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Multi-input/multi-output Control of Cellular Processes by PROCISiR

Novel combination AAV-based gene therapy simultaneously treats several age-related diseasesCells react to multiple environmental or autologous inputs by various behaviors. These biological processes are difficult to investigate because of their complexity. Small molecules are widely used as extrinsic inputs to investigate biological process in vitro, ex vivo and in vivo. While most systems allow single molecule input and single output that limits their applications. Recently, a research paper “Multi-input chemical control of protein dimerization for programming graded cellular responses” was published in Nature Biotechnology in October 2019. The paper developed a post-translational control system that can be used to detect diverse cellular responses. It utilizes the NS3a protease as a central receiver protein that is the target of multiple clinically approved drugs. Computationally designed reader proteins are used to translate different drug-NS3a reactions into diverse outputs.

Computational design of NS3a readers

With the Rosetta interface design method, they developed protein readers that selectively recognize NS3a-drug complex. They designed some candidate scaffolds recognizing danoprevir/NS3a, including LRRs, DHRs, ferredoxins and helical bundles. One of DHR designs that showed modest drug-dependent binding to NS3a was used for further optimization. The final construct of danoprevir/NS3 complex reader, DNCR2, showed apparent affinity for the NS3a/danoprevir complex and no binding to apo NS3a or free danoprevir. Further biochemical analysis confirmed that DNCR2/danoprevir/NS3a form a 1:1:1 complex. These results proved that DNCR2 is a perfect danoprevir/NS3a reader protein. Other two readers, GNCR1 for grazoprevir/NS3 complex and ANR for apo NS3a, were developed with similar method.

Validation of PROCISiR in mammalian cells

They next evaluated the function of PROCISIR in detecting multi-input/multi-output behaviors in mammalian cells. They observed that grazoprevir exclusively colocalized NS3a-mCherry with membrane-targeted GNCR1, while only danoprevir led to colocalization with mitochondrially targeted DNCR2. Likewise, membrane-targeted ANR prelocalized NS3a-mCherry to the plasma membrane, and danoprevir treatment recruited NS3a to the nucleus with nuclear targeted DNCR2. These colocalization experiments validated that the DNCR2, GNCR1 and ANR readers are selective for their targeted state of NS3a and can be used in concert.

PROCISiR enables programmable transcriptional control

Two fusion proteins, DNCR2-VPR (a transcriptional activator) and NS3a-dCas9, functioned together to promote endogenous gene expressions in HEK293 cells. Danoprevir acted as agonist and grazoprevir as antagonist to temporally and proportionally control the transcription. Addition of danoprevir activated CXCR4 expression, while addition of grazoprevir rapidly reversed this expression. Moreover, treatment with danoprevir and increasing grazoprevir proportions produced graded transcription outputs. Besides, they achieved proportional and graded control of two transcriptional outputs with two inputs. Thus the PROCISiR architecture can be used for temporal, graded and multiple transcriptional control.

Proportional control of cell signaling with PROCISiR

Confocal microscopy of EGFP-DNCR2 and BFP-GNCR1 validated that the concentrations of danoprevir and grazoprevir provided graded and proportional regimes of DNCR2 and GNCR1 colocalization with NS3a. They next used danoprevir/grazoprevir combinations with DNCR2-TIAM (Rac1 guanine nucleotide exchange factor) and GNCR1-LARG (RhoA/B/C guanine nucleotide exchange factor) to control the activations of Rac and Rho GTPases, which work reversely in actin cytoskeleton and cell morphology. In cells coexpressing DNCR2-TIAM and GNCR1-LARG with membrane-localized NS3a, danoprevir treatment resulted in cell expansion due to the activity of Rac. Grazoprevir treatment caused cell contraction and stress fibers because of the Rho activation. With PROCISiR, they found that concurrent variation in Rho and Rac activity does not result in an average phenotype, but one closer to the Rho-only.

Conclusion

PROCISiR can control switching behavior and provide graded, proportional control over two cellular outputs at once. The control modalities in PROCISiR can be used to manipulate mammalian cellular processes and potentially engineer drug regulated cell therapies.

In this paper, Grazoprevir/NS3a reader designs were synthesized and cloned into pETCON plasmids by Genscript. As the leader in gene synthesis, Genscript have completed over 600,000 gene synthesis projects for scientists around the world. GenScript's gene synthesis service saves both time and money that makes your research easy.


Foight, G.W., Wang, Z., Wei, C.T., Greisen, P.J., Warner, K.M., Cunningham-Bryant, D., Park, K., Brunette, T.J., Sheffler, W., Baker, D., & Maly, D.J. (2019). Multi-input chemical control of protein dimerization for programming graded cellular responses. Nature Biotechnology, 1-9.

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