Engineered mischarged transfer RNAs for correcting pathogenic missense mutations.

Missense mutations account for approximately 50% of pathogenic mutations in human genetic diseases, and most lack effective treatments. Gene therapies, gene editing, and RNA therapies, including transfer RNA (tRNA) modalities, are common strategies for genetic disease treatments. However, reported tRNA therapies are for nonsense mutations only. It has not been explored how tRNAs can be engineered to correct missense mutations. Here, we describe missense-correcting tRNAs (mc-tRNAs) as a potential therapeutic for correcting pathogenic missense mutations. Mc-tRNAs are engineered tRNAs charged with one amino acid, but read codons of another in translation. We first developed a series of fluorescent protein-based reporters that indicate the successful correction of missense mutations via restoration of fluorescence. We engineered mc-tRNAs that effectively corrected serine and arginine missense mutations in the reporters and confirmed the amino acid substitution by mass spectrometry and mc-tRNA expression by sequencing. We examined the transcriptome response to mc-tRNA expression and found some mc-tRNAs induced minimum transcriptomic changes. Furthermore, we applied an mc-tRNA to rescue a pathogenic CAPN3 Arg-to-Gln mutant involved in LGMD2A. These results establish a versatile pipeline for mc-tRNA engineering and demonstrate the potential of mc-tRNA as an alternative therapeutic platform for the treatment of genetic disorders.

Absolute configuration determination of pharmaceutical crystalline powders by MicroED via chiral salt formation.

Microcrystal electron diffraction (MicroED) has established its complementary role alongside X-ray diffraction in crystal structure elucidation. Unfortunately, kinematical refinement of MicroED data lacks the differentiation power to assign the absolute structure solely based on the measured intensities. Here we report a method for absolute configuration determination via MicroED by employing salt formation with chiral counterions.

Small Molecule Microcrystal Electron Diffraction for the Pharmaceutical Industry-Lessons Learned From Examining Over Fifty Samples.

The emerging field of microcrystal electron diffraction (MicroED) is of great interest to industrial researchers working in the drug discovery and drug development space. The promise of being able to routinely solve high-resolution crystal structures without the need to grow large crystals is very appealing. Despite MicroED's exciting potential, adoption across the pharmaceutical industry has been slow, primarily owing to a lack of access to specialized equipment and expertise. Here we present our experience building a small molecule MicroED service pipeline for members of the pharmaceutical industry. In the past year, we have examined more than fifty small molecule samples submitted by our clients, the majority of which have yielded data suitable for structure solution. We also detail our experience determining small molecule MicroED structures of pharmaceutical interest and offer some insights into the typical experimental outcomes. This experience has led us to conclude that small molecule MicroED adoption will continue to grow within the pharmaceutical industry where it is able to rapidly provide structures inaccessible by other methods.

An ER translocon for multi-pass membrane protein biogenesis.

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

Cell membranes are structures that separate the interior of the cell from its environment and determine the cell's shape and the structure of its internal compartments. Nearly 25% of human genes encode transmembrane proteins that span the entire membrane from one side to the other, helping the membrane perform its roles. Transmembrane proteins are synthesized by ribosomes ­ protein-making machines ­ that are on the surface of a cell compartment called the endoplasmic reticulum. As the new protein is made by the ribosome, it enters the endoplasmic reticulum membrane where it folds into the correct shape. This process is best understood for proteins that span the membrane once. Despite decades of work, however, much less is known about how multi-pass proteins that span the membrane multiple times are made. A study from 2017 showed that a protein called TMCO1 is related to a group of proteins involved in making membrane proteins. TMCO1 has been linked to glaucoma, and mutations in it cause cerebrofaciothoracic dysplasia, a human disease characterized by severe intellectual disability, distinctive facial features, and bone abnormalities. McGilvray, Anghel et al. ­ including several of the researchers involved in the 2017 study ­ wanted to determine what TMCO1 does in the cell and begin to understand its role in human disease. McGilvray, Anghel et al. discovered that TMCO1, together with other proteins, is part of a new 'translocon' ­ a group of proteins that transports proteins into the endoplasmic reticulum membrane. Using a combination of biochemical, genetic and structural techniques, McGilvray, Anghel et al. showed that the translocon interacts with ribosomes that are synthesizing multi-pass proteins. The experiments revealed that the translocon is required for the production of a multi-pass protein called EAAT1, and it provides multiple ways for proteins to be inserted into and folded within the membrane. The findings of McGilvray, Anghel et al. reveal a previously unknown cellular machinery which may be involved in the production of hundreds of human multi-pass proteins. This work provides a framework for understanding how these proteins are correctly made in the membrane. Additionally, it suggests that human diseases caused by mutations in TMCO1 result from a defect in the production of multi-pass membrane proteins.

