Acceptor Proteins Research Articles

Interrogation of skeletal muscle activation in an intact system via a myofilament incorporated FRET biosensor.

It is important to understand the mechanisms involved in regulating skeletal muscle activation and contraction for advancing insights into acquired and inherited muscle disorders and for identifying potential targets for therapeutics. The field has elucidated many sarcomeric components needed for activation of contraction. We seek to expand this work using a fluorescent sarcomere biosensor that allows for monitoring of sarcomeric activation in live, intact skeletal muscles. This biosensor fuses fluorescent donor and acceptor proteins to the N- and C-terminals of fast skeletal troponin C protein, respectively. Using the biosensor, skeletal muscle activation is monitored in real time through Förster Resonance Energy Transfer (FRET) fluorescence. This system maintains all essential, physiological features of the muscle, including excitation-contraction coupling and load, which was not possible in reconstituted systems. Using a transgenic mouse line with the biosensor, we have demonstrated FRET fluorescence in intact muscles from transgenic animals, with the time to peak of the fluorescence preceding the force development and the biosensor kinetics uncoupled from intracellular calcium kinetics. Of note, the FRET signal has a prolonged return to baseline as compared to force in intact EDL muscles, something that is not observed in unloaded FDB myofibers, and which may be evidence of a “primed state” of myofilament activation. The “primed state” is uncoupled from Ca2+ and is modified using fast skeletal muscle specific small molecules that target either the thin filament or thick filament. As thick filament modulating small molecules could be detected by the TnC localized biosensor during single twitch contractions, this demonstrates intermyofilament signaling in live muscle. Additionally, the “primed state” duration can be impacted through varying stimulation protocols, including dual pulse stimulations. This unique phenomenon will be further investigated using mechanical and pharmacological manipulations.

Modular and genetically encodable RNA-based bioluminescence resonance energy transfer (BRET) sensors for cellular target detection.

Genetically encodable bioluminescent sensors have been broadly used for highly sensitive, low-background, low-phototoxicity bioanalysis and imaging in living biological systems. To achieve real-time quantitative measurement and high-throughput screening, powerful bioluminescence resonance energy transfer (BRET) sensors have been designed, especially based on interactions between a luciferase/luciferin donor and a fluorescent protein acceptor. While fluorescent protein-based BRET sensors have been widely applied in cellular and in vivo studies, more modular and easy-to-engineer RNA-based BRET sensors have never been reported. In this project, we report, for the first time, genetically encoded fluorogenic RNA-based BRET sensors for cellular imaging and quantification of different target analytes and RNA-protein interactions. Fluorogenic RNAs are single-stranded RNA aptamers that can bind specific small-molecule fluorophores and activate the fluorescence signals. By coupling a NanoLuc luciferase donor with different types of fluorogenic RNA acceptor, without reliance on external light, sensitive BRET signals are generated in response to target metabolite, signaling molecule, or RNA levels inside living cells. We envision that this novel genetically encoded RNA-based BRET system can be widely used in bioanalysis and nanomedicine.

A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli.

Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli.

The preparation of recombinant arginyltransferase 1 (ATE1) for biophysical characterization.

Arginyltransferases (ATE1s) are eukaryotic enzymes that catalyze the non-ribosomal, post-translational addition of the amino acid arginine to an acceptor protein. While understudied, post-translation arginylation and ATE1 have major impacts on eukaryotic cellular homeostasis through both degradative and non-degradative effects on the intracellular proteome. Consequently, ATE1-catalyzed arginylation impacts major eukaryotic biological processes including the stress response, cellular motility, cardiovascular maturation, and even neurological function. Despite this importance, there is a lack of information on the structural and biophysical characteristics of ATE1, prohibiting a comprehensive understanding of the mechanism of this post-translational modification, and hampering efforts to design ATE1-specific therapeutics. To that end, this chapter details a protocol designed for the expression and the purification of ATE1 from Saccharomyces cerevisiae, although the approaches described herein should be generally applicable to other eukaryotic ATE1s. The detailed procedures afford high amounts of pure, homogeneous, monodisperse ATE1 suitable for downstream biophysical analyses such as X-ray crystallography, small angle X-ray scattering (SAXS), and cryo-EM techniques.

