(110e) Harnessing Protein-Protein Interactions for Programmable Control of Protein Secretion in Mammalian Cells
AIChE Annual Meeting
2023
2023 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Faculty Candidate Session: Food, Pharmaceuticals, and Bioengineering I
Monday, November 6, 2023 - 1:42pm to 2:00pm
Methods: To harness these protein-protein interactions with 14-3-3 proteins to create programmable outputs, we fused a small peptide binder of 14-3-3 proteins to the C-terminus of the RELEASE construct, effectively blocking its retention capabilities. By placing a protease cut site upstream of the 14-3-3 recruiting peptide, we could manipulate interactions with 14-3-3 proteins and implement NOT Gate logic control of protein secretion (Fig. 1B). This new design (Compact RELEASE) can function as both the processing and output module of protein circuits and achieves functional completeness of every possible logic operation.
Results: The Compact RELEASE suite overcomes the need to use additional proteases to confer logic iteration or quantitative processing through manipulating protein-protein interactions with native 14-3-3 scaffold proteins. For example, Compact RELEASE variants can be designed to have conditional activation of protein secretion in response to multiple inputs (i.e. NIMPLY gate â Fig. 1C). Through manipulating the cleavage efficiencies of different protease cut sites, Compact RELEASE also can be used for quantitative activation of protein secretion. One such example is bandpass activation, where protein secretion only occurs within a specific concentration range of input (Fig. 1D). Furthermore, by layering different RELEASE variants (i.e. standard, NOT and bandpass), we can enable concentration-dependent activation of multiple proteins for sequential activation of protein secretion, without requiring additional proteases to process the inputs. Pilot studies are underway to subcutaneously deliver engineered cells to control the local concentration of secreted proteins using small-molecule activators.
Conclusion: The Compact RELEASE suite enables functional complete programmable control of all Boolean logic gates and quantitative control of protein secretion. This platform expands the programmable capabilities of synthetic protein circuits in a compact manner, enabling their delivery using pre-existing viral vector methods such as adeno-associated viruses or adenoviruses.
Figure Caption: A) Schematic of RELEASE for controlling protein secretion in response to protease activity. B) Recruitment of 14-3-3 proteins via small peptides antagonizes the activity of the ER retention motif, resulting in constitutive protein secretion. Removal of the peptide via protease activation restores ER retention activity, implementing NOT gate logic. C) Conditional activation of protein secretion with multiple outputs. D) By creating a variant with two protease cut sites of different cutting efficiencies (Kcat), we can implement bandpass activation of protein secretion. Protein secretion (output) will only occur when the concentration of the input is within a specific range.