Discovery of Borosin Catalytic Strategies and Function through Bioinformatic Profiling.

Borosins are ribosomally synthesized and post-translationally modified peptides (RiPPs) containing backbone α-N-methylations. These modifications confer favorable pharmacokinetic properties including increased membrane permeability and resistance to proteolytic degradation. Previous studies have biochemically and bioinformatically explored several borosins, revealing (1) numerous domain architectures and (2) diverse core regions lacking conserved sequence elements. Due to these characteristics, large-scale computational identification of borosin biosynthetic genes remains challenging and often requires additional, time-intensive manual inspection. This work builds upon previous findings and updates the genome-mining tool RODEO to automatically evaluate borosin biosynthetic gene clusters (BGCs) and identify putative precursor peptides. Using the new RODEO module, we provide an updated analysis of borosin BGCs identified in the NCBI database. From our data set, we bioinformatically predict and experimentally characterize a new fused borosin domain architecture, in which the modified natural product core is encoded N-terminal to the methyltransferase domain. Additionally, we demonstrate that a borosin precursor peptide is a native substrate of shewasin A, a reported aspartyl peptidase with no previously identified substrates. Shewasin A requires post-translational modification of the leader peptide for proteolytic maturation, a feature not previously observed in RiPPs. Overall, this work provides a user-friendly and open-access tool for the analysis of borosin BGCs and we demonstrate its utility to uncover additional biosynthetic strategies within the borosin class of RiPPs.

Computationally guided exploration of borosin biosynthetic strategies.

Borosins are ribosomally synthesized and post-translationally modified peptides containing backbone α- N -methylations. Identification of borosin precursor peptides is difficult because (1) there are no conserved sequence elements among borosin precursor peptides and (2) the biosynthetic gene clusters contain numerous domain architectures and peptide fusions. To tackle this problem, we updated the genome mining tool RODEO to automatically evaluate putative borosin BGCs and identify precursor peptides. Enabled by the new borosin module, we analyzed all borosin BGCs found in available sequence data and assigned precursor peptides to previously orphan borosin methyltransferases. Additionally, we bioinformatically predict and experimentally characterize a new fused borosin domain architecture, in which the modified core is N-terminal to the methyltransferase domain. Finally, we demonstrate that a borosin precursor peptide is the native substrate of shewasin A, a previously characterized pepsin-like aspartic peptidase whose native biological function was unknown.

Readily available tissue-engineered vascular grafts.

Autologous or synthetic vascular grafts are used routinely for providing access in hemodialysis or for arterial bypass in patients with cardiovascular disease. However, some patients either lack suitable autologous tissue or cannot receive synthetic grafts. Such patients could benefit from a vascular graft produced by tissue engineering. Here, we engineer vascular grafts using human allogeneic or canine smooth muscle cells grown on a tubular polyglycolic acid scaffold. Cellular material was removed with detergents to render the grafts nonimmunogenic. Mechanical properties of the human vascular grafts were similar to native human blood vessels, and the grafts could withstand long-term storage at 4 °C. Human engineered grafts were tested in a baboon model of arteriovenous access for hemodialysis. Canine grafts were tested in a dog model of peripheral and coronary artery bypass. Grafts demonstrated excellent patency and resisted dilatation, calcification, and intimal hyperplasia. Such tissue-engineered vascular grafts may provide a readily available option for patients without suitable autologous tissue or for those who are not candidates for synthetic grafts.