Measuring NLR Oligomerization III: Detection of NLRP3 and NLRC4 Complex by Bioluminescence Resonance Energy Transfer.

Bioluminescent resonance energy transfer (BRET) is a natural phenomenon resulting from a non-radiative energy transfer between a bioluminescent donor (Renilla luciferase) and a fluorescent protein acceptor. BRET signal is dependent on the distance and the orientation between the donor and the acceptor and could be used to study protein-protein interactions and conformational changes within proteins at real-time in living cells. This protocol describes the use of BRET technique to study NLRP3 oligomerization in living cells before and during NLRP3 inflammasome activation.

Disruption of nucleosomes by DNA groove binders of clinical significance and implications for chromatin remodeling

Small molecules that bind in the minor groove of DNA are in clinical use as antibiotics and antitumor drugs. Two members of this class of molecules, netropsin and chromomycin, are shown here to displace DNA from the nucleosome and promote transfer of the histone octamer to an acceptor protein. The effects of these groove-binding molecules are exploited to address an outstanding problem in the mechanism of the RSC chromatin remodeling complex. RSC and other remodeling complexes are DNA translocases, acting near the center of the nucleosomal DNA, but translocation is apparently impossible because DNA cannot slide across the histone surface in the nucleosome. Netropsin and chromomycin promote the release of DNA from the histone surface, enhance the formation of a RSC-nucleosome complex, and synergize with RSC in chromatin remodeling. These findings are in keeping with an involvement of bulge translocation in chromatin remodeling.

Discovery and characterization of a new class of O-linking oligosaccharyltransferases from the Moraxellaceae family.

Bacterial protein glycosylation is commonly mediated by oligosaccharyltransferases (OTases) that transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Natively, O-linking OTases usually transfer a single repeat unit of the O-antigen or capsular polysaccharide to the side chains of serine or threonine on acceptor proteins. Three major families of bacterial O-linking OTases have been described: PglL, PglS, and TfpO. TfpO is limited to transferring short oligosaccharides both in its native context and when heterologously expressed in glycoengineered Escherichia coli. On the other hand, PglL and PglS can transfer long-chain polysaccharides when expressed in glycoengineered E. coli. Herein, we describe the discovery and functional characterization of a novel family of bacterial O-linking OTases termed TfpM from Moraxellaceae bacteria. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in distinct clades from known bacterial OTases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We further demonstrated that TfpM has broad substrate tolerance and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. Last, we find that a TfpM-derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. The glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox.

Intestinal Epithelial STAT6 Activation Rescues the Defective Anti-Helminth Responses Caused by Ogt Deletion.

Dynamic regulation of intestinal epithelial cell (IEC) proliferation and differentiation is crucial for maintaining mucosa homeostasis and the response to helminth infection. O-GlcNAc transferase (OGT), an enzyme catalyzing the transfer of GlcNAc from the donor substrate UDP-GlcNAc onto acceptor proteins, has been proposed to promote intestinal epithelial remodeling for helminth expulsion by modifying and activating epithelial STAT6, but whether the IEC intrinsic OGT-STAT6 axis is involved in anti-helminth responses has not been tested in vivo. Here, we show that the inducible deletion of Ogt in IECs of adult mice leads to reduced tuft and goblet cell differentiation, increased crypt cell proliferation, and aberrant Paneth cell localization. By using a mouse model with concurrent Ogt deletion and STAT6 overexpression in IECs, we provide direct in vivo evidence that STAT6 acts downstream of OGT to control tuft and goblet cell differentiation in IECs. However, epithelial OGT regulates crypt cell proliferation and Paneth cell differentiation in a STAT6-independent pathway. Our results verify that protein O-GlcNAcylation in IECs is crucial for maintaining epithelial homeostasis and anti-helminthic type 2 immune responses.

PARP1: Liaison of Chromatin Remodeling and Transcription.

