal cells
• B: blank control, under low oxygen concentration, sw1990 cell status is normal cells
• C: Negative control, at high oxygen concentration, after binding to empty exosomes, the sw1990 cell status is normal cells.
• D: Negative control, at low oxygen concentration, after binding to empty exosomes, the sw1990 cell status is normal cells.
• E: In the experimental group, after high oxygen concentration, after binding to the exosomes after transformation, the state of sw1990 cells was more early apoptosis.
• F: In the experimental group, after the combined oxygen concentration in the medium, the sw1990 cell status was more early apoptosis.
• G: In the experimental group, after low-oxygen concentration, after binding to the exosomes after transformation, the sw1990 cell status was more late apoptosis.

The experimental results show that activation of the Casp3 pathway can effectively kill pancreatic cancer cells.

Usage and Biology

Sequence and Features