Understanding protease selectivity for protein linker sequences

Background.

Selective processing of polypeptide sequences by proteases is of outstanding importance for the function and activity of many proteins. For example, many snake venom proteins exhibit protease activity (Tokarz, 2018), while insulin is activated through cellular proteases trimming of its N-terminus and removing a central region (Boldrini-Franca, 2020). Such proteolytic activation often occurs at interfaces between protein domains known as linkers. Understanding the selectivity of proteases can help to gain knowledge about cellular and disease mechanisms. Ultimately it facilitates the rational development of innovative medicines that, for example, allow a targeted release of an active drug substance in specific cellular compartments based on properly designed linker sequences (Lingg, 2022; Knodler, 2023).

Hypothesis.

Peptide sequence libraries can be used to establish and quantify protease selectivity towards linker sequences and to design protease resistance or sensitivity into such sequences.

Methods.

A set of relevant protein sequences known to undergo protease cleavage as well as a set of proteases covering a wide range of sub-cellular localizations and selectivities will be established based on literature research. The protease-sensitive sequences will be diversified using methods such as error-prone PCR (Laurent, 2021) and subsequently displayed on yeast (Zajc, 2020). In parallel, proteases will be recombinantly produced and purified in a suitable high-throughput compatible system such as E. coli, yeast or plant cell packs (PCPs) (Gengenbach, 2020). The highly diverse library of yeast displayed peptides (109 variants) can subsequently be screened for protease sensitivity or resistance by detecting the absence or presence of a C-terminally expressed tag, respectively. Sequences covering a wide range of protease sensitivity/resistance that have been identified during yeast surface display screening will be engineered as linkers into fusion proteins containing distinct fluorescent domains. The fusion proteins will be expressed in different eukaryotic systems and targeted to different sub-cellular compartments. Suitable cellular/technical assays (e.g., Fluorescence Activated Cell Sorting (FACS) applied to acute myeloid leukemia (AML) model cell cultures (Knodler, 2023)) will be used to monitor and quantify protease resistance/sensitivity in vivo/in situ.

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