Simple SummaryPoly(ADP-ribose) polymerase 1 (PARP1) is perhaps the most studied member of the PARP superfamily and participates in numerous cellular processes. PARP1 inhibitors have been approved as drugs to treat various cancers in clinics, based on its role in DNA repair. Yet, there is a growing body of evidence showing multitasking function of PARP1 in regulation of gene expression. In this review article, we discuss the current knowledge of PARP1 and its conducted enzymatic process, i.e., PARylation, with an emphasis on gene expression by the interaction with transcription factors and regulation of chromatin conformation, dependent or independent of DNA damage. The molecular action mode of PARP1 in gene transcription may present as a potential target for therapeutic intervention of inflammation-related diseases and also for cancer therapy.Poly(ADP-ribosyl)ation (PARylation) is a covalent post-translational modification and plays a key role in the immediate response of cells to stress signals. Poly(ADP-ribose) polymerase 1 (PARP1), the founding member of the PARP superfamily, synthesizes long and branched polymers of ADP-ribose (PAR) onto acceptor proteins, thereby modulating their function and their local surrounding. PARP1 is the most prominent of the PARPs and is responsible for the production of about 90% of PAR in the cell. Therefore, PARP1 and PARylation play a pleotropic role in a wide range of cellular processes, such as DNA repair and genomic stability, cell death, chromatin remodeling, inflammatory response and gene transcription. PARP1 has DNA-binding and catalytic activities that are important for DNA repair, yet also modulate chromatin conformation and gene transcription, which can be independent of DNA damage response. PARP1 and PARylation homeostasis have also been implicated in multiple diseases, including inflammation, stroke, diabetes and cancer. Studies of the molecular action and biological function of PARP1 and PARylation provide a basis for the development of pharmaceutic strategies for clinical applications. This review focuses primarily on the role of PARP1 in the regulation of chromatin remodeling and transcriptional activation.

Abstract 2463: 18F-Talazoparib: A novel potential PET imaging agent of PARP

Abstract Background: Poly-(ADP-ribose) polymerases (PARPs) are an enzyme family that catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to an acceptor protein. PARP family members play fundamental roles in single-strand DNA break (SSB) repair, cell signaling of DNA damage and inflammation, cell death, and cellular replication. Various PARP inhibitors have been developed and PARP1 inhibitors are currently used to treat subsets of breast and ovarian cancer patients. Of all the PARP inhibitors studied, Talazoparib (Pfizer), appears to be the most stable in pre-clinical and clinical studies, affords nanomolar affinity to PARP1, and demonstrates the highest affinity trapping of PARP on the DNA-protein complex. Imaging of PARP using a radiolabeled inhibitor has been proposed for patient selection, therapy dose optimization and validation of target engagement. Several PARP imaging agents have been developed and studied as possible tracers, by modifying the structure of an original PARP inhibitor to accommodate an imaging or therapeutic isotope. Herein we used a copper-mediated 18F-radio-fluorination strategy with a novel aryl boronic ester precursor to access 18F-Talazoparib (18F-TZ), a 18F-radiolabeled form of the structurally identical Talazoparib. Methods: A novel boronic ester precursor was synthetized in six steps synthesis with an overall yield of 3.3% and characterized by NMR and mass spectrometry. Radiofluorination was performed using an automatic GE TracerLab system and analytical radioHPLC was used for tracer characterization and for stability analysis. A panel of tumor cell lines representing a variety of PARP expression levels (Hela, SUM149, SUM1315, MDA-MB-436, MDA-MB-231, PSN1, MiaPaca2, Jurkat) were tested for time-dependent uptake and specific binding, utilizing both 4 °C and excess cold TZ blocking. Results: 18F-TZ was synthetized in 5.0±0.80% (n=20) radiochemical yield and a molar activity of 18.9-98.0 GBq/µmol by a copper-mediated radiofluorination of the corresponding pinacolic boronic ester, followed by purification via semi-preparative HPLC and reformulation in ethanol. 18F-TZ was obtained in >99% radiochemical purity, displaying up to 4 hr shelf stability (25 °C and 4 °C) in PBS (10% EtOH). 18F-TZ was also stable in human plasma up to 4 hr at 37 °C. Both shelf and plasma stability tests showed a decrease of the radioHPLC peak consistent with decay half-life, but did not show the formation of secondary species. All cell lines selected for the in cellulo study showed uptake of radioactive 18F-TZ after 30 min of incubation at 37 °C. Cell uptake was blocked >98% by excess cold TZ or incubation of cells at 4 °C. Conclusion: 18F-Talazoparib was synthesized by copper-mediated radiofluorination from the corresponding pinacol ester and demonstrates both uptake and target specificity in PARP-expressing tumor cells. Citation Format: Riccardo Muzzioli, Federica Pisaneschi, Yi Rao, Seth Gammon, David Piwnica-Worms. 18F-Talazoparib: A novel potential PET imaging agent of PARP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2463.

