Abstract:Proteases with reprogrammed specificity for nonnative
substrates
are highly desired in synthetic biology and biomedicine. However,
generating reprogrammed proteases that are orthogonal and highly specific
for a new target has been a major challenge. In this work, we sought
to expand the versatility of protease systems by engineering an orthogonal
botulinum neurotoxin serotype B (BoNT/B) protease that recognizes
an orthogonal substrate. We designed and validated an orthogonal BoNT/B
protease system in mammalian… Show more
“…When evolving proteases for switched specificity, selecting against undesired cleavage sequences is imperative to avoid variants with relaxed specificity. Cleveland et al aimed to overcome this limitation by designing a proteolysis‐dependent transcription factor inspired by hormone‐inducible synthetic TFs (Cleveland et al, 2022). Their best design incorporated a counterselection substrate in the following fusion protein: estrogen receptor ligand binding domain‐Gal4 binding domain‐counterselection substrate‐VP16 activation domain‐selection substrate‐estrogen receptor ligand binding domain, abbreviated as ER‐LBD‐GAL4BD‐CS‐VP16‐SS‐ER‐LBD (Figure 3b).…”
Section: Proteolysis‐mediated Protein Activation: Cytoplasmic Sequest...mentioning
Enzymes that catalyze posttranslational modifications (PTMs) of peptides and proteins (PTM–enzymes)—proteases, protein ligases, oxidoreductases, kinases, and other transferases—are foundational to our understanding of health and disease and empower applications in chemical biology, synthetic biology, and biomedicine. To fully harness the potential of PTM–enzymes, there is a critical need to decipher their enzymatic and biological mechanisms, develop molecules that can probe and modulate them, and endow them with improved and novel functions. These objectives are contingent upon implementation of high‐throughput functional screens and selections that interrogate large sequence libraries to isolate desired PTM–enzyme properties. This review discusses the principles of Saccharomyces cerevisiae organelle sequestration to study and engineer PTM–enzymes. These include outer membrane sequestration, specifically methods that modify yeast surface display, and cytoplasmic sequestration based on enzyme‐mediated transcription activation. Furthermore, we present a detailed discussion of yeast endoplasmic reticulum sequestration for the first time. Where appropriate, we highlight the major features and limitations of different systems, specifically how they can measure and control enzyme catalytic efficiencies. Taken together, yeast‐based high‐throughput sequestration approaches significantly lower the barrier to understanding how PTM–enzymes function and how to reprogram them.
“…When evolving proteases for switched specificity, selecting against undesired cleavage sequences is imperative to avoid variants with relaxed specificity. Cleveland et al aimed to overcome this limitation by designing a proteolysis‐dependent transcription factor inspired by hormone‐inducible synthetic TFs (Cleveland et al, 2022). Their best design incorporated a counterselection substrate in the following fusion protein: estrogen receptor ligand binding domain‐Gal4 binding domain‐counterselection substrate‐VP16 activation domain‐selection substrate‐estrogen receptor ligand binding domain, abbreviated as ER‐LBD‐GAL4BD‐CS‐VP16‐SS‐ER‐LBD (Figure 3b).…”
Section: Proteolysis‐mediated Protein Activation: Cytoplasmic Sequest...mentioning
Enzymes that catalyze posttranslational modifications (PTMs) of peptides and proteins (PTM–enzymes)—proteases, protein ligases, oxidoreductases, kinases, and other transferases—are foundational to our understanding of health and disease and empower applications in chemical biology, synthetic biology, and biomedicine. To fully harness the potential of PTM–enzymes, there is a critical need to decipher their enzymatic and biological mechanisms, develop molecules that can probe and modulate them, and endow them with improved and novel functions. These objectives are contingent upon implementation of high‐throughput functional screens and selections that interrogate large sequence libraries to isolate desired PTM–enzyme properties. This review discusses the principles of Saccharomyces cerevisiae organelle sequestration to study and engineer PTM–enzymes. These include outer membrane sequestration, specifically methods that modify yeast surface display, and cytoplasmic sequestration based on enzyme‐mediated transcription activation. Furthermore, we present a detailed discussion of yeast endoplasmic reticulum sequestration for the first time. Where appropriate, we highlight the major features and limitations of different systems, specifically how they can measure and control enzyme catalytic efficiencies. Taken together, yeast‐based high‐throughput sequestration approaches significantly lower the barrier to understanding how PTM–enzymes function and how to reprogram them.
“…When evolving proteases for switched specificity, selecting against undesired cleavage sequences is imperative to avoid variants with relaxed specificity. Tucker and coworkers aimed to overcome this limitation by designing a proteolysis-dependent transcription factor inspired by hormone-inducible synthetic TFs (Cleveland et al, 2022). Their best design incorporated a counterselection substrate in the following fusion protein: estrogen receptor ligand binding domain-Gal4 binding domaincounterselection substrate-VP16 activation domain-selection substrate-estrogen receptor ligand binding domain, abbreviated as ER-LBD-GAL4BD-CS-VP16-SS-ER-LBD (Figure 2B).…”
Enzymes that catalyze post-translational modifications of peptides and
proteins (PTM-enzymes) – proteases, protein ligases, oxidoreductases,
kinases, and other transferases - are foundational to our understanding
of health and disease and empower applications in chemical biology,
synthetic biology, and biomedicine. To fully harness the potential of
PTM-enzymes, there is a critical need to decipher their enzymatic and
biological mechanisms, develop molecules that can probe and reprogram
them, and endow them with improved and novel functions. These objectives
are contingent upon implementation of high-throughput functional screens
and selections that interrogate large sequence libraries to isolate
desired PTM-enzyme properties. This review discusses the principles of
S. cerevisiae organelle sequestration to study and engineer
PTM-enzymes. These include methods that modify yeast surface display and
employ enzyme-mediated transcription activation to evolve the activity
and substrate specificity of proteases and protein ligases. We also
present a detailed discussion of yeast endoplasmic reticulum (ER)
sequestration for the first time. Where appropriate, we highlight the
major features and limitations of different systems, specifically how
they can measure and control enzyme catalytic efficiencies. Taken
together, yeast-based high-throughput sequestration approaches
significantly lower the barrier to understanding how PTM-enzymes
function and how to reprogram them.
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