Saccharomyces cervisiae, or bakers' yeast, are utilized in the biotechnology industry to express, fold, and assemble foreign protein therapeutics [1].  Simply modifying yeast to express high levels of heterologous protein does not necessarily maximize production and secretion, as this action triggers the Unfolded Protein Response (UPR), which eliminates quantities of desired protein through promoted ER-Associated Degradation (ERAD).  The yeast UPR may even retard transcription and/or translation of the desired protein, thus decreasing yields.  Consequently, a combined approach of mathematical modeling and experiments has been employed in an attempt to obtain a thorough understanding of the yeast UPR, which will be utilized to forward engineer the system to maximize foreign protein therapeutic production. The yeast UPR has been traditionally modeled as a negative feedback loop in which low levels of the chaperone BiP (Binding Protein) relative to unfolded protein (UP) in the ER signal the need for increased production of UPR component proteins--including BiP--that help cells cope with these high ER UP levels.  The UPR signal is transduced from the ER to the nucleus and cytoplasm by the ER membrane-spanning endonuclease Ire1p, which enhances translation of Hac1p, a transcription factor that upregulates BiP and other UPR-related gene expression.  Traditional modeling identifies BiP as the primary regulator of Ire1p:  BiP typically binds Ire1p but is sequestered exclusively by UP when UP is in excess;  unbound Ire1p is then free to dimerize, trans-autophosphorylate, and transduce the UPR signal across the nuclear ER membrane [2,3].  However, recent experimental evidence suggests BiP may simply serve as an Ire1p adjustor, and that UP directly regulates Ire1p activation [4,5]. This work sought to identify and define fundamental differences, using mathematical modeling and systems analysis tools, between the traditional and newer Ire1p activation models where BiP serves as the primary activation regulator, UP does alone, and UP does combined with modulation by BiP.  These three activation models are the BiP-Ire1p (BI), UP-Ire1p (UPI), and BiP-modulated (BM) models, respectively.  With the differences identified, they could be compared against existing experimental data, or new experiments could be designed to select the candidate(s) that best represent the biological system. A mechanistic, deterministic mathematical model of the yeast UPR has been developed and implemented in the Matlab Simulink environment using the ode15s solver.  This model contained 32 states that described the interactions between the major UPR components--UP, Hac1p, and BiP--as well as two critical UPR inducers--heterologous scFv and DTT.  The three Ire1p activation regulation models were substituted into this larger UPR framework, and their ability to reproduce currently available UPR data was evaluated. All three models were similarly capable of reproducing this data, so the systems analysis tool, sensitivity analysis, was employed to comprehensively scan the models for less-apparent discrimination criteria.  This analysis was performed using the BioSens software package for BioSpice, which runs .bsn versions of the model in DASPK, and found that the models responded significantly different to changes in BiP-scFv binding and UP-ER entry rates, which are practically alterable experimentally.  The BI UPR was positively and UPI/BM UPRs were negatively correlated to changes in the BiP-scFv binding rate, which could be manipulated in vivo by mutating BiP-binding sites on the scFv.  The UPI/BM UPRs had a stronger correlation to the UP-ER entry rate than the BI UPR.  Further discrimination between the UPI and BM models comes from the mechanistic knowledge that BiP binds Ire1p when the UPR is inactive and releases it when the UPR is activated [4]. References [1]        A. S. Robinson and D. A. Lauffenburger.  Model for ER Chaperone Dynamics and Secretory Protein Interactions.  AIChE Journal, 42 (5):  1443-1453, 1996.

[2]        A. A. Welihinda and R. J. Kaufman.  The unfolded protein response pathway in Saccharomyces cerevisiae. Oligomerization and trans-phosphorylation of Ire1p (Ern1p) are required for kinase activation.  J. Biol. Chem.  271 (30):  18181-18187, 1996.

[3]        K. Okamura, Y. Kimata, H. Higashio, A. Tsuru, and K. Kohno.  Dissociation of Kar2p/BiP from an ER sensory molecule, Ire1p, triggers the unfolded protein response in yeast.  Biochem. Biophys. Res. Commun.  279 (2):  445-450, 2000.

[4]        Y. Kimata, D. Oikawa, Y. Shimizu, Y. Ishiwata-Kimata, and K. Kohno.  A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1.  J. Biol. Chem., 167 (3):  445-456, 2004.

[5]        J. J. Credle, J. S. Finer-Moore, F. R. Papa, R. M. Stroud, and P. Walter.  On the mechanism of sensing unfolded protein in the endoplasmic reticulum.  PNAS 102 (52):  18773-18784, 2005.