Interplay between symmetric arginine dimethylation and ubiquitylation regulates TDP1 proteostasis for the repair of topoisomerase I-DNA adducts.

Tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety and is implicated in the repair of trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation of TDP1 at the residues R361 and R586. Here, we establish mechanistic crosstalk between TDP1 arginine methylation and ubiquitylation, which is critical for TDP1 homeostasis and cellular responses to Top1 poisons. We show that R586 methylation promotes TDP1 ubiquitylation, which facilitates ubiquitin/proteasome-dependent TDP1 turnover by impeding the binding of UCHL3 (deubiquitylase enzyme) with TDP1. TDP1-R586 also promotes TDP1-XRCC1 binding and XRCC1 foci formation at Top1cc-damage sites. Intriguingly, R361 methylation enhances the 3'-phosphodiesterase activity of TDP1 in real-time fluorescence-based cleavage assays, and this was rationalized using structural modeling. Together, our findings establish arginine methylation as a co-regulator of TDP1 proteostasis and activity, which modulates the repair of trapped Top1cc.

Towards a metabolomic approach to investigate iron-sulfur cluster biogenesis.

Iron–sulfur clusters are prosthetic groups that are assembled on their acceptor proteins through a complex machine centered on a desulfurase enzyme and a transient scaffold protein. Studies to establish the mechanism of cluster formation have so far used either in vitro or in vivo methods, which have often resulted in contrasting or non‐comparable results. We suggest, here, an alternative approach to study the enzymatic reaction, that is based on the combination of genetically engineered bacterial strains depleted of specific components, and the detection of the enzymatic kinetics in cellular extracts through metabolomics. Our data prove that this ex vivo approach closely reproduces the in vitro results while retaining the full complexity of the system. We demonstrate that co‐presence of bacterial frataxin and iron is necessary to observe an inhibitory effect of the enzymatic activity of bacterial frataxin. Our approach provides a new powerful tool for the study of iron–sulfur cluster biogenesis.

Computational Analysis and Molecular Design of Aromatic Stacking System Between Human ARTD2 Catalytic Domain and its Small-Molecule Inhibitors and Peptidic Ligands

ADP-ribosyltransferase 2 (ARTD2) acts as a DNA break sensor and catalyzes the synthesis of polymers of ADP-ribose covalently attached to acceptor proteins at DNA damage sites. Its catalytic domain (CAT) contains a variety of aromatic amino aid residues that can form multiple [Formula: see text]-stacking interactions with substrates and inhibitors. Here, we systematically examined the intermolecular interaction of ARTD2 CAT domain with its cognate small-molecule inhibitors using hybrid quantum mechanics calculations and found that [Formula: see text]-stacking plays a critical role in the ARTD2-inhibitor recognition and association, where a number of ARTD2 inner aromatic residues contribute significantly to the inhibitor binding. A further molecular design of peptidic ligands was performed based on the structural basis and energetic property of the [Formula: see text]-stacking system involved in ARTD2/inhibitor interaction. These peptidic ligands are all [Formula: see text] electron-rich and composed of diverse unnatural aromatic amino acids. Interaction analysis revealed that the relative contribution of the [Formula: see text]-stacking system to ARTD2 affinity was improved from small-molecule inhibitors to peptidic ligands, although their total binding energies are roughly comparable. Subsequently, the computational findings were substantiated with biochemical assays, where several representative peptidic ligands were determined to have satisfactory inhibitory profiles against ARTD2 with biological activity at the nanomolar level. Structural analysis of ARTD2/peptidic ligand complexes observed a multiple [Formula: see text]-stacking networks at the computationally modeled complex interface of ARTD2 CAT domain with designed peptidic ligands, which can work with other nonbonded interactions such as hydrogen bonds and hydrophobic contacts to confer stability and selectivity for the complex recognition and association.

Distinct phosphorylation signals drive acceptor versus free ubiquitin chain targeting by parkin.