Identification of Oxa1 Homologs Operating in the Eukaryotic Endoplasmic Reticulum.

Members of the evolutionarily conserved Oxa1/Alb3/YidC family mediate membrane protein biogenesis at the mitochondrial inner membrane, chloroplast thylakoid membrane, and bacterial plasma membrane, respectively. Despite their broad phylogenetic distribution, no Oxa1/Alb3/YidC homologs are known to operate in eukaryotic cells outside the endosymbiotic organelles. Here, we present bioinformatic evidence that the tail-anchored protein insertion factor WRB/Get1, the "endoplasmic reticulum (ER) membrane complex" subunit EMC3, and TMCO1 are ER-resident homologs of the Oxa1/Alb3/YidC family. Topology mapping and co-evolution-based modeling demonstrate that Get1, EMC3, and TMCO1 share a conserved Oxa1-like architecture. Biochemical analysis of human TMCO1, the only homolog not previously linked to membrane protein biogenesis, shows that it associates with the Sec translocon and ribosomes. These findings suggest a specific biochemical function for TMCO1 and define a superfamily of proteins-the "Oxa1 superfamily"-whose shared function is to facilitate membrane protein biogenesis.

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane.

Development of a Drosophila cell-based error correction assay.

Accurate transmission of the genome through cell division requires microtubules from opposing spindle poles to interact with protein super-structures called kinetochores that assemble on each sister chromatid. Most kinetochores establish erroneous attachments that are destabilized through a process called error correction. Failure to correct improper kinetochore-microtubule (kt-MT) interactions before anaphase onset results in chromosomal instability (CIN), which has been implicated in tumorigenesis and tumor adaptation. Thus, it is important to characterize the molecular basis of error correction to better comprehend how CIN occurs and how it can be modulated. An error correction assay has been previously developed in cultured mammalian cells in which incorrect kt-MT attachments are created through the induction of monopolar spindle assembly via chemical inhibition of kinesin-5. Error correction is then monitored following inhibitor wash out. Implementing the error correction assay in Drosophila melanogaster S2 cells would be valuable because kt-MT attachments are easily visualized and the cells are highly amenable to RNAi and high-throughput screening. However, Drosophila kinesin-5 (Klp61F) is unaffected by available small molecule inhibitors. To overcome this limitation, we have rendered S2 cells susceptible to kinesin-5 inhibitors by functionally replacing Klp61F with human kinesin-5 (Eg5). Eg5 expression rescued the assembly of monopolar spindles typically caused by Klp61F depletion. Eg5-mediated bipoles collapsed into monopoles due, in part, to kinesin-14 (Ncd) activity when treated with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC). Furthermore, bipolar spindles reassembled and error correction was observed after STLC wash out. Importantly, error correction in Eg5-expressing S2 cells was dependent on the well-established error correction kinase Aurora B. This system provides a powerful new cell-based platform for studying error correction and CIN.

Insights from an erroneous kinetochore-microtubule attachment state.

Faithful distribution of the genome requires that sister kinetochores, which assemble on each chromatid during cell division, interact with dynamic microtubules from opposite spindle poles in a configuration called chromosome biorientation. Biorientation produces tension that increases the affinity of kinetochores for microtubules via ill-defined mechanisms. Non-bioriented kinetochore-microtubule (kt-MT) interactions are prevalent but short-lived due to an error correction pathway that reduces the affinity of kinetochores for microtubules. Interestingly, incorrect kt-MT interactions can be stabilized by experimentally applying force to misoriented chromosomes. Here, a live-cell force assay is utilized to characterize the molecular composition of a common type of improper kt-MT attachment. Our force-related studies are also discussed in the context of current models for tension-dependent stabilization of kt-MT interactions.