Detection and quantification of Na,K-ATPase dimers in the plasma membrane of living cells by FRET-FCS.

The sodium potassium pump, Na,K-ATPase (NKA), is an integral plasma membrane protein, expressed in all eukaryotic cells. It is responsible for maintaining the transmembrane Na+ gradient and is the major determinant of the membrane potential. Self-interaction and oligomerization of NKA in cell membranes has been proposed and discussed but is still an open question. Here, we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET-FCS, to analyze NKA in the plasma membrane of living cells. Click chemistry was used to conjugate the fluorescent labels Alexa 488 and Alexa 647 to non-canonical amino acids introduced in the NKA α1 and ß1 subunits. We demonstrate that FRET-FCS can detect an order of magnitude lower concentration of green-red labeled protein pairs in a single-labeled red and green background than what is possible with cross-correlation (FCCS). We show that a significant fraction of NKA is expressed as a dimer in the plasma membrane. We also introduce a method to estimate not only the number of single and double labeled NKA, but the number of unlabeled, endogenous NKA and estimate the density of endogenous NKA at the plasma membrane to 1400 ± 800 enzymes/µm2.

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery.

Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.

Generation of Amber Suppression Cell Lines Using CRISPR-Cas9.

Genetic code expansion via amber suppression allows cotranslational, site-specific introduction of nonnatural chemical groups into proteins in the living cell. The archaeal pyrrolysine-tRNA/pyrrolysine-tRNA synthetase (PylT/RS) pair from Methanosarcina mazei (Mma) has been established for incorporation of a wide range of noncanonical amino acids (ncAAs) in mammalian cells. Once integrated in an engineered protein, ncAAs allow for simple click-chemistry derivatization, photo-cage control of enzyme activity, and site-specific placement of posttranslational modifications. We previously described a modular amber suppression plasmid system for generating stable cell lines via piggyBac transposition in a range of mammalian cells. Here we detail a general protocol for the generation of CRISPR-Cas9 knock-in cell lines using the same plasmid system. The knock-in strategy relies on CRISPR-Cas9-induced double-strand breaks (DSBs) and nonhomologous end joining (NHEJ) repair to target the PylT/RS expression cassette to the AAVS1 safe harbor locus in human cells. MmaPylRS expression from this single locus is sufficient for efficient amber suppression when the cells are subsequently transfected transiently with a PylT/gene of interest plasmid.

Translational readthrough of nonsense mutant TP53 by mRNA incorporation of 5-Fluorouridine.

TP53 nonsense mutations in cancer produce truncated inactive p53 protein. We show that 5-FU metabolite 5-Fluorouridine (FUr) induces full-length p53 in human tumor cells carrying R213X nonsense mutant TP53. Ribosome profiling visualized translational readthrough at the R213X premature stop codon and demonstrated that FUr-induced readthrough is less permissive for canonical stop codon readthrough compared to aminoglycoside G418. FUr is incorporated into mRNA and can potentially base-pair with guanine, allowing insertion of Arg tRNA at the TP53 R213X UGA premature stop codon and translation of full-length wild-type p53. We confirmed that full-length p53 rescued by FUr triggers tumor cell death by apoptosis. FUr also restored full-length p53 in TP53 R213X mutant human tumor xenografts in vivo. Thus, we demonstrate a novel strategy for therapeutic rescue of nonsense mutant TP53 and suggest that FUr should be explored for treatment of patients with TP53 nonsense mutant tumors.

A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling.

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

Engineered Human Induced Pluripotent Cells Enable Genetic Code Expansion in Brain Organoids.

Human induced pluripotent stem cell (hiPSC) technology has revolutionized studies on human biology. A wide range of cell types and tissue models can be derived from hiPSCs to study complex human diseases. Here, we use PiggyBac-mediated transgenesis to engineer hiPSCs with an expanded genetic code. We demonstrate that genomic integration of expression cassettes for a pyrrolysyl-tRNA synthetase (PylRS), pyrrolysyl-tRNA (PylT) and the target protein of interest enables site-specific incorporation of a non-canonical amino acid (ncAA) in response to an amber stop codon. Neural stem cells, neurons and brain organoids derived from the engineered hiPSCs continue to express the amber suppression machinery and produce ncAA-bearing reporter. The incorporated ncAA can serve as a minimal bioorthogonal handle for further modifications by labeling with fluorescent dyes. Site-directed ncAA mutagenesis will open a wide range of applications to probe and manipulate proteins in brain organoids and other hiPSC-derived cell types and complex tissue models.

