PubMed · 1108764
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H E Sutton, R P Wagner. 1975. Mutation and enzyme function in humans.. https://doi.org/10.1146/annurev.ge.09.120175.001155
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Post-translational control enables rapid and precise regulation of cell behavior. Despite these advantages, general strategies to build phosphorylation-based synthetic circuits are limited. Here we reasoned that engineered allostery, a technique that has been applied to design light- and chemically gated protein switches, could also be used to engineer phosphorylation-controlled protein switches (phospho-switches). Using an allosterically controllable Gal4 transcription factor as a scaffold, we show that a classic kinase Förster resonance energy transfer biosensor architecture can be used as a starting point for phospho-switch design. We optimize all features of the phospho-switch to develop an ERK-controlled transcription factor with a 20-fold phosphorylation-dependent change in transcriptional output. The resulting synthetic ERK-responsive transcription factor responds with comparable sensitivity to the c-fos promoter and reveals spatial ERK signaling patterns in mammalian developmental organoids. We further show that our switch architecture can be generalized to other input kinases and allosterically controlled targets. This work provides a general platform for a new generation of kinase-responsive tools for biosensing and synthetic biology applications.
TAR RNA represents an attractive target for the intervention of human immunodeficiency virus type 1 (HIV-1) replication by small molecules. We now describe three small molecule inhibitors of the HIV-1 Tat-TAR interaction that target the RNA, not the protein. The chemical structures and RNA binding characteristics of these inhibitors are unique for each molecule. Results from various biochemical and spectroscopic methods reveal that each of the three Tat-TAR inhibitors recognizes a different structural feature at the bulge, lower stem, or loop region of TAR. Furthermore, one of these Tat-TAR inhibitors has been demonstrated, in cellular environments, to inhibit (a) a TAR-dependent, Tat-activated transcription and (b) the replication of HIV-1 in a latently infectious model.
The ADPglucose pyrophosphorylase (EC 2.7.7.27) from Escherichia coli is allosterically activated by fructose 1,6-bisphosphate and inhibited by AMP. Proteolysis of the enzyme with proteinase K causes loss of activity and generates two peptides, 21 and 28 kDa, from the 49.7-kDa subunit. The presence of ADPglucose, Mg2+, and fructose 1, 6-bisphosphate during the incubation with proteinase K protected the enzyme activity and prevented cleavage at sites Met181-Ala182 and Phe192-Val193. Proteolysis of the protected enzyme removed 10 to 13 amino acids from the N-terminal and 2 amino acids from the C-terminal. The resulting enzyme was almost independent of the need for fructose 1,6-bisphosphate for maximal activity and insensitive to inhibition by AMP. The apparent affinity for the substrates was similar to that of the fully-activated wild-type enzyme. These data suggest that amino acid residues in the N-terminal portion and possibly the C-terminal portion of ADPglucose pyrophosphorylase are part of the regulatory domain of the enzyme, critical for allosteric regulation of the enzyme.