Hundreds of bacterial species produce proteinaceous microcompartments (MCPs) that act as simple organelles by confining the enzymes of metabolic pathways that have toxic or volatile intermediates. A fundamental unanswered question about bacterial MCPs is how enzymes are packaged within the protein shell that forms their outer surface. Here, we report that a short N-terminal peptide is necessary and sufficient for packaging enzymes into the lumen of an MCP involved in B 12 -dependent 1,2-propanediol utilization (Pdu MCP). Deletion of 10 or 14 amino acids from the N terminus of the propionaldehyde dehydrogenase (PduP) enzyme, which is normally found within the Pdu MCP, substantially impaired packaging, with minimal effects on its enzymatic activity. Fusion of the 18 N-terminal amino acids from PduP to GFP, GST, or maltose-binding protein resulted in their encapsulation within MCPs. Bioinformatic analyses revealed N-terminal extensions in two additional Pdu proteins and three proteins from two unrelated MCPs, suggesting that N-terminal peptides may be used to package proteins into diverse MCPs. The potential uses of MCP assembly principles in nature and in biotechnology are discussed.

Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (~50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.

Mutations of lamin A/C (LMNA) cause a wide range of human disorders, including progeria, lipodystrophy, neuropathies and autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD). EDMD is also caused by X-linked recessive loss-of-function mutations of emerin, another component of the inner nuclear lamina that directly interacts with LMNA. One model for disease pathogenesis of LMNA and emerin mutations is cell-specific perturbations of the mRNA transcriptome in terminally differentiated cells. To test this model, we studied 125 human muscle biopsies from 13 diagnostic groups (125 U133A, 125 U133B microarrays), including EDMD patients with LMNA and emerin mutations. A Visual and Statistical Data Analyzer (VISDA) algorithm was used to statistically model cluster hierarchy, resulting in a tree of phenotypic classifications. Validations of the diagnostic tree included permutations of U133A and U133B arrays, and use of two probe set algorithms (MAS5.0 and MBEI). This showed that the two nuclear envelope defects (EDMD LMNA, EDMD emerin) were highly related disorders and were also related to fascioscapulohumeral muscular dystrophy (FSHD). FSHD has recently been hypothesized to involve abnormal interactions of chromatin with the nuclear envelope. To identify disease-specific transcripts for EDMD, we applied a leave-one-out (LOO) cross-validation approach using LMNA patient muscle as a test data set, with reverse transcription-polymerase chain reaction (RT-PCR) validations in both LMNA and emerin patient muscle. A high proportion of top-ranked and validated transcripts were components of the same transcriptional regulatory pathway involving Rb1 and MyoD during muscle regeneration (CRI-1, CREBBP, Nap1L1, ECREBBP/p300), where each was specifically upregulated in EDMD. Using a muscle regeneration time series (27 time points) we develop a transcriptional model for downstream consequences of LMNA and emerin mutations. We propose that key interactions between the nuclear envelope and Rb and MyoD fail in EDMD at the point of myoblast exit from the cell cycle, leading to poorly coordinated phosphorylation and acetylation steps. Our data is consistent with mutations of nuclear lamina components leading to destabilization of the transcriptome in differentiated cells.

Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of non-canonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing non-canonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that regulates numerous cellular processes through the attachment of O-linked N-acetylglucosamine (O-GlcNAc) residues to nuclear and cytoplasmic proteins. Its targets include kinases, phosphatases, transcription factors, histones, and many other intracellular proteins. The biology of O-GlcNAc modification is still not well understood and cell-permeable inhibitors of OGT are needed both as research tools and for validating OGT as a therapeutic target. Here we report a small molecule OGT inhibitor, OSMI-1, developed from a high-throughput screening hit. It is cell-permeable and inhibits protein O-GlcNAcylation in several mammalian cell lines without qualitatively altering cell surface N- or O-linked glycans. The development of this molecule validates high-throughput screening approaches for the discovery of glycosyltransferase inhibitors, and further optimization of this scaffold may lead to yet more potent OGT inhibitors useful for studying OGT in animal models.