Team:Tianjin/Modeling

Our modelling is based on the following signal transduction network of TOR protein. As we discussed before, the major part of signaling transduction is regulated by the rapamycin-sensitive TORC1 complex either via the Tap42-Sit4/PPA2c or the recently identified Sch9 branches. Nevertheless, Sch9 branch appears to have overlapping functions with cAMP-PKA pathway,and be additionally regulated by proteins not contained in central TOR pathway. Besides, as most of the downstream transription factors are definitely regulated to Tap42-Sit4/PPA2c, we decide to simplify the modelling part to this branch only.

1. Original object of modeling (based on mechanism) In normal yeast cell, TORC1 (Tor complex 1) with phosphorylated Tor2 is in a functional state, which is able to phosphorylate Tip41 and Tap42 which would bind together when dephosphorylated. [Tap42p]+[Tor1/2p] ←→21KK[Tap42p•Tor1/2p] →3K [Tap42p~p]+[Tor1/2p] [Tip41p]+[Tor1/2p] ←→1514KK [Tip41p•Tor1/2p] →3K [Tip41p~p]+[Tor1/2p] [Tip41p]+[Tap42p] ←→2120KK [Tip41p•Tap42p] PP2A1 and PP2A2 belong to a PP2A family that has the ability to dephosphorylate other proteins. PP2A1 consists of Pph21/22 and Cdc55/Tpd3, which are catalytic and regulatory subunits respectively. PP2A2 consists of Sit4 and Sap, which are catalytic and regulatory subunits respectively. [Cdc55p/Tpd3p]+[Pph21/22p] ←→109KK [PP2A1] [Sit4p]+[Sap] ←→1013KK [PP2A2] However, phosphorylated Tap42 is more likely to bind catalytic subunits of PP2As, like Pph21/22 or Sit4, which means phosphorylated Tap42 suppress the activities of PP2A1 and PP2A2. [Tap42p~p]+[Pph21/22p] ←→54KK [Tap42p~p •Pph21/22p] [Tap42p~p]+[Sit4p] ←→511KK [Tap42p~p•Sit4p] PP2As could dephosphorylate some transcription factors, like phosphorylated Rtg1/3, Gcn4, Gln3, etc, as well as phosphorylated Tap42 and Tip41. [Tap42p~p]+[PP2A1] ←→76KK [Tap42p~p •PP2A1] →8K [Tap42p]+[PP2A1] [Tap42p~p]+[PP2A2] ←→712KK [Tap42p~p•PP2A2] →8K [Tap42p]+[PP2A2] [Tip41p~p]+[PP2A1] ←→1716KK [Tip41p~p•PP2A1] →8K [Tip41p]+[PP2A1] [Tip41p~p]+[PP2A2] ←→1918KK [Tip41p~p •PP2A2] →8K [Tip41p]+[PP2A2] [Rtg1/3p~p]+[PP2A1] ←→104103KK[Rtg1/3•PP2A1]→8K [Rtg1/3p]+[PP2A1] [Rtg1/3p~p]+[PP2A2] ←→106105KK[Rtg1/3•PP2A2]→8K [Rtg1/3p]+[PP2A2]

Partial conclusion: The activity of TORC1 can suppress the activities of PP2As. In our project this year, multiple inhibitors ”FAP”, which are short for furans, acetic acid and phenol could inhibit the activity of TOR protein. When FAP exists in vivo, TORC1 (Tor complex 1) will be dephosphorylated, leading to an inactivated state without the ability to phosphorylate downstream proteins (Notice: The equilibrium constant 1012102TorkKk= can reflect the resistance of Tor2 to FAP to some degree). Another simplification here is that, before we fully understand the mechanism of how FAP inhibit Tor2, we could just treat the interaction between them as complex formation. When Tor2 is bound to FAP, it no longer fulfills the downstream phosphorylation. [FAP]+[Tor1/2p] ←→102101KK [FAP•Tor1/2p] As a result, dephosphorylation of Tor2 leads to increased activity of PP2As. Then PP2As turn to be functional again and dephosphorylate a series of transcription factors (Here we use Rtg1/3 as example in the rest modeling part). [Rtg1/3p~p]+[PP2A1] ←→104103KK[Rtg1/3•PP2A1]→8K [Rtg1/3p]+[PP2A1] [Rtg1/3p~p]+[PP2A2] ←→106105KK[Rtg1/3•PP2A2]→8K [Rtg1/3p]+[PP2A2] Those dephosphorylated transcription factors move into the nucleus (they use to be excluded out of nucleus when phosphorylated), and then activate specific genes. First, dephosphorylated transcription factor could bind with promoter of specific sequences. Here transcription factor Rtg1/3 can activate gene CIT2 by binding its promoter pCIT2. Once bound with transcription factor Rtg1/3, pCIT2 turns into an activated state - pCIT2*. Only activated promoters pCIT2* are able to initiate the transcription process. After transcription, pCIT2* break down into pCIT2, Rtg1/3 and mRNA. These specific mRNAs would complete translation, during which mTOR2p (short for mutant Tor2 protein) would exist in cytoplasm. [pCIT2]+[Rtg1/3p] ←→108107KK[pCIT2*] [pCIT2*]→109K[pCIT2]+[mRNA-mTOR2]+ [Rtg1/3p] [mRNA-mTOR2] →110K[mRNA-mTOR2]+[mTOR2p] For mutant Tor2 protein, it has the identical functions of original Tor2 protein, which means it can phosphorylate Tip41 and Tap42 with the same reaction rate. [Tap42p]+[mTOR2p] ←→112111KK[Tap42p•mTOR2p]→113K [Tap42p~p]+[mTOR2p] [Tip41p]+[mTOR2p] ←→115114KK[Tip41p•mTOR2p]→113K [Tip41p~p]+[mTOR2p] However, our mutations give mTor2 protein improved resistance to FAP, which can be demonstrated from the value of reaction rate: 1162117mTorkKk= is much more greater than 1012102TorkKk=. [FAP]+[mTOR2p] ←→117116KK[FAP•mTOR2p]

Notes: 1. As [Tap42p] [Tip41p][Tor1/2p] [PP2A1][PP2A2][RTG1/3p][pCIT2] are treated as factors in signaling transduction, we assume that their total amount would remain unchanged. There only exist different states. For example: the total amount of Tap42 in cytoplasm is unchanged, but Tap42 had two different states, phosphorylated and dephosphorylated. Only certain amount of protein will take part into this gene circuit and transduction loop. 2. Our main mission is about the regulation on transduction of a signal (FAP existing in cytoplasm), thus other complicated mechanism are ignored. Certainly there must be some Tap42 proteins having interactions with other substances, but the small amount of proteins leaked are not taken into our consideration. 3. We assume that mRNA, mutant Tor2 protein and FAP will degrade in a constant rate during normal metabolism in yeast cell. [mRNA•mTORC1] 118K→● [mTORC1] 118K→● [FAP] 119K→● 1. Simplified object of modeling To demonstrate the above complicated model more clearly, we simplify the original model and devide the whole network into four levels, which could form a feedback loop. After the simplification, it's much easier for reader without much professional knowledge to understand and more convenient to set parameters.