The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of the Ubl domain in parkin, both mediated by the OMM kinase, PINK1. How an OMM protein is selected for ubiquitination is unclear. Parkin targeted OMM proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. Here, we used chimeric proteins, tagged with ubiquitin (Ub), to evaluate parkin ubiquitination of mitochondrial acceptor proteins pre-ligated to Ub. We find that pUb tethered to the mitochondrial target proteins, Miro1 or CISD1, is necessary for parkin recruitment and essential for target protein ubiquitination. Surprisingly, phosphorylation of parkin is not necessary for the ubiquitination of either Miro1 or CISD1. Thus, parkin lacking its Ubl domain efficiently ubiquitinates a substrate tethered to pUb. Instead, phosphorylated parkin appears to stimulate free Ub chain formation. We also demonstrate that parkin ubiquitination of pUb-tethered substrates occurs on the substrate, rather than the pUb modification. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb on the OMM protein and subsequent parkin phosphorylation triggers free Ub chain formation. This finding accounts for the broad spectrum of OMM proteins ubiquitinated by parkin and has implications on target design for therapeutics.

Tankyrase Regulates Neurite Outgrowth through Poly(ADP-ribosyl)ation-Dependent Activation of β-Catenin Signaling.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins by transferring poly(ADP-ribose) (PAR) to acceptor proteins by the action of poly(ADP-ribose) polymerase (PARP). Two tankyrase (TNKS) isoforms, TNK1 and TNK2 (TNKS1/2), are ubiquitously expressed in mammalian cells and participate in diverse cellular functions, including wnt/β-catenin signaling, telomere maintenance, glucose metabolism and mitosis regulation. For wnt/β-catenin signaling, TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, a key component of the β-catenin degradation complex, which allows Axin’s ubiquitination and subsequent degradation, thereby activating β-catenin signaling. In the present study, we focused on the functions of TNKS1/2 in neuronal development. In primary hippocampal neurons, TNKS1/2 were detected in the soma and neurites, where they co-localized with PAR signals. Treatment with XAV939, a selective TNKS1/2 inhibitor, suppressed neurite outgrowth and synapse formation. In addition, XAV939 also suppressed norepinephrine uptake in PC12 cells, a rat pheochromocytoma cell line. These effects likely resulted from the inhibition of β-catenin signaling through the stabilization of Axin, which suggests TNKS1/2 enhance Axin degradation by modifying its poly(ADP-ribosyl)ation, thereby stabilizing wnt/β-catenin signaling and, in turn, promoting neurite outgrowth and synapse formation.

A FRET-based method for monitoring structural transitions in protein self-organization

SummaryProteins assemble into a variety of dynamic and functional structures. Their structural transitions are often challenging to distinguish inside cells, particularly with a high spatiotemporal resolution. Here, we present a fluorescence resonance energy transfer (FRET)-based method for continuous and high-throughput monitoring of protein self-assemblies to reveal well-resolved transient intermediate states. Intermolecular FRET with both the donor and acceptor proteins at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We apply this method to monitor self-assembly of three proteins. We show that the mutant Huntingtin exon1 (mHttex1) first forms less-ordered assemblies, which develop into fibril-like aggregates, and demonstrate that the chaperone protein DNAJB6b increases the critical saturation concentration of mHttex1. We also monitor the structural changes in fused in sarcoma (FUS) condensates. This method adds to the toolbox for protein self-assembly structure and kinetics determination, and implementation with native or non-native proteins can inform studies involving protein condensation or aggregation.

PARP1 is activated by membrane damage and is involved in membrane repair through poly(ADP-ribosyl)ation.

Mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation are posttranslational modifications evolutionarily conserved in prokaryotes and eukaryotes. They entail transfer of one or more ADP-ribose moieties from NAD+ to acceptor proteins with the simultaneous release of nicotinamide. The resultant ADP-ribosylated acceptor proteins regulate diverse cellular functions. For instance, ADP-ribosyltransferase 1 (ART1) catalyzes mono(ADP-ribosyl)ation of arginine residues in Trim72, a protein specifically expressed in muscle cells and involved in cell membrane repair, which is enhanced upon its ADP-ribosylation. By contrast, the contribution made by ADP-ribosylation to membrane repair in epithelial cells remains unclear. In this study, we investigated the involvement of ADP-ribosylation in cell membrane repair in HEK293T and HeLa cells. We found that upon induction of membrane damage using streptolysin-O, poly(ADP-ribose) polymerase 1 (PARP1) catalyzed poly(ADP-ribosyl)ation. In scratch assays, inhibition of PARP1 activity using the nonspecific PARP inhibitor PJ34 or shRNA targeting PARP1 delayed wound healing, suggesting that PARP1-catalyzed poly(ADP-ribosyl)ation plays a key role in membrane repair in epithelial cells.

