Team:BGU Israel/Parts1

Heat shock proteins (HSP) are a key part of the heat shock response in almost all organisms, from bacteria to humans. Their expression is induced drastically when cells expose to stress such as elevated temperature, heavy metals or starvation. The 70 kDa heat shock proteins (Hsp70s) are a family of conserved ubiquitously expressed heat shock proteins. Like many other HSPs, HSP70 is an important part of the cell's machinery of protein folding. Its role is to prevent the aggregation of partially synthesized peptides or damaged proteins.

The HSP70 promoter can be regulated by at list two distinct domains, a distal domain which contains sequences responsive to heat shock and cadmium stress, and proximal domain necessary for transcription stimulated by serum. The activity of the promoter can be induced by moderate hyperthermia (39°C to 43°C), metal induction or by addition of serum after starvation (Wu, Kingston, & Morimoto, 1986).

Using this promoter allows thermal induced expression in human cell lines.
This promoter’s sequence was taken from Invivogen pDRIVE-HSP70 (http://www.invivogen.com/pdrive-hsp70). Since it contained two forbidden restriction sites, we changed the second base from C to G and the 339th base from T to A.

References:

Wu, B., Kingston, R., & Morimoto, R. (1986). Human HSP70 promoter contains at least two distinct regulatory domains. Proceedings of the National Academy of Sciences of the United States of America, 83(February), 629–633. Retrieved from http://www.pnas.org/content/83/3/629.short

Brief introduction Of DsbA-L:

DsbA-L is a protein present in the ER (endoplasmic reticulum). Its main action is a Glutathione S-Transferase action on the hormone adiponectin. It has the ability to catalyze the conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for the purpose of detoxification, and it helps with binding two substrates with to s-s bonds and cellular detoxification.
DsbA-L is expressed mostly in adipose tissue, and its expression level is negatively correlated with the obesity of mice and humans.
DsbA-L has been shown to bind adiponectin and increase the production of the HMW species, for that it can become a novel therapeutic and industrial target.

Modularization of DsbA-L:

The DsbA-L sequence had iGEM Restriction sites that had to be change according to iGEM protocol.
In position 131 G was changed to A to avoid Pst1 Site.
In position 446 A was changed to T to avoid Pst1 Site.
In position 467 A was changed to T to avoid Pst1 Site.
In position 545 C was changed to G to avoid Pst1 Site.

All changes in nucleotides was done according to an amino acid chart to maintain the same amino acids at the protein sequence Level.

References:

1. A disulfide-bond A oxidoreductase-like protein (DsbA-L) regulates adiponectin multimerization, Meilian Liua, Lijun Zhoub.  18302–18307PNAS ,November 25, 2008 vol. 105  no. 47.
2. Fat-Specific DsbA-L Overexpression Promotes Adiponectin Multimerization and Protects Mice From Diet-Induced Obesity and Insulin Resistance. Meilian Liu, Ruihua Xiang, DIABETES, VOL. 61, NOVEMBER 2012.
3. Role of the Endoplasmic Reticulum Chaperone DsbA-L Glutathione S-Transferase Activity in the Assembly of Adipocyte Hormone Adiponectin . Dr Tsu-Shuen Taso, kartchner, lavrel, Brianne, The University of Arizona library release, may 2011.
4. Human GSTK1 on NCBI:
   http://www.ncbi.nlm.nih.gov/gene/373156

Brief introduction Of AdiopQ:

The AdipoQ gene which codes for the protein adiponectin has domains similar to collagens X and VIII and complement factor C1q. Adiponectin forms multimeric complexes that combine via its collagen domains to create three major oligomeric forms: a low molecular weight (LMW) trimer, a middle - molecular weight (MMW) hexamer, and high molecular weight (HMW) 12-18- mer Adiponectin.
Normally it is expressed exclusively in adipose tissues from which it is secreted to the blood stream. It directly sensitizes the body to insulin and has an important role in lipid and glucose metabolism regulation. Therefore adiponectin represents a potential therapeutic target to fighting obesity-linked diseases characterized by insulin resistance and the Metabolic Syndrom.

Furthermore low levels of serum Adiponectin is highly related with occurrence of abdominal obesity, insulin resistance, type 2 diabetes, and the metabolic syndrome

References:

1. Kadowaki, T., Yamauchi, T., Kubota, N., Hara, K., Ueki, K., & Tobe, K. (2006). Review series Adiponectin and adiponectin receptors in insulin resistance , diabetes , and the metabolic syndrome. J Clin Invest., 116(7), 1784–1792. doi:10.1172/JCI29126.1784
2. Human adiponectin on NCBI : http://www.ncbi.nlm.nih.gov/gene/9370

Brief introduction Of UCP1

A transmembrane protein naturally present in mitochondria of brown adipose tissue. The gene itself is found in the nuclear genome, and after translation it is sent to the mitochondria.

UCP1 increases the permeability of the inner mitochondrial membrane to protons, allowing them to move from the inner membrane space to the mitochondrial matrix, bypassing ATP synthase.This process weakens the coupling of the electron transport chain to the production of ATP. In an attempt to raise the pH gradient negatively affected by UCP1, a fast oxidation of substrates occurs while the synthesis rate of ATP stays low and oxidation energy is released as heat instead.

UCP1 is activated in the brown fat cell by fatty acids and is inhibited by nucleotides.

References:

1. Bugianesi, E., Moscatiello, S., Ciaravella, M.F. & Marchesini, G. (2010).  Insulin resistance in nonalcoholic fatty liver disease.  Curr Pharm Des 16: 1941-1951
2. Kozak, L.P. & Anunciado-Koza, R. (2008). UCP1: its involvement and utility in obesity. Int. J. Obes., 32 (Suppl. 7), pp. S32–S38
3. Wu, B.J., Kingston, R.E. & Morimoto, R.I. (1986). Human HSP70 promoter contains at least two distinct regulatory domains