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Team:UCL/Project/Degradation
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In order for MMP-9 to be expressed in mammalian chassis, we ligated CMV to our BBa_K1018001 biobrick. Below shows gel photo indicates our successfully ligated plasmid. | In order for MMP-9 to be expressed in mammalian chassis, we ligated CMV to our BBa_K1018001 biobrick. Below shows gel photo indicates our successfully ligated plasmid. | ||
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Revision as of 00:28, 5 October 2013
Matrix
Metalloproteinase-9
For β-amyloid Degradation
The pathological hallmark of Alzheimer's disease (AD) is the senile plaques primarily composed of tightly aggregated β-amyloid (Aβ) fibrils. Matrix metalloproteinase-9 (MMP-9) can degrade Aβ fibrils and soluble Aβ, an ability that is not shared by other soluble Aβ degrading enzymes, including endothelin-converting enzyme, insulin-degrading enzyme, and neprilysin (Yan et al. 2006), all of which we had been considering for our circuit. Therefore, using MMP-9 satisfies both those theories that hold fibrillar Aβ senile plaques responsible for AD. This also supports some of the newer ideas that amyloid plaque forms protectively to seal away more soluble Aβ. By expressing it in inactive microglia, we can engender plaque removal without neuroinflammation. To characterise MMP-9, we carried out a β-amyloid degradation assay.
MMP-9 is a Zinc II dependent endopeptidase that in humans is encoded by the MMP9 gene. MMP-9 is readily synthesised and secreted by microglia upon activation. The primary function of MMP-9 is to degrade proteins in the extracellular matrix. Physiologically, MMP-9 in coordination with other MMPs, play a role in normal tissue remodelling events such as neurite growth, embryonic development, angiogenesis, ovulation, mammary gland involution and wound healing. Initially synthesised as an inactive proenzyme, pro-MMP-9 is cleaved into an active form upon cellular release by other proteases. (Nagase & Woessner Jr., 1996) This property puts MMP-9 in a unique position to regulate extracellular Aβ levels. For ease of demonstration, we have our cells synthesise the already active version of the enzyme.
Constructing The BioBrick
We created a BioBrick containing the active form of Matrix Metallopeptidase-9 (BBa_K1018000)
We ligated CMV promoter with BBa_K1018001 to enable expression in mammalian cells. This biobrick could not be submitted to the registry in time.
In order for MMP-9 to be expressed in mammalian chassis, we ligated CMV to our BBa_K1018001 biobrick. Below shows gel photo indicates our successfully ligated plasmid.
7U - Uncut
7C - EcoRI single digest
7D - EcoRI and PstI double digest
Amyloid Degradation Assay
The 42-amino acid peptide (Aβ1-42), the predominant peptide length found in senile plaques, has a remarkable propensity to aggregate at high concentrations to form a β-pleated sheet structure. While plaques and amyloid fibrils have been viewed by some as resistant to proteolytic degradation, it is possible that certain proteases, such as MMP-9 may contribute to endogenous mechanisms leading to plaque clearance. Our assay, inspired by Yan et al., demonstrates our BioBrick's capability to do this.
EXPERIMENTS AND RESULTS
Protocol: Preparation
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.
Protocol: Amyloid Degradation Assay
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with (BBa_K1018000), as it contains the active form.
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.
c. After digestion, analyse the reaction by Congo red assay.
At the time of wiki freeze, the amyloid fibrils necessary for this assay had not yet arrived from SynoBiologicals despite being ordered a month prior. We expect to be able to present the results from our assay after wiki thaw.