The cytosolic Arabidopsis thaliana cysteine desulfurase ABA3 delivers sulfur to the sulfurtransferase STR18

The biosynthesis of many sulfur-containing molecules depends on cysteine as a sulfur source. Both the cysteine desulfurase (CD) and rhodanese (Rhd) domain–containing protein families participate in the trafficking of sulfur for various metabolic pathways in bacteria and human, but their connection is not yet described in plants. The existence of natural chimeric proteins containing both CD and Rhd domains in specific bacterial genera, however, suggests a general interaction between these proteins. We report here the biochemical relationships between two cytosolic proteins from Arabidopsis thaliana, a Rhd domain–containing protein, the sulfurtransferase 18 (STR18), and a CD isoform referred to as ABA3, and compare these biochemical features to those of a natural CD–Rhd fusion protein from the bacterium Pseudorhodoferax sp. We observed that the bacterial enzyme is bifunctional exhibiting both CD and STR activities using l-cysteine and thiosulfate as sulfur donors but preferentially using l-cysteine to catalyze transpersulfidation reactions. In vitro activity assays and mass spectrometry analyses revealed that STR18 stimulates the CD activity of ABA3 by reducing the intermediate persulfide on its catalytic cysteine, thereby accelerating the overall transfer reaction. We also show that both proteins interact in planta and form an efficient sulfur relay system, whereby STR18 catalyzes transpersulfidation reactions from ABA3 to the model acceptor protein roGFP2. In conclusion, the ABA3–STR18 couple likely represents an uncharacterized pathway of sulfur trafficking in the cytosol of plant cells, independent of ABA3 function in molybdenum cofactor maturation.

A novel violet fluorescent protein contains a unique oxidized tyrosine as the simplest chromophore ever reported in fluorescent proteins.

We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP-like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high-resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and Ec 14,300 M-1 cm-1 ) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors.

Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe.

Dolichol diphosphate-linked oligosaccharides (LLO) are the sugar donors in N -glycosylation, a fundamental protein post-translational modification of the eukaryotic secretory pathway. Defects in LLO biosynthesis produce human Congenital Disorders of Glycosylation Type I. The synthesis of LLOs and the transfer reactions to their protein acceptors is highly conserved among animal, plant, and fungi kingdoms, making the fission yeast Schizosaccharomyces pombe a suitable model to study these processes. Here, we present a protocol to determine the LLO patterns produced in vivo by S. pombe cells that may be easily adapted to other cell types. First, exponentially growing cultures are labeled with a pulse of [ 14 C]-glucose. LLOs are then purified by successive extractions with organic solvents, and glycans are separated from the lipid moieties in mild acid hydrolysis and a new solvent extraction. The purified glycans are then run on paper chromatography. We use a deconvolution process to adjust the profile obtained to the minimal number of Gaussian functions needed to fit the data and determine the proportion of each species with respect to total glycan species present in the cell. The method we provide here might be used without any expensive or specialized equipment. The deconvolution process described here might also be useful to analyze species in non-completely resolved chromatograms. Graphical abstract: Workflow for the labeling, extraction, separation, and identification of LLO species in S. pombe . (A) Radioactive pulse of S. pombe cells with [ 14 C]-glucose for 15 min at 28 °C. (B) Organic extraction of LLOs from labeled yeasts sequentially using methanol, chloroform, H 2 O, chloroform:methanol:H 2 O (1:1:0.3), 0.02 M HCl (to separate glycans from dolichol), and chloroform:methanol:H 2 O (1:16:16). (C) Preparation of the sample for chromatography on paper: drying by airflow and radioactivity check. (D) Loading of samples in chromatographic paper and descendent chromatography in a glass chamber. The obtained plots (CPM versus running distance) need to be analyzed to identify single glycan species.