Multianalyte serology in home-sampled blood enables an unbiased assessment of the immune response against SARS-CoV-2.

Serological testing is essential to curb the consequences of the COVID-19 pandemic. However, most assays are still limited to single analytes and samples collected within healthcare. Thus, we establish a multianalyte and multiplexed approach to reliably profile IgG and IgM levels against several versions of SARS-CoV-2 proteins (S, RBD, N) in home-sampled dried blood spots (DBS). We analyse DBS collected during spring of 2020 from 878 random and undiagnosed individuals from the population in Stockholm, Sweden, and use classification approaches to estimate an accumulated seroprevalence of 12.5% (95% CI: 10.3%-14.7%). This includes 5.4% of the samples being IgG+IgM+ against several SARS-CoV-2 proteins, as well as 2.1% being IgG-IgM+ and 5.0% being IgG+IgM- for the virus' S protein. Subjects classified as IgG+ for several SARS-CoV-2 proteins report influenza-like symptoms more frequently than those being IgG+ for only the S protein (OR = 6.1; p < 0.001). Among all seropositive cases, 30% are asymptomatic. Our strategy enables an accurate individual-level and multiplexed assessment of antibodies in home-sampled blood, assisting our understanding about the undiagnosed seroprevalence and diversity of the immune response against the coronavirus.

Site-Specific Incorporation of Two ncAAs for Two-Color Bioorthogonal Labeling and Crosslinking of Proteins on Live Mammalian Cells.

The pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase (PylT/RS) pair from the archaeon Methanosarcina mazei (Mma) is widely used in protein engineering to site-specifically introduce noncanonical amino acids (ncAAs) through nonsense codon suppression. Here, we engineer the PylT/RS pair encoded by Methanogenic archaeon ISO4-G1 (G1) to be orthogonal to Mma PylT/RS and alter the G1 PylRS active site to accept a complementary ncAA spectrum. We combine the resulting mutual orthogonal pairs for site-specific dual ncAA incorporation of two lysine analogs with high selectivity and efficiency. Demonstrating the robustness of the system, we incorporate two ncAAs with compatible bioorthogonal reactivity into a Notch receptor, as well as a G protein-coupled receptor. We show that selective and site-specific incorporation of two ncAAs allows for two-color bioorthogonal labeling as well as chemical-controlled crosslinking of surface proteins on live mammalian cells.

Methanomethylophilus alvus Mx1201 Provides Basis for Mutual Orthogonal Pyrrolysyl tRNA/Aminoacyl-tRNA Synthetase Pairs in Mammalian Cells.

Genetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded noncanonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei ( Mma PylRS/PylT) being the most active and versatile to date. We found a pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that this PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its noncognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

The t6A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in Streptococcus mutans.

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNALysUUU in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t6A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t6A-deficient yeast derivatives, it is shown that t6A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t6A synthesis gene tsaE (brpB) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the S. mutans tsaE mutant was devoid of t6A. However, we describe here a novel and a more sensitive hybridization-based t6A detection method (compared to HPLC) that showed t6A was still present in the S. mutans ΔtsaE, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other S. mutans t6A synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of t6A modification ratios and of t6A synthesis genes mRNA levels in S. mutans suggest they may be regulated by growth phase.

Structural basis for Pan3 binding to Pan2 and its function in mRNA recruitment and deadenylation.

The conserved eukaryotic Pan2-Pan3 deadenylation complex shortens cytoplasmic mRNA 3' polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C-terminal domain and via an N-terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N-terminal WD40 domain to the C-terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high-affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2-Pan3 complex.

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair.

PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases. These secreted ribotoxins are assimilated by Saccharomyces cerevisiae, wherein they arrest growth by depleting specific tRNAs. Toxicity can be recapitulated by induced intracellular expression of PaOrf2 or γ-toxin in S. cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, we report that PaOrf2 residues Glu9 and His287 (putative counterparts of γ-toxin Glu9 and His209) are essential for toxicity. Our results suggest a similar basis for RNA transesterification by PaOrf2 and γ-toxin, despite their dissimilar primary structures and distinctive tRNA target specificities. PaOrf2 makes two sequential incisions in tRNA, the first of which occurs 3' from the mcm(5)s(2)U wobble nucleoside and depends on mcm(5). A second incision two nucleotides upstream results in the net excision of a di-nucleotide. Expression of phage and plant tRNA repair systems can relieve PaOrf2 toxicity when tRNA cleavage is restricted to the secondary site in elp3 cells that lack the mcm(5) wobble U modification. Whereas the endogenous yeast tRNA ligase Trl1 can heal tRNA halves produced by PaOrf2 cleavage in elp3 cells, its RNA sealing activity is inadequate to complete the repair. Compatible sealing activity can be provided in trans by plant tRNA ligase. The damage-rescuing ability of tRNA repair systems is lost when PaOrf2 can break tRNA at both sites. These results highlight the logic of a two-incision mechanism of tRNA anticodon damage that evades productive repair by tRNA ligases.

Structure-function relations in the NTPase domain of the antiviral tRNA ribotoxin Escherichia coli PrrC.

Breakage of tRNA by Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection. Expression of EcoPrrC is cytocidal in yeast, signifying that PrrC ribotoxicity crosses phylogenetic domain boundaries. EcoPrrC consists of an N-terminal NTPase module that resembles ABC transporters and a C-terminal nuclease module that is sui generis. PrrC homologs are prevalent in many other bacteria. Here we report that Haemophilus influenzae PrrC is toxic in E. coli and yeast. To illuminate structure-activity relations, we conducted a new round of mutational analysis of EcoPrrC guided by primary structure conservation among toxic PrrC homologs. We indentify 17 candidate active site residues in the NTPase module that are essential for toxicity in yeast when EcoPrrC is expressed at high gene dosage. Their functions could be educed by integrating mutational data with the atomic structure of the transition-state complex of a homologous ABC protein.

Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair.

Breakage of tRNA(Lys(UUU)) by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection that is ultimately thwarted by a virus-encoded RNA repair system. PrrC homologs are prevalent in other bacteria, but their activities and substrates are not defined. We find that induced expression of EcoPrrC is toxic in Saccharomyces cerevisiae and E. coli, whereas the Neisseria meningitidis PrrC (NmePrrC) is not. PrrCs consist of an N-terminal NTPase module and a C-terminal nuclease module. Domain swaps identified the EcoPrrC nuclease domain as decisive for toxicity when linked to either the Eco or Nme NTPase. Indeed, a single arginine-to-tryptophan change in the NmePrrC nuclease domain (R316W) educed a gain-of-function and rendered NmePrrC toxic to yeast, with genetic evidence for tRNA(Lys(UUU)) being the relevant target. The reciprocal Trp-to-Arg change in EcoPrrC (W335R) abolished its toxicity. Further mutagenesis of the EcoPrrC nuclease domain highlighted an ensemble of 15 essential residues and distinguished between hypomorphic alleles and potential nuclease-nulls. We report that the RNA repair phase of the bacterial virus-host dynamic is also portable to yeast, where coexpression of the T4 enzymes Pnkp and Rnl1 ameliorated the toxicity of NmePrrC-R316W. Plant tRNA ligase AtRNL also countered NmePrrC-R316W toxicity, in a manner that depended on AtRNL's 5'-kinase and ligase functions.

RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo.

