Tuning protein half-life in mouse using sequence-defined biopolymers functionalized with lipids.

The use of biologics in the treatment of numerous diseases has increased steadily over the past decade due to their high specificities, low toxicity, and limited side effects. Despite this success, peptide- and protein-based drugs are limited by short half-lives and immunogenicity. To address these challenges, we use a genomically recoded organism to produce genetically encoded elastin-like polypeptide-protein fusions containing multiple instances of para-azidophenylalanine (pAzF). Precise lipidation of these pAzF residues generated a set of sequence-defined synthetic biopolymers with programmable binding affinity to albumin without ablating the activity of model fusion proteins, and with tunable blood serum half-lives spanning 5 to 94% of albumin's half-life in a mouse model. Our findings present a proof of concept for the use of genetically encoded bioorthogonal conjugation sites for multisite lipidation to tune protein stability in mouse serum. This work establishes a programmable approach to extend and tune the half-life of protein or peptide therapeutics and a technical foundation to produce functionalized biopolymers endowed with programmable chemical and biophysical properties with broad applications in medicine, materials science, and biotechnology.

Chemoselective restoration of para-azido-phenylalanine at multiple sites in proteins.

The site-specific incorporation of nonstandard amino acids (nsAAs) during translation has expanded the chemistry and function of proteins. The nsAA para-azido-phenylalanine (pAzF) encodes a biorthogonal chemical moiety that facilitates "click" reactions to attach diverse chemical groups for protein functionalization. However, the azide moiety is unstable in physiological conditions and is reduced to para-amino-phenylalanine (pAF). Azide reduction decreases the yield of pAzF residues in proteins to 50%-60% per azide and limits protein functionalization by click reactions. Here, we describe the use of a pH-tunable diazotransfer reaction that converts pAF to pAzF at >95% efficiency in proteins. The method selectively restores pAzF at multiple sites per protein without introducing off-target modifications. This work addresses a key limitation in the production of pAzF-containing proteins by restoring azides for multi-site functionalization with diverse chemical moieties, setting the stage for the production of genetically encoded biomaterials with broad applications in biotherapeutics, materials science, and biotechnology.

Protein nanowires with tunable functionality and programmable self-assembly using sequence-controlled synthesis.

Advances in synthetic biology permit the genetic encoding of synthetic chemistries at monomeric precision, enabling the synthesis of programmable proteins with tunable properties. Bacterial pili serve as an attractive biomaterial for the development of engineered protein materials due to their ability to self-assemble into mechanically robust filaments. However, most biomaterials lack electronic functionality and atomic structures of putative conductive proteins are not known. Here, we engineer high electronic conductivity in pili produced by a genomically-recoded E. coli strain. Incorporation of tryptophan into pili increased conductivity of individual filaments >80-fold. Computationally-guided ordering of the pili into nanostructures increased conductivity 5-fold compared to unordered pili networks. Site-specific conjugation of pili with gold nanoparticles, facilitated by incorporating the nonstandard amino acid propargyloxy-phenylalanine, increased filament conductivity ~170-fold. This work demonstrates the sequence-defined production of highly-conductive protein nanowires and hybrid organic-inorganic biomaterials with genetically-programmable electronic functionalities not accessible in nature or through chemical-based synthesis.

Natural circularly permuted group II introns in bacteria produce RNA circles.

Group II self-splicing introns are large structured RNAs that remove themselves from transcripts while simultaneously sealing the resulting gaps. Some representatives can subsequently reverse splice into DNA, accounting for their pervasive distribution in bacteria. The catalytically active tertiary structure of each group II intron is assembled from six domains that are arranged in a conserved order. Here, we report structural isomers of group II introns, called CP group II ribozymes, wherein the characteristic order of domains has been altered. Domains five and six, which normally reside at the 3' end of group II introns, instead occupy the 5' end to form circularly permuted variants. These unusual group II intron derivatives are catalytically active and generate large linear branched and small circular RNAs, reaction products that are markedly different from those generated by canonical group II introns. The biological role of CP group II ribozymes is currently unknown.

Next-generation genetic code expansion.

Engineering of the translation apparatus has permitted the site-specific incorporation of nonstandard amino acids (nsAAs) into proteins, thereby expanding the genetic code of organisms. Conventional approaches have focused on porting tRNAs and aminoacyl-tRNA synthetases (aaRS) from archaea into bacterial and eukaryotic systems where they have been engineered to site-specifically encode nsAAs. More recent work in genome engineering has opened up the possibilities of whole genome recoding, in which organisms with alternative genetic codes have been constructed whereby codons removed from the genetic code can be repurposed as new sense codons dedicated for incorporation of nsAAs. These advances, together with the advent of engineered ribosomes and new molecular evolution methods, enable multisite incorporation of nsAAs and nonstandard monomers (nsM) paving the way for the template-directed production of functionalized proteins, new classes of polymers, and genetically encoded materials.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy type 1: a light on molecular mechanisms.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited cardiomyopathy associated with cardiac arrhythmias originating in the right ventricle, heart failure, and sudden cardiac death. Development of ARVD/C type 1 has been attributed to differential expression of transforming growth factor beta 3 (TGF ß 3). Several mechanisms underlying the molecular basis of ARVD/C type 1 have been proposed. Evaluating previously described mechanisms might elucidate how TGF ß 3 contributes to disease progression in ARVD/C type 1. Here we review how TGF ß 3 can induce fibrogenesis through Smad and/or ß -catenin signaling. Moreover, the role of apoptosis is addressed. Finally the extent to which the immune system has been demonstrated to be a modulating and amplifying agent in the onset and progression of ARVD/C in general is discussed.