4 research outputs found
Mini-Intein Structures from Extremophiles Suggest a Strategy for Finding Novel Robust Inteins
Inteins are prevalent among extremophiles. Mini-inteins with robust splicing properties are of particular interest for biotechnological applications due to their small size. However, biochemical and structural characterization has still been limited to a small number of inteins, and only a few serve as widely used tools in protein engineering. We determined the crystal structure of a naturally occurring Pol-II mini-intein from Pyrococcus horikoshii and compared all three mini-inteins found in the genome of P. horikoshii. Despite their similar sizes, the comparison revealed distinct differences in the insertions and deletions, implying specific evolutionary pathways from distinct ancestral origins. Our studies suggest that sporadically distributed mini-inteins might be more promising for further protein engineering applications than highly conserved mini-inteins. Structural investigations of additional inteins could guide the shortest path to finding novel robust mini-inteins suitable for various protein engineering purposes
Mini-Intein Structures from Extremophiles Suggest a Strategy for Finding Novel Robust Inteins
Inteins are prevalent among extremophiles. Mini-inteins with robust splicing properties are of particular interest for biotechnological applications due to their small size. However, biochemical and structural characterization has still been limited to a small number of inteins, and only a few serve as widely used tools in protein engineering. We determined the crystal structure of a naturally occurring Pol-II mini-intein from Pyrococcus horikoshii and compared all three mini-inteins found in the genome of P. horikoshii. Despite their similar sizes, the comparison revealed distinct differences in the insertions and deletions, implying specific evolutionary pathways from distinct ancestral origins. Our studies suggest that sporadically distributed mini-inteins might be more promising for further protein engineering applications than highly conserved mini-inteins. Structural investigations of additional inteins could guide the shortest path to finding novel robust mini-inteins suitable for various protein engineering purposes
PTCHD1 Binds Cholesterol but Not Sonic Hedgehog, Suggesting a Distinct Cellular Function
Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1−/− mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function
PTCHD1 Binds Cholesterol but Not Sonic Hedgehog, Suggesting a Distinct Cellular Function
Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1−/− mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function