Team:Queens Canada/Contribution


Our
Contributions

We’ve made a number of contributions with this year’s project – not just to the past work of the iGEM community, but other communities including; protein engineering, microfluidics, and clinical nephrology.

BBa_K3260024, a remarkable part

BBa_K3260024, a part first characterized in 2019 by a joint team with Princeton, Stanford, and Brown, is Teriparatide – the 1-34 residue fragment of parathyroid hormone (PTH). While their aim was to use this fragment to treat osteoporosis, addition of our fluorophore and competition assay incorporating this PTH fragment may help clinicians monitor the level of PTH in the body, and would provide help with the treatment of osteoporosis, ensuring information guided dosing.

Methodology

How did we perform nanoscale molecular dynamic simulations?

All the simulations were performed on the software GROMACS 2020.2 with the NVIDA CUDA toolkit. The methodology employed to generate our models was adapted from Justin Lemkul’s GROMACS Tutorials.

Parathyroid hormone receptor competitive fluorescent assay. Protein structures were obtained from the RCSB Protein Data Bank. All protein residues are shown in a cartoon preset with the mNeonGreen, mCherry, parathyroid hormone receptor, and parathyroid hormone (1-34) as green, red, blue, and grey, respectively, using Chimera software. A. Parathyroid receptor hormone with mNeonGreen and parathyroid hormone with mCherry interaction. Fluorescent is expected to occur. B. Parathyroid hormone from the interstitial fluid enters the biosensor and competes with the parathyroid hormone bound to mCherry. Interaction with parathyroid hormone receptor with mNeonGreen. Quantifiable drop in fluorescent intensity expected.

Here’s the information we added to the part on the registry:

“Our group used a structural approach to measure the concentrations of PTH in the interstitial fluid. This may improve this teams part of providing 1-34 PTH for therapeutics. With a PTH protein based assay, clinicians may make better decisions regarding dosing of PTH and treatment. A recent study did assess the use of PTHR and FRET to measure the concentrations of PTH. This method did not go as far as to include the detection of PTH in a biosensor. Using the 60 amino acid N- terminal, extracellular domain, of PTHR to bind PTH, this would allow for easier protein expression. We added a mNeonGreen fluorophore to the N-termini of this extracellular domain. This domain had one cysteine and so adding our E/K coiled coil system that immobilizes the protein was not a large concern.
The issue at hand is rather detecting the concentration of PTH in the body. While PTH is difficult to express, a PTH fragment (residues 1-34) is easier to express (or obtain). This PTH fragment is attached to a mCherry fluorophore through its one lysine residues amino group. Allowing the PTH fragment with mCherry and PTHR with mNeonGreen to associate produces a FRET signal. When PTH from the body enters the biosensor, it displaces the PTH with mCherry and a drop in FRET signal is observed. You may consider this as a competition assay or similar to an antibody detection system.

” Part:BBa K3260024 - parts.igem.org [Internet]. [cited 2020 Jul 21]. Available from: http://parts.igem.org/Part:BBa_K3260024

The Protein Engineering Contribution: Mutaguide

Much of our protein construct work involved removing cysteines and ensuring one cysteine remained on the proteins surface, away from the fluorophores and active site. This is an intensive process involving protein homology, modelling, and ultimately user-based decisions. Should I mutate the cysteine in an active site? Do I add a cysteine to an intrinsically disordered region? (Aside: Don’t do either) What if there was a program that could pick your residues to replace cysteines and identify the ideal cysteine anchoring location? While locating ideal attachment point is still being developed, and requires some very intensive artificial intelligence, we’ve developed a program to find the ideal residue to replace a cysteine with.

By analyzing intensive sequence homology outputs, we can rank alternative residues based on their repetitiveness at distinct cysteine positions. Not only will this make choosing a cysteine replacement easier, but ensures the protein designer is considering homology, ultimately, we aim to protect the way our native protein folds – and functions.

A nephrology contribution: FGF23 and PTH competitive assay

In 2011/2012 in Ontario, Canada, 149,690 parathyroid hormone (PTH) tests were completed at a cost of about $62 each. A test for fibroblast growth factor 23 (FGF23) is tougher to come by. Samples must be shipped all over North America to MayoClinic for an antibody test at a cost unknown to us (unfortunately, we don’t have a MayoClinic account). Both of these tests are typically antibody based due to the concentration of peptide hormones in the body. However, after consultation with experts – in patients with CKD – concentrations of PTH and FGF23 are elevated several folds placing them in the range of FRET detection.

FGF23 is believed regulate phosphate and vitamin D metabolism, while evidence points to PTH regulating calcium and phosphate. Because of the complexity of mineral homeostasis, both FGF23 and PTH would be great biomarkers for CKD diagnostics as evidenced through our conversation with nephrologists.

Our FRET competition assay lets FGF23 or PTH in the body compete with a fluorophore tagged FGF23 or PTH bound to its native fluorophore tagged receptor. As FGF23 or PTH from the interstitial fluid enters the biosensor, a drop in fluorescent intensity is detected and levels of FGF23 or PTH is readily quantifiable.

Alpha-Klotho (FGF23 receptor) competitive fluorescent assay. Protein structures were obtained from the RCSB Protein Data Bank. All protein residues are shown in a cartoon preset with the mNeonGreen, mCherry, alpha-klotho ternary complex, and FGF23 as green, red, wheat, and grey, respectively, using Chimera software. A. Alpha-Klotho with mNeonGreen and FGF23with mCherry interaction. Fluorescent is expected to occur. B. FGF23 from the interstitial fluid enters the biosensor and competes with the FGF23bound to mCherry. Interaction with alpha-klotho with mNeonGreen. Quantifiable drop in fluorescent intensity expected.


Parathyroid Hormone - Health Quality Ontario (HQO) [Internet]. [cited 2020 Jul 21]. Available from: https://www.hqontario.ca/Evidence-to-Improve-Care/Health-Technology-Assessment/Reviews-And-Recommendations/Parathyroid-Hormone
FGF23 - Overview: Fibroblast Growth Factor 23, Plasma [Internet]. [cited 2020 Jul 21]. Available from: https://www.mayocliniclabs.com/test-catalog/Overview/88662
Smit MA, van Kinschot CMJ, van der Linden J, van Noord C, Kos S. Clinical Guidelines and PTH Measurement: Does Assay Generation Matter? Endocr Rev. 2019 01;40(6):1468–80.
Rodelo-Haad C, Santamaria R, Muñoz-Castañeda JR, Pendón-Ruiz de Mier MV, Martin-Malo A, Rodriguez M. FGF23, Biomarker or Target? Toxins. 2019 22;11(3).