RtcB enzymes are novel RNA ligases that join 2',3'-cyclic phosphate and 5'-OH ends. The phylogenetic distribution of RtcB points to its candidacy as a tRNA splicing/repair enzyme. Here we show that Escherichia coli RtcB is competent and sufficient for tRNA splicing in vivo by virtue of its ability to complement growth of yeast cells that lack the endogenous "healing/sealing-type" tRNA ligase Trl1. RtcB also protects yeast trl1Δ cells against a fungal ribotoxin that incises the anticodon loop of cellular tRNAs. Moreover, RtcB can replace Trl1 as the catalyst of HAC1 mRNA splicing during the unfolded protein response. Thus, RtcB is a bona fide RNA repair enzyme with broad physiological actions. Biochemical analysis of RtcB highlights the uniqueness of its active site and catalytic mechanism. Our findings draw attention to tRNA ligase as a promising drug target.

Determinants of eukaryal cell killing by the bacterial ribotoxin PrrC.

tRNA damage inflicted by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies an antiviral response to phage T4 infection. PrrC homologs are present in many bacterial proteomes, though their biological activities are uncharted. PrrCs consist of two domains: an N-terminal NTPase module related to the ABC family and a distinctive C-terminal ribonuclease module. In this article, we report that the expression of EcoPrrC in budding yeast is fungicidal, signifying that PrrC is toxic in a eukaryon in the absence of other bacterial or viral proteins. Whereas Streptococcus PrrC is also toxic in yeast, Neisseria and Xanthomonas PrrCs are not. Via analysis of the effects of 118 mutations on EcoPrrC toxicity in yeast, we identified 22 essential residues in the NTPase domain and 11 in the nuclease domain. Overexpressing PrrCs with mutations in the NTPase active site ameliorated the toxicity of wild-type EcoPrrC. Our findings support a model in which EcoPrrC toxicity is contingent on head-to-tail dimerization of the NTPase domains to form two composite NTP phosphohydrolase sites. Comparisons of EcoPrrC activity in a variety of yeast genetic backgrounds, and the rescuing effects of tRNA overexpression, implicate tRNA(Lys(UUU)) as a target of EcoPrrC toxicity in yeast.

Structure-activity relationships in Kluyveromyces lactis gamma-toxin, a eukaryal tRNA anticodon nuclease.

tRNA anticodon damage inflicted by secreted ribotoxins such as Kluyveromyces lactis gamma-toxin and bacterial colicins underlies a rudimentary innate immune system that distinguishes self from nonself species. The intracellular expression of gamma-toxin (a 232-amino acid polypeptide) arrests the growth of Saccharomyces cerevisiae by incising a single RNA phosphodiester 3' of the modified wobble base of tRNA(Glu). Fungal gamma-toxin bears no primary structure similarity to any known nuclease and has no plausible homologs in the protein database. To gain insight to gamma-toxin's mechanism, we tested the effects of alanine mutations at 62 basic, acidic, and polar amino acids on ribotoxin activity in vivo. We thereby identified 22 essential residues, including 10 lysines, seven arginines, three glutamates, one cysteine, and one histidine (His209, the only histidine present in gamma-toxin). Structure-activity relations were gleaned from the effects of 44 conservative substitutions. Recombinant tag-free gamma-toxin, a monomeric protein, incised an oligonucleotide corresponding to the anticodon stem-loop of tRNA(Glu) at a single phosphodiester 3' of the wobble uridine. The anticodon nuclease was metal independent. RNA cleavage was abolished by ribose 2'-H and 2'-F modifications of the wobble uridine. Mutating His209 to alanine, glutamine, or asparagine abolished nuclease activity. We propose that gamma-toxin catalyzes an RNase A-like transesterification reaction that relies on His209 and a second nonhistidine side chain as general acid-base catalysts.

The outer membrane form of the mitochondrial protein Mcr1 follows a TOM-independent membrane insertion pathway.

The yeast gene MCR1 encodes two isoforms of the mitochondrial NADH-cytochrome b5 reductase. One form is embedded in the outer membrane whereas the other is located in the intermembrane space (IMS). In the present work we investigated the biogenesis of the outer membrane form. We demonstrate that while the IMS form crosses the outer membrane via the translocase of the outer mitochondrial membrane (TOM) complex, the other form is integrated into the outer membrane by a process that does not require any of the known import components at the outer membrane. Thus, the import pathways of the two forms diverge in a stage before the encounter with the TOM complex and their mechanism of biogenesis represents a unique example how to achieve dual localization within one organelle.