Team:UCL/Project/Degradation

From 2013.igem.org

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<p class="major_title">OXIDATIVE STRESS PROMOTER</p>
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<p class="major_title">Matrix metalloproteinase-9</p>
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<p class="minor_title">For Plaque Specific Expression</p>
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<p class="minor_title">For β-amyloid Degradation</p>
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In order to direct microglia activity to <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank"> senile plaques </a>, we needed to find a way to detect these plaques. Several possible routes were explored; initial focus was on a plaque binding protein, such as the B10 antibody <a href="http://www.ncbi.nlm.nih.gov/pubmed/21059358" target="_blank">(Haupt et al. 2011)</a>. However, there was no easy way for plaque binding to transduce changes in gene expression. Therefore, alternatives were explored, where plaque proximity could be indirectly detected via a proxy. One such proxy is oxidative stress - free radical production <a href="http://www.ncbi.nlm.nih.gov/pubmed/10863548" target="_blank">(Colton et al., 2000)</a> which is generated by plaques. Microglia are naturally attracted to plaques, and upon reaching plaques, a standard immune response follows, which also includes free radical production. Therefore, we have designed a promoter which will initiate transcription in response to the oxidative stress generated by native microglia and plaques already present in the brain.
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The pathological hallmark of <a href="http://2013.igem.org/Team:UCL/Background/Alzheimers" target="_blank">Alzheimer's disease (AD)</a> is the <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank"> senile plaques </a> 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 <a href="http://www.ncbi.nlm.nih.gov/pubmed/16787929" target="_blank">(Yan et al. 2006)</a>. Therefore, using MMP-9 satisfies both those theories that hold fibrillar Aβ senile plaques responsible for AD and some of the newer ideas that plaque form 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.
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This promoter is an improvement of a yeast minimal promoter <a href="http://parts.igem.org/Part:BBa_K105027" target="_blank">cyc1001</a> already in the registry. Although from yeast, parts of this promoter show homology to the consensus sequences of mammalian core promoter elements, notably the TATA box and initiator element <a href="http://www.ncbi.nlm.nih.gov/pubmed/17486122" target="_blank">(Sandelin et al. 2007)</a>. NF-κB is a transcription factor which translocates to the nucleus under oxidative stress <a href="http://www.ncbi.nlm.nih.gov/pubmed/12730877" target="_blank">(Shi et al., 2003)</a>, and binds to the sequence GGGAATTT <a href="http://www.ncbi.nlm.nih.gov/pubmed/19435890" target="_blank">(Park et al., 2009)</a>. Thus, by placing this site upstream of the yeast minimal promoter, we created a novel mammalian promoter which initiates transcription in response to oxidative stress.
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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 <a href="http://www.jbc.org/content/274/31/21491.full?ijkey=875a77f6b9239459a92e85c272d0a2e6cd4170f7&keytype2=tf_ipsecsha" target="_blank">(Nagase & Woessner Jr., 1996)</a>; 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.
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To construct this, we firstly created a BioBrick which consists of 5 copies of the NF-κB binding site (5NFKB [link to parts page]). This was done using linkers - short overlapping primers were ordered, and allowed to anneal, and then ligated together.  
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<p class="minor_title">Creating The BioBrick</p>
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<p class="minor_title">Amyloid Degradation Assay</p>
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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 demonstrates our BioBrick's capability to do this.
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Revision as of 11:18, 7 September 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). Therefore, using MMP-9 satisfies both those theories that hold fibrillar Aβ senile plaques responsible for AD and some of the newer ideas that plaque form 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.

Creating The BioBrick

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 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. Analysis with Tris-Tricine gels indicated that the vast majority of Aβ in this preparation (referred to as sAβ) was in the monomer form.

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: Congo Red Assay

a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use.

b. For fAβ digestions (employing Congo red), 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.

Results.

Results.