Part:BBa_K2583002

ADH1_promoter-HRH4-ADH1_terminator

This part heterologously expresses Histamine Receptor H4 in yeast. HRH4 locates at the plasma membrane, binds to extracellular histamine, and couples to the endogenous pheromone sensing pathway in yeast Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2157
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 180
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 830
  Illegal SapI site found at 2307
  

Usage

In our project, this part is used to express functional human histamine receptor H4 (HRH4) under the trigger of a constitutive promoter—ADH1 promoter in yeast, so that yeast can use this receptor to bind extracellular histamine and activate its own pheromone pathway—a G-protein coupled signal transduction in yeast.

Biology

HRH4 is a G-protein coupled receptor (GPCR)from Homo sapiens. H4 receptor is primarily expressed in mast cells and eosinophils, but is also found on human neutrophils and basophils, and plays an important role in modulating eosinophil chemotaxis. H4 receptor acts in concert with H2 receptor to enhance IL-16 production in lymphocytes. In mast cells, selective H4 receptor activation increases calcium influx, degranulation, cytokine release and leukotriene production (cysteinyl and LTB4). In comparison to the H1 receptor, the H4 receptor is a relatively new therapeutic target for inflammation [3,8,39]. Selective H4 receptor antagonists are in development for potential clinical use, in particular for the treatment of Th2-dependent dermal inflammation such as atopic dermatitis (see for example H4 antagonist 48), and asthma. [1]

Characterization

The induce of modified yeast with histamine This part’s function is tested by our well modified yeast--successfully transformed the plasmid ADH1-HRH4-Ter (Part:BBa_K2583002) and pFUS1-EGFP (Part: BBa_K775004) into CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △ura3 Saccharomyces cerevisiae. The test of this part is using the histamine to induce the modified yeast, then activate the reporter expression. According to medical research, the histamine concentration in normal human blood is around 30 ng/ml to 60ng/ml, while in allergic people, this concentration would reach 100 ng/ml or even more. We set three group of histamine concentration: 0 ng/ml as control group, 50 ng/ml to simulate the normal concentration and 200 ng/ml to simulate allergic concentration. According to the test of reporter plasmid, we estimated the expression peak would be between 5 to 10 hours, so we choose 5h, 8h and 11h as three time points to run flow cytometry. One thing worth mentioning is that due to the low concentration of yeast, we could only set 10 group so as to meet the criterion concentration of FCS. The concentration of yeast is around 100000 cell/ml (measured by OD600). We pick 10000 events inside the gate for each sample during FCS. The FCS result is as showed in figure 2.

Fig 1. Design of the pathway in modified yeast Fig.2 The relative fluorescence intensity of histamine induced yeast

Reference

[1] Paul Chazot, Hiroyuki Fukui, C. Robin Ganellin, Helmut L. Haas, Stephen J. Hill, Rebecca Hills, Roberto Levi, Walter Schunack, Jean-Charles Schwartz, Nigel P. Shankley, Henk Timmerman, J. Michael Young.Histamine receptors: H4 receptor. Last modified on 04/04/2018. Accessed on 18/10/2018. IUPHAR/BPS Guide to PHARMACOLOGY, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=265.